The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

PubMed Central

Vergis, James M.; Wiener, Michael C.

2011-01-01

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase, Factor Xa, Human Rhinovirus 3C Protease, SUMOstar, Tobacco Etch Virus Protease, and Thrombin) by use of a panel of ninety-four individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and Factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

PubMed

Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

2016-03-01

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Reversible chemoselective tagging and functionalization of methionine containing peptides.

PubMed

Kramer, Jessica R; Deming, Timothy J

2013-06-07

Reagents were developed to allow chemoselective tagging of methionine residues in peptides and polypeptides, subsequent bioorthogonal functionalization of the tags, and cleavage of the tags when desired. This methodology can be used for triggered release of therapeutic peptides, or release of tagged protein digests from affinity columns.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a smallmore » change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.« less

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Optimal use of tandem biotin and V5 tags in ChIP assays

PubMed Central

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications.

PubMed

Young, Carissa L; Britton, Zachary T; Robinson, Anne S

2012-05-01

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology.

PubMed

Peccerella, Teresa; Lukan, Nadine; Hofheinz, Ralf; Schadendorf, Dirk; Kostrezewa, Markus; Neumaier, Michael; Findeisen, Peter

2010-02-01

The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant." Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

PubMed

Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

2014-11-01

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

PubMed

Besbrugge, Nienke; Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; Kulkarni, Shubhada R; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Bontinck, Michiel; Aesaert, Stijn; Impens, Francis; Gevaert, Kris; Van Damme, Daniel; Van Lijsebettens, Mieke; Inzé, Dirk; Vandepoele, Klaas; Nelissen, Hilde; De Jaeger, Geert

2018-06-01

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS yellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS yellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS yellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research. © 2018 American Society of Plant Biologists. All rights reserved.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: Insights into their use as tools for the removal of affinity tags

PubMed Central

Austin, Brian P.; Tözsér, József; Bagossi, Péter; Tropea, Joseph E.; Waugh, David S.

2012-01-01

Carboxypeptidases may serve as tools for removal for C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint. PMID:21073956

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor

PubMed Central

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G.M.

2017-01-01

Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. Here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker. The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. PMID:27867058

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

HaloTag Technology: A Versatile Platform for Biomedical Applications

PubMed Central

2015-01-01

Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications. PMID:25974629

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tykvart, J.; Sacha, P.; Barinka, C.

2012-02-07

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo.more » We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.« less

Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

PubMed

Sun, Ying-Chieh; Hsu, Wen-Chi; Hsu, Chia-Jen; Chang, Chia-Ming; Cheng, Kai-Hsiang

2015-11-01

Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcal mol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcal mol(-1). The present TI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0 kcal mol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5 kcal mol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcal mol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ΔG values. The error bars in the computed ΔG values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/dλ. The estimated correlation time was ~0.5 ps and the error bar in the ΔG values estimated using nanosecond-scale simulations was ±0.3 kcal mol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy.

Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.

PubMed

Moldes, Cristina; García, José L; García, Pedro

2004-08-01

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo.

PubMed

Gatterdam, Karl; Joest, Eike F; Gatterdam, Volker; Tampé, Robert

2018-05-29

Small chemical/biological interaction pairs are at the forefront in tracing proteins' function and interaction at high signal-to-background ratio in cellular pathways. Pharma ventures have eager plans to develop trisNTA probes for in vitro and in vivo screening of His-tagged protein targets. However, the optimal design of scaffold, linker, and chelator head yet deserves systematic investigations to achieve highest affinity and kinetic stability for in vitro and especially cell applications. In this study, we report on a library of N-nitrilotriacetic acid (NTA) based multivalent chelator heads (MCHs) built up on linear, cyclic, and dendritic scaffolds and contrast these with regard to their binding affinity and stability for labeling of cellular His-tagged proteins. Furthermore, we assign a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we describe fundamental differences between the MCH scaffold and define a cyclic trisNTA chelator, which displays the highest affinity and kinetic stability of all reversible, low-molecular weight interaction pairs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

Impact of an N-terminal Poly Histidine Tag on Protein Thermal Stability

USDA-ARS?s Scientific Manuscript database

For years, the use of polyhistidine tags (His-tags) have been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a cons...

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

PubMed Central

2013-01-01

Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient. PMID:23687945

The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

PubMed

Amarasinghe, Chinthaka; Jin, Jian-Ping

2015-01-01

Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads. PMID:22688388

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

PubMed

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

PubMed Central

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible. PMID:23874971

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

PubMed

Chen, Jia-Liang; Zhao, Yu; Gong, Yan-Jun; Pan, Bin-Bin; Wang, Xiao; Su, Xun-Cheng

2018-02-01

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

Self-Powered Wireless Affinity-Based Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled RFID Antennas.

PubMed

Yuan, Mingquan; Alocilja, Evangelyn C; Chakrabartty, Shantanu

2016-08-01

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

PubMed

Zilio, Nicola; Boddy, Michael N

2017-03-01

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins. © 2017 Cold Spring Harbor Laboratory Press.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

PubMed

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.

PubMed

Imanaka, Hiroyuki; Yamadzumi, Daisuke; Yanagita, Keisuke; Ishida, Naoyuki; Nakanishi, Kazuhiro; Imamura, Koreyoshi

2016-03-01

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016. © 2016 American Institute of Chemical Engineers.

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

PubMed

Li, Junhua; Zhang, Yang; Yang, Yanjun

2013-03-01

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Tianqing; Ming, Hongyan; Deng, Lili

Recombinant scorpion anti-excitation peptide (rANEP) has previously been expressed using the pET32a system and purified via affinity chromatography. However, rANEP is expressed in BL21(DE3) cells as an inclusion body, and the affinity tag can not be removed. To overcome this problem, we used a variety of protein, DsbA, MBP, TrxA, intein, and affinity tags in fusion and co-expression to achieve soluble and functional rANEP without any affinity tag. In the pCIT-ANEP expression vector, the highest soluble expression level was approximately 90% of the total cellular proteins in E. coli, and the rANEP was cleaved by the intein protein and subsequently purifiedmore » to obtain rANEP, which had the same activity as the natural ANEP. The purity of rANEP obtained using this method was over 95%, with a quantity of 5.1 mg from of purified rANEP from 1 L of culture. This method could expand the application of the soluble expression of disulfide-rich peptides in E. coli.« less

Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

USDA-ARS?s Scientific Manuscript database

Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

Overview of the Purification of Recombinant Proteins

PubMed Central

Wingfield, Paul T.

2015-01-01

When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

Overview of the purification of recombinant proteins.

PubMed

Wingfield, Paul T

2015-04-01

When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

PubMed

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G M

2017-03-01

Human cannabinoid receptor CB 2 belongs to the class A of G protein-coupled receptor (GPCR). CB 2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB 2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB 2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB 2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB 2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB 2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB 2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. Published by Elsevier Inc.

Bidirectional immobilization of affinity-tagged cytochrome c on electrode surfaces.

PubMed

Schröper, Florian; Baumann, Arnd; Offenhäusser, Andreas; Mayer, Dirk

2010-08-07

Here, we report a new strategy for the directed bivalent immobilization of cyt c on or between gold electrodes. C-terminal modification with cys- or his-tag did not affect the functional integrity of the protein. In combination with electrostatic protein binding, these tags enable a bifunctional immobilization between two electrodes or alternatively one electrode and interacting enzymes.

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

PubMed Central

Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

2014-01-01

SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

PubMed Central

Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

2014-01-01

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

PubMed Central

Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

2012-01-01

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

PubMed

Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

2004-06-01

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

Barley as a green factory for the production of functional Flt3 ligand.

PubMed

Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

2010-02-01

Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

PubMed

Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

2015-08-20

For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

PubMed

Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

2015-09-01

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular Targets of Dietary Polyphenol Resveratrol

DTIC Science & Technology

2005-03-01

attempts to generate affinity columns tagged with other polyphenols, e.g., epigallocatechin gallate ( EGCG ). Conceivably such columns, if generated, would...Similar affinity chromatography with the related polyphenol Epigallocatechin gallate does not produce similar results.” Answer: We did not make...addition, the PI does not provid expression. If there is “increased ex many bind the resveratrol affinity co related polyphenol Epigallocatechin Response

High-throughput purification of recombinant proteins using self-cleaving intein tags.

PubMed

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

PubMed

Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

2016-03-15

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

PubMed

London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

2015-04-01

Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel strategy using MASCOT Distiller for analysis of cleavable isotope-coded affinity tag data to quantify protein changes in plasma.

PubMed

Leung, Kit-Yi; Lescuyer, Pierre; Campbell, James; Byers, Helen L; Allard, Laure; Sanchez, Jean-Charles; Ward, Malcolm A

2005-08-01

A novel strategy consisting of cleavable Isotope-Coded Affinity Tag (cICAT) combined with MASCOT Distiller was evaluated as a tool for the quantification of proteins in "abnormal" patient plasma, prepared by pooling samples from patients with acute stroke. Quantification of all light and heavy cICAT-labelled peptide ion pairs was obtained using MASCOT Distiller combined with a proprietary software. Peptides displaying differences were selected for identification by MS. These preliminary results show the promise of our approach to identify potential biomarkers.

Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

PubMed

Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

2014-07-18

This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

In vivo expression and purification of aptamer-tagged small RNA regulators

PubMed Central

Said, Nelly; Rieder, Renate; Hurwitz, Robert; Deckert, Jochen; Urlaub, Henning; Vogel, Jörg

2009-01-01

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria. PMID:19726584

DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

PubMed Central

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

Purification of bacteriophage lambda repressor

PubMed Central

Gao, Ning; Shearwin, Keith; Mack, John; Finzi, Laura; Dunlap, David

2013-01-01

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops. PMID:23831434

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

PubMed Central

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface. PMID:29360877

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

PubMed

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.

PubMed

Giusti, Fabrice; Kessler, Pascal; Hansen, Randi Westh; Della Pia, Eduardo A; Le Bon, Christel; Mourier, Gilles; Popot, Jean-Luc; Martinez, Karen L; Zoonens, Manuela

2015-12-14

Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

Jing, Chaoran; Cornish, Virginia W

2013-01-01

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

Enhanced Reactive Oxygen Species Scavenging by Overproduction of Superoxide Dismutase and Catalase Delays Postharvest Physiological Deterioration of Cassava Storage Roots1[C][W][OA

PubMed Central

Xu, Jia; Duan, Xiaoguang; Yang, Jun; Beeching, John R.; Zhang, Peng

2013-01-01

Postharvest physiological deterioration (PPD) of cassava (Manihot esculenta) storage roots is the result of a rapid oxidative burst, which leads to discoloration of the vascular tissues due to the oxidation of phenolic compounds. In this study, coexpression of the reactive oxygen species (ROS)-scavenging enzymes copper/zinc superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) in transgenic cassava was used to explore the intrinsic relationship between ROS scavenging and PPD occurrence. Transgenic cassava plants integrated with the expression cassette p54::MeCu/ZnSOD-35S::MeCAT1 were confirmed by Southern-blot analysis. The expression of MeCu/ZnSOD and MeCAT1 was verified by quantitative reverse transcription-polymerase chain reaction and enzymatic activity analysis both in the leaves and storage roots. Under exposure to the ROS-generating reagent methyl viologen or to hydrogen peroxide (H2O2), the transgenic plants showed higher enzymatic activities of SOD and CAT than the wild-type plants. Levels of malondialdehyde, chlorophyll degradation, lipid peroxidation, and H2O2 accumulation were dramatically reduced in the transgenic lines compared with the wild type. After harvest, the storage roots of transgenic cassava lines show a delay in their PPD response of at least 10 d, accompanied by less mitochondrial oxidation and H2O2 accumulation, compared with those of the wild type. We hypothesize that this is due to the combined ectopic expression of Cu/ZnSOD and CAT leading to an improved synergistic ROS-scavenging capacity of the roots. Our study not only sheds light on the mechanism of the PPD process but also develops an effective approach for delaying the occurrence of PPD in cassava. PMID:23344905

Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots.

PubMed

Xu, Jia; Duan, Xiaoguang; Yang, Jun; Beeching, John R; Zhang, Peng

2013-03-01

Postharvest physiological deterioration (PPD) of cassava (Manihot esculenta) storage roots is the result of a rapid oxidative burst, which leads to discoloration of the vascular tissues due to the oxidation of phenolic compounds. In this study, coexpression of the reactive oxygen species (ROS)-scavenging enzymes copper/zinc superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) in transgenic cassava was used to explore the intrinsic relationship between ROS scavenging and PPD occurrence. Transgenic cassava plants integrated with the expression cassette p54::MeCu/ZnSOD-35S::MeCAT1 were confirmed by Southern-blot analysis. The expression of MeCu/ZnSOD and MeCAT1 was verified by quantitative reverse transcription-polymerase chain reaction and enzymatic activity analysis both in the leaves and storage roots. Under exposure to the ROS-generating reagent methyl viologen or to hydrogen peroxide (H2O2), the transgenic plants showed higher enzymatic activities of SOD and CAT than the wild-type plants. Levels of malondialdehyde, chlorophyll degradation, lipid peroxidation, and H2O2 accumulation were dramatically reduced in the transgenic lines compared with the wild type. After harvest, the storage roots of transgenic cassava lines show a delay in their PPD response of at least 10 d, accompanied by less mitochondrial oxidation and H2O2 accumulation, compared with those of the wild type. We hypothesize that this is due to the combined ectopic expression of Cu/ZnSOD and CAT leading to an improved synergistic ROS-scavenging capacity of the roots. Our study not only sheds light on the mechanism of the PPD process but also develops an effective approach for delaying the occurrence of PPD in cassava.

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

PubMed

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

PubMed

Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

2014-12-01

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

PubMed Central

Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

2007-01-01

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

ERIC Educational Resources Information Center

Bailey, Cheryl P.

2009-01-01

This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

PubMed

Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

2011-04-01

Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs.

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

PubMed

Ryan, Daniel P; Tremethick, David J

2016-04-01

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C. Copyright © 2015 Elsevier Inc. All rights reserved.

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

PubMed

Ferguson, A D; Breed, J; Diederichs, K; Welte, W; Coulton, J W

1998-07-01

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

PubMed

Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

2010-01-01

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui

2013-05-01

The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-bindingmore » pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.« less

Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

PubMed Central

De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

2014-01-01

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation. PMID:24599037

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

PubMed

De Souza, Colin P; Hashmi, Shahr B; Osmani, Aysha H; Osmani, Stephen A

2014-01-01

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco.

PubMed

Nagaki, Kiyotaka; Shibata, Fukashi; Kanatani, Asaka; Kashihara, Kazunari; Murata, Minoru

2012-04-01

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres. © Springer-Verlag 2011

Host Immune Selection of Rumen Bacteria through Salivary Secretory IgA

PubMed Central

Fouhse, Janelle M.; Smiegielski, Luke; Tuplin, Melanie; Guan, Le Luo; Willing, Benjamin P.

2017-01-01

The rumen microbiome is integral to efficient production in cattle and shows strong host specificity, yet little is known about what host factors shape rumen microbial composition. Secretory immunoglobulin A (SIgA) is produced in large amounts in the saliva, can coat both commensal and pathogenic microbes within the gut, and presents a plausible mechanism of host specificity. However, the role salivary SIgA plays in commensal bacteria selection in ruminants remains elusive. The main objectives of this study were to develop an immuno-affinity benchtop method to isolate SIgA-tagged microbiota and to determine if salivary SIgA preferentially binds selected bacteria. We hypothesized that SIgA-tagged bacteria would differ from total bacteria, thus supporting a potential host-derived mechanism in commensal bacterial selection. Whole rumen (n = 9) and oral secretion samples (n = 10) were incubated with magnetic beads conjugated with anti-secretory IgA antibodies to enrich SIgA-tagged microbiota. Microbial DNA from the oral secretion, whole rumen, SIgA-tagged oral secretion, and SIgA-tagged rumen was isolated for amplicon sequencing of V1–V3 region of 16S rDNA genes. Whole rumen and oral secretion had distinctive (P < 0.05) bacterial compositions indicated by the non-parametric multidimensional scaling plot using Euclidean distance metrics. The SIgA-tagged microbiota from rumen and oral secretion had similar abundance of Bacteroidetes, Actinobacteria, Fibrobacter, candidate phyla TM7, and Tenericutes and are clustered tightly. Composition of SIgA-tagged oral secretion microbiota was more similar to whole rumen microbiota than whole oral secretion due to enrichment of rumen bacteria (Lachnospiraceae) and depletion of oral taxa (Streptococcus, Rothia, Neisseriaceae, and Lactobacillales). In conclusion, SIgA-tagged oral secretion microbiota had an increased resemblance to whole rumen microbiota, suggesting salivary SIgA-coating may be one host-derived mechanism impacting commensal colonization. Further studies, to explore the variations in antibody affinity between different animals as a driver of microbial composition are warranted. PMID:28553275

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

PubMed

Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

2006-10-01

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

PubMed

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2016-02-01

Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

Strep-Tagged Protein Purification.

PubMed

Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

2015-01-01

The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts.

PubMed

von Haller, Priska D; Yi, Eugene; Donohoe, Samuel; Vaughn, Kelly; Keller, Andrew; Nesvizhskii, Alexey I; Eng, Jimmy; Li, Xiao-jun; Goodlett, David R; Aebersold, Ruedi; Watts, Julian D

2003-07-01

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules.

PubMed

Tsunehiro, Masaya; Meki, Yuma; Matsuoka, Kanako; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2013-04-15

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol. Copyright © 2013 Elsevier B.V. All rights reserved.

System and method for a parallel immunoassay system

DOEpatents

Stevens, Fred J.

2002-01-01

A method and system for detecting a target antigen using massively parallel immunoassay technology. In this system, high affinity antibodies of the antigen are covalently linked to small beads or particles. The beads are exposed to a solution containing DNA-oligomer-mimics of the antigen. The mimics which are reactive with the covalently attached antibody or antibodies will bind to the appropriate antibody molecule on the bead. The particles or beads are then washed to remove any unbound DNA-oligomer-mimics and are then immobilized or trapped. The bead-antibody complexes are then exposed to a test solution which may contain the targeted antigens. If the antigen is present it will replace the mimic since it has a greater affinity for the respective antibody. The particles are then removed from the solution leaving a residual solution. This residual solution is applied a DNA chip containing many samples of complimentary DNA. If the DNA tag from a mimic binds with its complimentary DNA, it indicates the presence of the target antigen. A flourescent tag can be used to more easily identify the bound DNA tag.

Quantitative interaction proteomics using mass spectrometry.

PubMed

Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

2009-03-01

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

PubMed

Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

2015-12-01

To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

A General Strategy for Targeting Drugs to Bone.

PubMed

Jahnke, Wolfgang; Bold, Guido; Marzinzik, Andreas L; Ofner, Silvio; Pellé, Xavier; Cotesta, Simona; Bourgier, Emmanuelle; Lehmann, Sylvie; Henry, Chrystelle; Hemmig, René; Stauffer, Frédéric; Hartwieg, J Constanze D; Green, Jonathan R; Rondeau, Jean-Michel

2015-11-23

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of the Mechanism of Electron Capture and Electron Transfer Dissociation of Peptides with a Covalently Attached Free Radical Hydrogen Atom Scavenger.

PubMed

Sohn, Chang Ho; Yin, Sheng; Peng, Ivory; Loo, Joseph A; Beauchamp, J L

2015-11-15

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQX TEMPO EEQQQTEDELQDK. The X TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (b H ) and y + H (y H ) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

In vivo phosphorylation of a peptide tag for protein purification.

PubMed

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

2016-05-01

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

NASA Technical Reports Server (NTRS)

Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

2000-01-01

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2017-01-01

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

PubMed

Chen, Gen-Hung; Yin, Li-Jung; Chiang, I-Hua; Jiang, Shann-Tzong

2008-12-01

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

Generic tags for Mn(ii) and Gd(iii) spin labels for distance measurements in proteins.

PubMed

Yang, Yin; Gong, Yan-Jun; Litvinov, Aleksei; Liu, Hong-Kai; Yang, Feng; Su, Xun-Cheng; Goldfarb, Daniella

2017-10-11

High-affinity chelating tags for Gd(iii) and Mn(ii) ions that provide valuable high-resolution distance restraints for biomolecules were used as spin labels for double electron-electron resonance (DEER) measurements. The availability of a generic tag that can bind both metal ions and provide a narrow and predictable distance distribution for both ions is attractive owing to their different EPR-related characteristics. Herein we introduced two paramagnetic tags, 4PSPyMTA and 4PSPyNPDA, which are conjugated to cysteine residues through a stable thioether bond, forming a short and, depending on the metal ion coordination mode, a rigid tether with the protein. These tags exhibit high affinity for both Mn(ii) and Gd(iii) ions. The DEER performance of the 4PSPyMTA and 4PSPyNPDA tags, in complex with Gd(iii) or Mn(ii), was evaluated for three double cysteine mutants of ubiquitin, and the Gd(iii)-Gd(iii) and Mn(ii)-Mn(ii) distance distributions they generated were compared. All three Gd(iii) complexes of the ubiquitin-PyMTA and ubiquitin-PyNPDA conjugates produced similar and expected distance distributions. In contrast, significant variations in the maxima and widths of the distance distributions were observed for the Mn(ii) analogs. Furthermore, whereas PyNPDA-Gd(iii) and PyNPDA-Mn(ii) delivered similar distance distributions, appreciable differences were observed for two mutants with PyMTA, with the Mn(ii) analog exhibiting a broader distance distribution and shorter distances. ELDOR (electron-electron double resonance)-detected NMR measurements revealed some distribution in the Mn(ii) coordination environment for the protein conjugates of both tags but not for the free tags. The broader distance distributions generated by 4PSPyMTA-Mn(ii), as compared with Gd(iii), were attributed to the distributed location of the Mn(ii) ion within the PyMTA chelate owing to its smaller size and lower coordination number that leave the pyridine nitrogen uncoordinated. Accordingly, in terms of distance resolution, 4PSPyNPDA can serve as an effective generic tag for Gd(iii) and Mn(ii), whereas 4PSPyMTA is efficient for Gd(iii) only. This comparison between Gd(iii) and Mn(ii) suggests that PyMTA model compounds may not predict sufficiently well the performance of PyMTA-Mn(ii) as a tag for high-resolution distance measurements in proteins because the protein environment can influence its coordination mode.

Cobalt carbonyl complexes as probes for alkyne-tagged lipids[S

PubMed Central

Tallman, Keri A.; Armstrong, Michelle D.; Milne, Stephen B.; Marnett, Lawrence J.; Brown, H. Alex; Porter, Ned A.

2013-01-01

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection. PMID:23307946

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

PubMed

White, Jim F; Grisshammer, Reinhard

2010-09-07

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

PSBinder: A Web Service for Predicting Polystyrene Surface-Binding Peptides.

PubMed

Li, Ning; Kang, Juanjuan; Jiang, Lixu; He, Bifang; Lin, Hao; Huang, Jian

2017-01-01

Polystyrene surface-binding peptides (PSBPs) are useful as affinity tags to build a highly effective ELISA system. However, they are also a quite common type of target-unrelated peptides (TUPs) in the panning of phage-displayed random peptide library. As TUP, PSBP will mislead the analysis of panning results if not identified. Therefore, it is necessary to find a way to quickly and easily foretell if a peptide is likely to be a PSBP or not. In this paper, we describe PSBinder, a predictor based on SVM. To our knowledge, it is the first web server for predicting PSBP. The SVM model was built with the feature of optimized dipeptide composition and 87.02% (MCC = 0.74; AUC = 0.91) of peptides were correctly classified by fivefold cross-validation. PSBinder can be used to exclude highly possible PSBP from biopanning results or to find novel candidates for polystyrene affinity tags. Either way, it is valuable for biotechnology community.

Affinity monolith-integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis.

PubMed

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T

2008-07-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Affinity Monolith-Integrated Poly(methyl Methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

PubMed Central

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T.

2008-01-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis. PMID:18479142

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

PubMed

Trachman, Robert J; Abdolahzadeh, Amir; Andreoni, Alessio; Cojocaru, Razvan; Knutson, Jay R; Ryckelynck, Michael; Unrau, Peter J; Ferré-D'Amaré, Adrian R

2018-05-24

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

ONE-STEP METAL-AFFINITY PURIFICATION OF HISTIDINE-TAGGED PROTEINS BY TEMPERATURE-TRIGGERED PRECIPITATION. (R829606)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

PubMed

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ocean Heat Content Reveals Secrets of Fish Migrations

PubMed Central

Luo, Jiangang; Ault, Jerald S.; Shay, Lynn K.; Hoolihan, John P.; Prince, Eric D.; Brown, Craig A.; Rooker, Jay R.

2015-01-01

For centuries, the mechanisms surrounding spatially complex animal migrations have intrigued scientists and the public. We present a new methodology using ocean heat content (OHC), a habitat metric that is normally a fundamental part of hurricane intensity forecasting, to estimate movements and migration of satellite-tagged marine fishes. Previous satellite-tagging research of fishes using archival depth, temperature and light data for geolocations have been too coarse to resolve detailed ocean habitat utilization. We combined tag data with OHC estimated from ocean circulation and transport models in an optimization framework that substantially improved geolocation accuracy over SST-based tracks. The OHC-based movement track provided the first quantitative evidence that many of the tagged highly migratory fishes displayed affinities for ocean fronts and eddies. The OHC method provides a new quantitative tool for studying dynamic use of ocean habitats, migration processes and responses to environmental changes by fishes, and further, improves ocean animal tracking and extends satellite-based animal tracking data for other potential physical, ecological, and fisheries applications. PMID:26484541

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.

PubMed

Pratt, Susan E; Speltz, Elizabeth B; Mochrie, Simon G J; Regan, Lynne

2016-09-02

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of the STAT3 interactome using in-situ biotinylation and SILAC.

PubMed

Blumert, Conny; Kalkhof, Stefan; Brocke-Heidrich, Katja; Kohajda, Tibor; von Bergen, Martin; Horn, Friedemann

2013-12-06

Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein-protein interaction effectively and comprehensively. The location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3. With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications. © 2013. Published by Elsevier B.V. All rights reserved.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein.

PubMed

Monteiro, Rose A; Souza, Emanuel M; Geoffrey Yates, M; Steffens, M Berenice R; Pedrosa, Fábio O; Chubatsu, Leda S

2003-02-01

The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription. Copyright 2002 Elsevier Science (USA)

Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease.

PubMed

Li, Junhua; Zhang, Yang; Shen, Fei; Yang, Yanjun

2012-10-15

A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups - carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

A new pH-responsive peptide tag for protein purification.

PubMed

Nonaka, Takahiro; Tsurui, Noriko; Mannen, Teruhisa; Kikuchi, Yoshimi; Shiraki, Kentaro

2018-06-01

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH. Copyright © 2018 Elsevier Inc. All rights reserved.

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

PubMed

Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

2002-12-01

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

PubMed

Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

2015-02-01

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-01-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle. PMID:23649778

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

PubMed

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

HaloTag technology for specific and covalent labeling of fusion proteins.

PubMed

Benink, Hélène A; Urh, Marjeta

2015-01-01

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

A two-step strategy to visually identify molecularly imprinted polymers for tagged proteins.

PubMed

Brandis, Alexander; Partouche, Eran; Yechezkel, Tamar; Salitra, Yoseph; Shkoulev, Vladimir; Scherz, Avigdor; Grynszpan, Flavio

2017-08-01

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope-like) approach has been developed. In our two-step method, we first challenge a previously obtained anti-tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low-affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope-like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti-biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of Fe3O4@nickel-silicate core-shell nanoparticles for His-tagged enzyme immobilizing agents

NASA Astrophysics Data System (ADS)

Shin, Moo-Kwang; Kang, Byunghoon; Yoon, Nam-Kyung; Kim, Myeong-Hoon; Ki, Jisun; Han, Seungmin; Ahn, Jung-Oh; Haam, Seungjoo

2016-12-01

Immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. Specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various pH and temperatures. Herein, we fabricated Fe3O4 encapsulated ‘sea-urchin’ shaped nickel-silicate nanoparticles with a facile synthetic route. The enzymes were then rapidly and easily immobilized with poly-histidine tags (His-tags) and nickel ion affinity. Porous nickel silicate covered nanoparticles achieved a high immobilization capacity (85 μg mg-1) of His-tagged tobacco etch virus (TEV) protease. To investigate immobilized TEV protease enzymatic activity, we analyzed the cleaved quantity of maltose binding protein-exendin-fused immunoglobulin fusion protein, which connected with the TEV protease-specific cleavage peptide sequence. Moreover, TEV protease immobilized nanocomplexes conveniently removed and recollected from the reactant by applying an external magnetic field, maintained their enzymatic activity after reuse. Therefore, our newly developed nanoplatform for His-tagged enzyme immobilization provides advantageous features for biotechnological industries including recombinant protein processing.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1.

PubMed

Tirard, Marilyn; Brose, Nils

2016-01-01

Protein SUMOylation is a posttranslational protein modification that is emerging as a key regulatory process in neurobiology. To date, however, SUMOylation in vivo has only been studied cursorily. Knock-in mice expressing His6-HA-SUMO1 from the Sumo1 locus allow for the highly specific localization and identification of endogenous SUMO1 substrates under physiological and pathophysiological conditions. By making use of the HA-tag and using wild-type mice for highly stringent negative control samples, SUMO1 targets can be specifically localized in and purified from cultured mouse nerve cells and mouse tissues.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

PubMed Central

Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

2017-01-01

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model. PMID:28873407

Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

NASA Astrophysics Data System (ADS)

Liu, Fang

Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

Aptamer facilitated purification of functional proteins.

PubMed

Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

2018-01-15

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

PubMed

Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

2014-01-16

Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli.

PubMed

Borgnia, M J; Agre, P

2001-02-27

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

PubMed

Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

2015-10-21

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

Tail proteins of phage T5: investigation of the effect of the His6-tag position, from expression to crystallisation.

PubMed

Noirclerc-Savoye, Marjolaine; Flayhan, Ali; Pereira, Cindy; Gallet, Benoit; Gans, Pierre; Ebel, Christine; Breyton, Cécile

2015-05-01

Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality. Copyright © 2015 Elsevier Inc. All rights reserved.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

PubMed

Wang, Jianhao; Qin, Yuqin; Qin, Haifang; Liu, Li; Ding, Shumin; Teng, Yiwan; Ji, Junling; Qiu, Lin; Jiang, Pengju

2016-08-01

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Laboratory Exercise to Illustrate Protein-Membrane Interactions

ERIC Educational Resources Information Center

Weers, Paul M. M.; Prenner, Elmar J.; Curic, Spomenka; Lohmeier-Vogel, Elke M.

2016-01-01

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6-tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV,…

Proteomic validation of protease drug targets: pharmacoproteomics of matrix metalloproteinase inhibitor drugs using isotope-coded affinity tag labelling and tandem mass spectrometry.

PubMed

Butler, G S; Overall, C M

2007-01-01

We illustrate the use of quantitative proteomics, namely isotope-coded affinity tag labelling and tandem mass spectrometry, to assess the targets and effects of the blockade of matrix metalloproteinases by an inhibitor drug in a breast cancer cell culture system. Treatment of MT1-MMP-transfected MDA-MB-231 cells with AG3340 (Prinomastat) directly affected the processing a multitude of matrix metalloproteinase substrates, and indirectly altered the expression of an array of other proteins with diverse functions. Therefore, broad spectrum blockade of MMPs has wide-ranging biological consequences. In this human breast cancer cell line, secreted substrates accumulated uncleaved in the conditioned medium and plasma membrane protein substrates were retained on the cell surface, due to reduced processing and shedding of these proteins (cell surface receptors, growth factors and bioactive molecules) to the medium in the presence of the matrix metalloproteinase inhibitor. Hence, proteomic investigation of drug-perturbed cellular proteomes can identify new protease substrates and at the same time provides valuable information for target validation, drug efficacy and potential side effects prior to commitment to clinical trials.

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

PubMed Central

Liu, Wenhao; Howarth, Mark; Greytak, Andrew B.; Zheng, Yi; Nocera, Daniel G.; Ting, Alice Y.; Bawendi, Moungi G.

2009-01-01

We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor’s local chemical environment. PMID:18177042

Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.

PubMed

Twerdochlib, Adriana L; Chubatsu, Leda S; Souza, Emanuel M; Pedrosa, Fábio O; Steffens, M Berenice R; Yates, M Geoffrey; Rigo, Liu U

2003-07-01

NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

PubMed Central

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

PubMed

Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

2016-01-01

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bio-inorganic synthesis of ZnO powders using recombinant His-tagged ZnO binding peptide as a promoter.

PubMed

Song, Lei; Liu, Yingying; Zhang, Zhifang; Wang, Xi; Chen, Jinchun

2010-10-01

Inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs), are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. Recent advances in nanotechnology and molecular biology allow the engineering of these peptides with specific affinity to inorganics, often used as molecular linkers or assemblers, to facilitate materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here we report a novel biosynthetic ZnO binding protein and its application in promoting bio-inorganic materials synthesis. In brief, the gene encoding a ZnO binding peptide(ZBP) was genetically fused with His(6)-tag and GST-tag using E.coli expression vector pET-28a (+) and pGEX-4T-3. The recombinant protein GST-His-ZBP was expressed, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Affinity adsorption test demonstrated that the fusion protein had a specific avidity for ZnO nanoparticles (NPs). Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)(2) sol. X-ray diffraction (XRD) analysis on the final products after calcining the precursor powders showed that hexagonal wurtzite ZnO crystals were obtained. Our work suggested a novel approach to the application about the organic-inorganic interactions.

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

PubMed

Sarpong, Kwabena; Bose, Ron

2017-03-15

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

Functionalized Nano-adsorbent for Affinity Separation of Proteins

NASA Astrophysics Data System (ADS)

Zou, Xueyan; Yang, Fengbo; Sun, Xin; Qin, Mingming; Zhao, Yanbao; Zhang, Zhijun

2018-05-01

Thiol-functionalized silica nanospheres (SiO2-SH NSs) with an average diameter of 460 nm were synthesized through a hydrothermal route. Subsequently, the prepared SiO2-SH NSs were modified by SnO2 quantum dots to afford SnO2/SiO2 composite NSs possessing obvious fluorescence, which could be used to trace the target protein. The SnO2/SiO2 NSs were further modified by reduced glutathione (GSH) to obtain SnO2/SiO2-GSH NSs, which can specifically separate glutathione S-transferase-tagged (GST-tagged) protein. Moreover, the peroxidase activity of glutathione peroxidase 3 (GPX3) separated from SnO2/SiO2-GSH NSs in vitro was evaluated. Results show that the prepared SnO2/SiO2-GSH NSs exhibit negligible nonspecific adsorption, high concentration of protein binding (7.4 mg/g), and good reused properties. In the meantime, the GST-tagged GPX3 separated by these NSs can retain its redox state and peroxidase activity. Therefore, the prepared SnO2/SiO2-GSH NSs might find promising application in the rapid separation and purification of GST-tagged proteins.

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

PubMed

Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

2016-10-01

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Construction of stabilized and tagged foot-and-mouth disease virus.

PubMed

Park, Jeong-Nam; Ko, Mi-Kyeong; Kim, Rae-Hyung; Park, Min-Eun; Lee, Seo-Yong; Yoon, Ji-Eun; Choi, Joo-Hyung; You, Su-Hwa; Park, Jung-Won; Lee, Kwang-Nyeong; Chun, Ji-Eun; Kim, Su-Mi; Tark, Dongseob; Lee, Hyang-Sim; Ko, Young-Joon; Kim, Byounghan; Lee, Myoung-Heon; Park, Jong-Hyeon

2016-11-01

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins

NASA Astrophysics Data System (ADS)

Liu, Jia-Wei; Yang, Ting; Ma, Lin-Yu; Chen, Xu-Wei; Wang, Jian-Hua

2013-12-01

Nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g-1 at 10 000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged Smt A from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni2+ column.

Analysis of Ethylene Receptor Interactions by Co-immunoprecipitation Assays.

PubMed

Gao, Zhiyong; Schaller, G Eric

2017-01-01

Ethylene receptors are predominantly localized to the endoplasmic reticulum (ER) membrane, and coordinate ethylene signal output through protein-protein interactions with each other and additional signaling components. Here, we describe a co-immunoprecipitation (Co-IP) assay based on the use of the Tandem Affinity Purification (TAP) tag to examine the interactions of ethylene receptors in plant extracts. Human IgG-agarose beads are used to pull down TAP-tagged versions of the protein of interest from detergent extracts of Arabidopsis membranes, and the precipitate then is analyzed immunologically for co-purification of the ethylene receptors. This method has been successfully used to examine interactions of the receptors with each other as well as with the Raf-like kinase CTR1.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.

PubMed

Korshun, Ludmila; Vudmaska, Mariya; Moysa, Larissa; Kovtonjuk, Galina; Mikhalap, Svetlana; Ganova, Larissa; Spivak, Nikolay

2013-12-01

In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

PubMed

Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

2012-04-01

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. Copyright © 2012 American Society for Bone and Mineral Research.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

PubMed

Kumada, Yoichi; Otsuki, Ryoko; Sakoda, Yumiko; Akai, Ryota; Matoba, Kazutaka; Katayama, Junko; Kishimoto, Michimasa; Horiuchi, Jun-Ichi

2016-10-20

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

PubMed Central

Borchert, Sophie; Czech-Sioli, Manja; Neumann, Friederike; Schmidt, Claudia; Wimmer, Peter; Dobner, Thomas

2014-01-01

ABSTRACT Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential. PMID:24371076

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

PubMed

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

PubMed Central

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

Mass spectrometry-based methods for detection and differentiation of botulinum neurotoxins

DOEpatents

Schmidt, Jurgen G [Los Alamos, NM; Boyer, Anne E [Atlanta, GA; Kalb, Suzanne R [Atlanta, GA; Moura, Hercules [Tucker, GA; Barr, John R [Suwannee, GA; Woolfitt, Adrian R [Atlanta, GA

2009-11-03

The present invention is directed to a method for detecting the presence of clostridial neurotoxins in a sample by mixing a sample with a peptide that can serve as a substrate for proteolytic activity of a clostridial neurotoxin; and measuring for proteolytic activity of a clostridial neurotoxin by a mass spectroscopy technique. In one embodiment, the peptide can have an affinity tag attached at two or more sites.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

PubMed

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

GLUT-3 expression in human skeletal muscle

NASA Technical Reports Server (NTRS)

Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

2000-01-01

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions

PubMed Central

Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

2015-01-01

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

Graphene Field-Effect Transistors for the Sensitive and Selective Detection of Escherichia coli Using Pyrene-Tagged DNA Aptamer.

PubMed

Wu, Guangfu; Dai, Ziwen; Tang, Xin; Lin, Zihong; Lo, Pik Kwan; Meyyappan, M; Lai, King Wai Chiu

2017-10-01

This study reports biosensing using graphene field-effect transistors with the aid of pyrene-tagged DNA aptamers, which exhibit excellent selectivity, affinity, and stability for Escherichia coli (E. coli) detection. The aptamer is employed as the sensing probe due to its advantages such as high stability and high affinity toward small molecules and even whole cells. The change of the carrier density in the probe-modified graphene due to the attachment of E. coli is discussed theoretically for the first time and also verified experimentally. The conformational change of the aptamer due to the binding of E. coli brings the negatively charged E. coli close to the graphene surface, increasing the hole carrier density efficiently in graphene and achieving electrical detection. The binding of negatively charged E. coli induces holes in graphene, which are pumped into the graphene channel from the contact electrodes. The carrier mobility, which correlates the gate voltage to the electrical signal of the APG-FETs, is analyzed and optimized here. The excellent sensing performance such as low detection limit, high sensitivity, outstanding selectivity and stability of the graphene biosensor for E. coli detection paves the way to develop graphene biosensors for bacterial detection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats.

PubMed

Pershad, Kritika; Sullivan, Mark A; Kay, Brian K

2011-05-15

Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a "drop-out" phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to "drop-out" the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments. Copyright © 2011 Elsevier Inc. All rights reserved.

Synthesis of poly(N-isopropylacrylamide) particles for metal affinity binding of peptides

PubMed Central

Tsai, Hsin-Yi; Lee, Alexander; Peng, Wei; Yates, Matthew Z.

2013-01-01

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgel particles with metal affinity ligands were prepared for selective binding of peptides containing the His6-tag (six consecutive histidine residues). The PNIPAM particles were copolymerized with the functional ligand vinylbenzyl iminodiacetic acid (VBIDA) through a two-stage dispersion polymerization using poly(N-vinyl pyrrolidone) (PVP) as a steric stabilizer. The resulting particles were monodisperse in size and colloidally stable over a wide range of temperature and ionic strength due to chemically grafted PVP chains. The particle size was also found to be sensitive to ionic strength and pH of the aqueous environment, likely due to the electrostatic repulsion between ionized VBIDA groups. Divalent nickel ions were chelated to the VBIDA groups, allowing selective metal affinity attachment of a His6-Cys peptide. The peptide was released upon the addition of the competitive ligand imidazole, demonstrating that the peptide attachment to the particles is reversible and selective. PMID:24176889

Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

NASA Astrophysics Data System (ADS)

Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

2017-07-01

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

PubMed

de Aquino, Luciana Cristina Lins; de Sousa, Heloisa Ribeiro Tunes; Miranda, Everson Alves; Vilela, Luciano; Bueno, Sônia Maria Alves

2006-04-13

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

PubMed Central

Fujishiro, Mitsuhiro; Okamura, Tomohisa; Fujio, Keishi; Okazaki, Hiroaki; Nomura, Seitaro; Takeda, Norifumi; Harada, Mutsuo; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Yamamoto, Kazuhiko; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays. PMID:26020647

Identification of novel lysosomal matrix proteins by proteome analysis.

PubMed

Kollmann, Katrin; Mutenda, Kudzai E; Balleininger, Martina; Eckermann, Ellen; von Figura, Kurt; Schmidt, Bernhard; Lübke, Torben

2005-10-01

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.

A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

PubMed

Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

2014-03-26

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

A Suite of "Minimalist" Photo-Crosslinkers for Live-Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors.

PubMed

Pan, Sijun; Jang, Se-Young; Wang, Danyang; Liew, Si Si; Li, Zhengqiu; Lee, Jun-Seok; Yao, Shao Q

2017-09-18

Affinity-based probes (AfBPs) provide a powerful tool for large-scale chemoproteomic studies of drug-target interactions. The development of high-quality probes capable of recapitulating genuine drug-target engagement, however, could be challenging. "Minimalist" photo-crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine-containing photo-crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of AfBPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug-target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC-based chemoproteomic studies have led to the discovery of a novel off-target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up-regulation of endogenous DNMT1 expression under normoxia conditions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

PubMed Central

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant. PMID:26539718

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

PubMed

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

PubMed

Nilsson, J; Bosnes, M; Larsen, F; Nygren, P A; Uhlén, M; Lundeberg, J

1997-04-01

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

Identification of Small RNA-Protein Partners in Plant Symbiotic Bacteria.

PubMed

Robledo, Marta; Matia-González, Ana M; García-Tomsig, Natalia I; Jiménez-Zurdo, José I

2018-01-01

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin. As proof of principle, we show recovery of the RNA chaperone Hfq associated to the strictly Hfq-dependent AbcR2 trans-sRNA. This method can be applied for the investigation of sRNA-protein interactions on a broad range of genetically tractable α-proteobacteria.

Decoration of silicon nanostructures with copper particles for simultaneous selective capture and mass spectrometry detection of His-tagged model peptide.

PubMed

Coffinier, Yannick; Kurylo, Ievgen; Drobecq, Hervé; Szunerits, Sabine; Melnyk, Oleg; Zaitsev, Vladimir N; Boukherroub, Rabah

2014-10-21

We present in this work a simple and fast preparation method of a new affinity surface-assisted laser/desorption ionization mass spectrometry (SALDI-MS) substrate based on silicon nanostructures decorated with copper particles. The silicon nanostructures were fabricated by the metal-assisted chemical etching (MACE) method. Then, superhydrophilic areas surrounded by superhydrophobic regions were formed through hydrosilylation reaction of 1-octadecene, followed by local degradation of the octadecyl layer. After that, copper particles were deposited in the hydrophilic areas by using the electroless method. We have demonstrated that these surfaces were able to perform high selective capture of model His-tag peptide even in a complex mixture such as serum solution. Then, the captured peptide was detected by mass spectrometry at a femtomolar level without the need of organic matrix.

Regulation of the catalytic behavior of pullulanases chelated onto nickel (II)-modified magnetic nanoparticles.

PubMed

Wang, Jianfeng; Liu, Zhongmei; Zhou, Zhemin

2017-06-01

Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior. When pullulanases were chelated onto the surface-modified MNPs, the thermostability of all pullulanase derivatives were lower than that of free counterpart, being not relevant to the protein orientation guided by the locality of the His6-tag, but related to the MNPs basal surface polarity and the grafted spacer length. After chelating of pullulanases onto MNPs, there were changes observed in the pH-activity profile and the apparent Michaelis constant toward pullulan. The changing tendencies were mainly dependent on the His6-tagged pullulanase orientation, and the changing extents were tuned by the spacer length. The reusability of pullulanase immobilized by N-terminal His6-tag was higher than that of pullulanase immobilized by C-terminal His6-tag. Moreover, the reusability of the immobilized pullulanase tested increased till grafting polyether amine-400 as spacer-arm, therefore the N-terminal His6-tagged pullulanase chelating MNPs grafted polyether amine-400 gave the best reusability, which retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h. Additionally, the correlation analysis of the catalyst behaviors indicated that the reusability was independent from other catalytic properties such as thermostability and substrate affinity. All the results revealed that the catalyst behavior can be mainly controlled by the His6-tagged pullulanase orientation than by the MNPs surface property which can tune the catalyst function. Copyright © 2017. Published by Elsevier Inc.

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.

PubMed

Tackett, Alan J; DeGrasse, Jeffrey A; Sekedat, Matthew D; Oeffinger, Marlene; Rout, Michael P; Chait, Brian T

2005-01-01

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.

2006-08-01

Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with amore » His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.« less

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

PubMed

Guillén-Chable, Francisco; Arenas-Sosa, Iván; Islas-Flores, Ignacio; Corzo, Gerardo; Martinez-Liu, Cynthia; Estrada, Georgina

2017-08-01

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

PubMed

Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

2017-05-28

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

PubMed Central

Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

PubMed

Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

PubMed Central

Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-01

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

PubMed Central

Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro

2016-01-01

Abstract Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies. PMID:27595817

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad Javad; Forouzandeh-Moghadam, Mehdi; Allameh, Abdol-Amir

2005-11-01

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

PubMed

Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-15

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

[Identification of the interacting proteins with S100A8 or S100A9 by affinity purification and mass spectrometry].

PubMed

Wang, Jing; Zhang, Xuemei; Li, Zheng; Li, Xiayu; Ma, Jian; Shen, Shourong

2017-04-28

To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS).
 Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins. Co-immunoprecipitation (Co-IP) was applied to confirm the proteins interacted with S100A8 or S100A9.
 Results: Fourteen proteins including pyruvate kinase, muscle (PKM), nucleophosmin (NPM1) and eukaryotic translation initiation factor 5A (EIF5A), which potentially interacted with S100A8, were successfully identified by Flag-tag affinity purification followed by LC-MS/MS analysis. Six proteins, such as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (14-3-3ε) and PKM, which potentially interacted with S100A9, were successfully identified. Gene ontology analysis of the identified proteins suggested that proteins interacted with S100A8 or S100A9 were involved in several biological pathways, including canonical glycolysis, positive regulation of NF-κB transcription factor activity, negative regulation of apoptotic process, cell-cell adhesion, etc. Co-IP experiment confirmed that PKM2 can interact with both S100A8 and S100A9, and 14-3-3ε can interact with S100A8.
 Conclusion: PKM2 is identified to interact with both S100A8 and S100A9, while 14-3-3ε can interact with S100A9. These results may provide a new clue for the role of S100A8 or S100A9 in the progression of colitis-associated colorectal cancer.

Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

PubMed

Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

2013-07-01

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

Identification of Crosslinked Peptides after Click-based Enrichment Using Sequential CID and ETD Tandem Mass Spectrometry

PubMed Central

Chowdhury, Saiful M.; Du, Xiuxia; Tolić, Nikola; Wu, Si; Moore, Ronald J.; Mayer, M. Uljana; Smith, Richard D.; Adkins, Joshua N.

2010-01-01

Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO2-) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO2- moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Click-enabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides. PMID:19496583

Studying Nuclear Receptor Complexes in the Cellular Environment.

PubMed

Schaufele, Fred

2016-01-01

The ligand-regulated structure and biochemistry of nuclear receptor complexes are commonly determined by in vitro studies of isolated receptors, cofactors, and their fragments. However, in the living cell, the complexes that form are governed not just by the relative affinities of isolated cofactors for the receptor but also by the cell-specific sequestration or concentration of subsets of competing or cooperating cofactors, receptors, and other effectors into distinct subcellular domains and/or their temporary diversion into other cellular activities. Most methods developed to understand nuclear receptor function in the cellular environment involve the direct tagging of the nuclear receptor or its cofactors with fluorescent proteins (FPs) and the tracking of those FP-tagged factors by fluorescence microscopy. One of those approaches, Förster resonance energy transfer (FRET) microscopy, quantifies the transfer of energy from a higher energy "donor" FP to a lower energy "acceptor" FP attached to a single protein or to interacting proteins. The amount of FRET is influenced by the ligand-induced changes in the proximities and orientations of the FPs within the tagged nuclear receptor complexes, which is an indicator of the structure of the complexes, and by the kinetics of the interaction between FP-tagged factors. Here, we provide a guide for parsing information about the structure and biochemistry of nuclear receptor complexes from FRET measurements in living cells.

Expression and affinity purification of recombinant proteins from plants

NASA Technical Reports Server (NTRS)

Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

2002-01-01

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

Systems Biology of Recombinant Protein Production in Bacillus megaterium

NASA Astrophysics Data System (ADS)

Biedendieck, Rebekka; Bunk, Boyke; Fürch, Tobias; Franco-Lara, Ezequiel; Jahn, Martina; Jahn, Dieter

Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

PubMed

Castañón, Jesús; Román, José Pablo; Jessop, Theodore C; de Blas, Jesús; Haro, Rubén

2018-06-01

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

PubMed

Massiah, Michael A; Wright, Katharine M; Du, Haijuan

2016-04-01

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay. Copyright © 2016 John Wiley & Sons, Inc.

Recombinant antigens for immunodiagnosis of cystic echinococcosis

PubMed Central

Li, Jun; Zhang, Wen-Bao

2004-01-01

Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections. PMID:15188015

Toward biophysical probes for the 5-HT3 receptor: structure-activity relationship study of granisetron derivatives.

PubMed

Vernekar, Sanjeev Kumar V; Hallaq, Hasan Y; Clarkson, Guy; Thompson, Andrew J; Silvestri, Linda; Lummis, Sarah C R; Lochner, Martin

2010-03-11

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

Toward Biophysical Probes for the 5-HT3 Receptor: Structure−Activity Relationship Study of Granisetron Derivatives

PubMed Central

2010-01-01

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT3A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT3A receptors in mammalian cells. PMID:20146481

Targeting the UPR to Circumvent Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2015-10-01

were submitted for the screen. Kinase assay protocol (DiscoverX): For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well...blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of...unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

PubMed

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

2013-12-01

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3)

PubMed Central

Austin, Brian P.; Waugh, David S.

2011-01-01

The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25 mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5 mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4 °C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration. PMID:22197595

Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification.

PubMed

Liu, Hu; Dong, Xiaoyan; Sun, Yan

2016-01-15

A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy. Copyright © 2015 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K

Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed bymore » liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.« less

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

PubMed Central

Campos, Magnólia de A; Silva, Marilia S; Magalhães, Cláudio P; Ribeiro, Simone G; Sarto, Rafael PD; Vieira, Eduardo A; Grossi de Sá, Maria F

2008-01-01

Background Heterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity. Results To validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions. Conclusion Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications. PMID:18334031

A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments.

PubMed

Moulder, Robert; Filén, Jan-Jonas; Salmi, Jussi; Katajamaa, Mikko; Nevalainen, Olli S; Oresic, Matej; Aittokallio, Tero; Lahesmaa, Riitta; Nyman, Tuula A

2005-07-01

The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed.

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

PubMed

Lee, Ji-Hye; Lee, Ji-Eun; Kang, Kyung-Jung; Jang, Young-Joo

2017-07-01

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H 6 ) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN 6 ) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 ) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.

PubMed

Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

2017-10-01

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rai, Devendra K.; Segundo, Fayna Diaz-San; Department of Pathobiology and Veterinary Science, CANR, University of Connecticut, Storrs, CT 06269

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co{sup 2+} affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni{sup 2+}-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDVmore » neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance. - Highlights: • 6xHis-tags in A{sub 24} FMDV enable purification and biding to adjuvants via metal ions. • 6xHis A{sub 24} FMDV:MPLA:NiNLP vaccine enhanced protective immunity against FMDV. • Surface exposed capsid tags allow distinction of infected from vaccinated animals.« less

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

PubMed

Boeneman, Kelly; Fossum, Solveig; Yang, Yanhua; Fingland, Nicholas; Skarstad, Kirsten; Crooke, Elliott

2009-05-01

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins. The library was screened for the ability to rescue dnaA(ts) mutants, and a candidate gfp-dnaA was used to replace the dnaA gene of wild-type cells. The resulting cells produce close to physiological levels of GFP-DnaA from the endogenous promoter as their only source of DnaA and somewhat under-initiate replication with moderate asynchrony. Visualization of GFP-tagged DnaA in living cells revealed that DnaA adopts a helical pattern that spirals along the long axis of the cell, a pattern also seen in wild-type cells by immunofluorescence with affinity purified anti-DnaA antibody. Although the DnaA helices closely resemble the helices of the actin analogue MreB, co-visualization of GFP-tagged DnaA and RFP-tagged MreB demonstrates that DnaA and MreB adopt discrete helical structures along the length of the longitudinal cell axis.

Thermal ecology of subadult and adult muskellunge in a thermally enriched reservoir

USGS Publications Warehouse

Cole, A. J.; Bettoli, Phillip William

2014-01-01

The movement of adult muskellunge, Esox masquinongy Mitchill, has been investigated in a variety of systems, but temperature selection by muskellunge has not been examined where well-oxygenated waters were available over a range of temperatures for much of the year. Thirty subadult and adult muskellunge tagged internally with temperature-sensing radio tags were tracked from March 2010 to March 2011 in a Tennessee reservoir. Mean tag temperatures were 18.9 °C in spring (March to May), 22.1 °C in summer (June to August), 16.5 °C in autumn and 9.8 °C in winter (December to February). When the greatest range in water temperatures was available (7.1–33.3 °C; May to early August 2010), their realised thermal niche (mean ± 1 SD) was 22.3 °C ± 1.8; the realised thermal niche was affected by fish size (smaller fish selected slightly warmer temperatures) but not sex. An electric generating steam plant discharging warm water resumed operation in January 2011, and most (86%) tagged fish occupied the plume where temperatures were ≈10 °C warmer than ambient water temperatures. No mortalities were observed 15 days later when plant operations ceased. Their affinity for the heated plume prompted concerns that muskellunge will be too easily exploited when the plant operates during winter.

Structural characterization of core-bradavidin in complex with biotin

PubMed Central

Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

2017-01-01

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

PubMed

Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

2014-05-15

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

PubMed

Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

2014-04-01

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

Bicarbonate Increases Binding Affinity of Vibrio cholerae ToxT to Virulence Gene Promoters

PubMed Central

Thomson, Joshua J.

2014-01-01

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription. PMID:25182489

Integrated amperometric affinity biosensors using Co2+-tetradentate nitrilotriacetic acid modified disposable carbon electrodes: application to the determination of β-lactam antibiotics.

PubMed

Conzuelo, Felipe; Gamella, María; Campuzano, Susana; Martínez-Ruiz, Paloma; Esteban-Torres, María; de las Rivas, Blanca; Reviejo, A Julio; Muñoz, Rosario; Pingarrón, José M

2013-03-19

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co(2+)-tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at -0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min.

Advances in identification and validation of protein targets of natural products without chemical modification.

PubMed

Chang, J; Kim, Y; Kwon, H J

2016-05-04

Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

PubMed

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-03-22

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

PubMed Central

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-01-01

A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

Simultaneously achieve soluble expression and biomimetic immobilization of Candida antarctica lipase B by introducing polyamine tags.

PubMed

Zhou, Xiaoxue; Han, Yu; Lv, Zheng; Tian, Xuemei; Li, Han; Xie, Panpan; Zheng, Liangyu

2017-05-10

Polyamine tags fused in Candida antarctica lipase B (CalB) can help achieve high soluble expression of CalB in E. coli and can directly mediate silicification, which leads to rapid formation of a CalB-silica particle complex through a one-step approach. After optimization experiments, the fused lipase CalB tagged with 6-histidine at the N terminal and 10-lysine at the C terminal (6His-CalB-10Lys) is effectively expressed with high solubility (0.1mg/mL) and specific activity (10.1U/mg), and easily cross-linked in silica particles with a high immobilization efficiency of 96.8% and activity recovery of 81.5%. The immobilized lipase 6His-CalB-10Lys exhibits excellent performance at broad temperature ranges, high thermal and storage stabilities, and superior reusability. Michaelis-Menten kinetics indicates that the affinity and enantioselectivity of the free and immobilized 6His-CalB-10Lys toward the substrate are better than that of commercial Novozym 435 in enantioselective resolution of (S)-N-(2-ethyl-6-methylphenyl) alanine ((S)-NEMPA). The strategies described in this paper are useful for the facile expression and construction of diverse enzyme systems with high efficiency and excellent recyclability. Copyright © 2017 Elsevier B.V. All rights reserved.

Dual-Valve and Counter-Flow Surface Plasmon Resonance.

PubMed

Wang, Xiaoying; Zhou, Feimeng

2018-04-17

Two six-port injector valves and one selector valve commonly used in flow injection analysis are combined with a surface plasmon resonance (SPR) instrument wherein solutions introduced from the two inlets counter-flow inside the flow cell. The system is versatile as the same or different solutions can be rapidly and repeatedly introduced to the two fluidic channels in series or in parallel. Unlike most commercial SPR instruments employing a single injector valve, solutions separately injected from the two injector valves can be readily exchanged (<1 s) between the two channels. This new method, referred to as the alternate injection mode, not only saves analysis time but also facilitates efficient and facile surface reactions for ligand immobilization and prevents immobilized species from desorbing. These advantages are demonstrated with the measurements of binding of acetazolamide (222.2 Da) to histidine-tagged human carbonic anhydrase II (his-tagged HCA). Amine-containing residues of his-tagged HCA molecules tethered at Ni-nitrilotriacetic acid (NTA) sensors were rapidly cross-linked to the underlying carboxymethylated dextran. The higher ligand densities and more stable surfaces are essential for SPR detection of small molecule binding. In a different application, microglobulin solutions of increasing concentrations were introduced for continuous binding to the preimmobilized antibody. The kinetic and affinity measurements can be conducted without performing repeated dissociation and surface regeneration reactions.

Comparing the Properties of Electrochemical-Based DNA Sensors Employing Different Redox Tags

PubMed Central

Kang, Di; Zuo, Xiaolei; Yang, Renqiang; Xia, Fan; Plaxco, Kevin W.; White, Ryan J.

2009-01-01

Many electrochemical biosensor approaches developed in recent years utilize redox labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported employing a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum. PMID:19810694

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

Expression and Production of SH2 Domain Proteins.

PubMed

Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

PubMed

Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

2013-01-01

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

Identification of secreted bacterial proteins by noncanonical amino acid tagging

PubMed Central

Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.

2014-01-01

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637

Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point

PubMed Central

Jeon, Won Bae

2015-01-01

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value. PMID:20510014

Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

PubMed

Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

2016-06-07

Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

PubMed Central

Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

2016-01-01

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Immuno-affinity Capture Followed by TMPP N-Terminus Tagging to Study Catabolism of Therapeutic Proteins.

PubMed

Kullolli, Majlinda; Rock, Dan A; Ma, Ji

2017-02-03

Characterization of in vitro and in vivo catabolism of therapeutic proteins has increasingly become an integral part of discovery and development process for novel proteins. Unambiguous and efficient identification of catabolites can not only facilitate accurate understanding of pharmacokinetic profiles of drug candidates, but also enables follow up protein engineering to generate more catabolically stable molecules with improved properties (pharmacokinetics and pharmacodynamics). Immunoaffinity capture (IC) followed by top-down intact protein analysis using either matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry analysis have been the primary methods of choice for catabolite identification. However, the sensitivity and efficiency of these methods is not always sufficient for characterization of novel proteins from complex biomatrices such as plasma or serum. In this study a novel bottom-up targeted protein workflow was optimized for analysis of proteolytic degradation of therapeutic proteins. Selective and sensitive tagging of the alpha-amine at the N-terminus of proteins of interest was performed by immunoaffinity capture of therapeutic protein and its catabolites followed by on-bead succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl N-terminus (TMPP-NTT) tagging. The positively charged hydrophobic TMPP tag facilitates unambiguous sequence identification of all N-terminus peptides from complex tryptic digestion samples via data dependent liquid chromatgraphy-tandem mass spectroscopy. Utility of the workflow was illustrated by definitive analysis of in vitro catabolic profile of neurotensin human Fc (NTs-huFc) protein in mouse serum. The results from this study demonstrated that the IC-TMPP-NTT workflow is a simple and efficient method for catabolite formation in therapeutic proteins.

Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.

PubMed

Challier, Emilse; Lisa, María-Natalia; Nerli, Bibiana B; Calcaterra, Nora B; Armas, Pablo

2014-01-01

Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advances made concerning CNBP biological roles, a full knowledge about the structure-function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here, we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan, which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and β-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding. Copyright © 2013 Elsevier Inc. All rights reserved.

Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin-Diaconescu, Vlad; Joseph, Crisjoe A.; Boer, Jodi L.

Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donormore » ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.« less

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

PubMed

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice

PubMed Central

Ding, Haiming; Carlton, Michelle M.; Povoski, Stephen P.; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H.; Colcher, David; Brody, Rich; Davis, Paul D.; Pokora, Alex; Phelps, Mitchell; Martin, Edward W.; Tweedle, Michael F.

2014-01-01

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2–4 kDa discrete, branched PEGylation reagents on mCC49 Fab′ (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab′ (Fab′-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 (124I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab′ with Mal-dPEG-A (Fab′-A) reduced the binding affinity of the non PEGylated Fab′ by 30%; however, in vivo, Fab′-A significantly lengthened the blood retention vs Fab′-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab′-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A. PMID:24175669

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

PubMed

Mainali, Dipak; Smith, Emily A

2013-04-01

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

PubMed Central

2011-01-01

Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge. PMID:21824404

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

PubMed

Chen, WeiYue; Avezov, Edward; Schlachter, Simon C; Gielen, Fabrice; Laine, Romain F; Harding, Heather P; Hollfelder, Florian; Ron, David; Kaminski, Clemens F

2015-03-10

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Application of histone modification-specific interaction domains as an alternative to antibodies.

PubMed

Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul; Jeltsch, Albert

2014-11-01

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. © 2014 Kungulovski et al.; Published by Cold Spring Harbor Laboratory Press.

Probing Cocaine-Antibody Interactions in Buffer and Human Serum

PubMed Central

Ramakrishnan, Muthu; Alves De Melo, Fernando; Kinsey, Berma M.; Ladbury, John E.; Kosten, Thomas R.; Orson, Frank M.

2012-01-01

Background Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. Results MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20–50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. Conclusions High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide valuable information for characterization of their interactions and thermodynamic properties. In addition MST measurements of antibody affinity in the presence of biological fluids will provide a better opportunity to make reliable decisions and facilitate the design of cocaine vaccines and immunization conditions. The methods should be more widely adopted in characterization of antibody complexes. PMID:22859949

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

PubMed Central

Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G.

2017-01-01

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays. PMID:28607052

Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

PubMed

Thomson, Joshua J; Withey, Jeffrey H

2014-11-01

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.

PubMed

Konziase, Benetode

2015-08-01

We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa. Copyright © 2015 Elsevier Inc. All rights reserved.

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

PubMed

Manuel-Cabrera, Carlos Alberto; Vallejo-Cardona, Alba Adriana; Padilla-Camberos, Eduardo; Hernández-Gutiérrez, Rodolfo; Herrera-Rodríguez, Sara Elisa; Gutiérrez-Ortega, Abel

2016-11-29

Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Preparation of silica coated cobalt ferrite magnetic nanoparticles for the purification of histidine-tagged proteins

NASA Astrophysics Data System (ADS)

Aygar, Gülfem; Kaya, Murat; Özkan, Necati; Kocabıyık, Semra; Volkan, Mürvet

2015-12-01

Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (-COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2-CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.

Unique self-assembly properties of a bridge-shaped protein dimer with quantum dots

NASA Astrophysics Data System (ADS)

Wang, Jianhao; Jiang, Pengju; Gao, Liqian; Yu, Yongsheng; Lu, Yao; Qiu, Lin; Wang, Cheli; Xia, Jiang

2013-09-01

How protein-protein interaction affects protein-nanoparticle self-assembly is the key to the understanding of biomolecular coating of nanoparticle in biological fluids. However, the relationship between protein shape and its interaction with nanoparticles is still under-exploited because of lack of a well-conceived binding system and a method to detect the subtle change in the protein-nanoparticle assemblies. Noticing this unresolved need, we cloned and expressed a His-tagged SpeA protein that adopts a bridge-shaped dimer structure, and utilized a high-resolution capillary electrophoresis method to monitor assembly formation between the protein and quantum dots (QDs, 5 nm in diameter). We observed that the bridge-shaped structure rendered a low SpeA:QD stoichiometry at saturation. Also, close monitoring of imidazole (Im) displacement of surface-bound protein revealed a unique two-step process. High-concentration Im could displace surface-bound SpeA protein and form a transient QD-protein intermediate, through a kinetically controlled displacement process. An affinity-driven equilibrium step then followed, resulting in re-assembling of the QD-protein complex in about 1 h. Through a temporarily formed intermediate, Im causes a rearrangement of His-tagged proteins on the surface. Thus, our work showcases that the synergistic interplay between QD-His-tag interaction and protein-protein interaction can result in unique properties of protein-nanoparticle assembly for the first time.

Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis.

PubMed

Wang, Huping; Han, Wenyu; Takagi, Junichi; Cong, Yao

2018-05-11

Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

PubMed

Zhang, Mengjie; Martin, Jody L; Kumar, Mohit; Khairallah, Ramzi J; de Tombe, Pieter P

2015-11-01

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner. Copyright © 2015 the American Physiological Society.

Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor

PubMed Central

Ramsay, Douglas; Bevan, Nicola; Rees, Stephen; Milligan, Graeme

2001-01-01

The wild-type β2-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant β2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant β2-adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC50 for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the β2-adrenoceptor. Co-expression of the wild-type β2-adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. Co-expression of the constitutively active mutant β2-adrenoceptor-Renilla luciferase and an equivalent mutant of the α1b-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors. PMID:11350868

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag

PubMed Central

Zhang, Mengjie; Martin, Jody L.; Kumar, Mohit; de Tombe, Pieter P.

2015-01-01

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1′ cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner. PMID:26386113

Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification.

PubMed

Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan

2017-03-24

In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc) 2 .4H 2 O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg -1 (protein/MnFe 2 O 4 @SiO 2 @Ni-Salen complex). Collectively, purification process with MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

PubMed Central

Pratt, Christopher P.; Kuljis, Dika A.; Homanics, Gregg E.; He, Jianjun; Kolodieznyi, Dmytro; Dudem, Srikanth; Hollywood, Mark A.; Barth, Alison L.; Bruchez, Marcel P.

2017-01-01

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking. PMID:29163049

Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

PubMed Central

Gokulan, Kuppan; Varughese, Kottayil I.

2013-01-01

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, KD = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy. PMID:24349338

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins.

PubMed

Baumann, Pascal; Bluthardt, Nicolai; Renner, Sarah; Burghardt, Hannah; Osberghaus, Anna; Hubbuch, Jürgen

2015-04-20

Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag™ technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag™ was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis.

PubMed

Kim, Young Hwan; Cho, Kun; Yun, Sung-Ho; Kim, Jin Young; Kwon, Kyung-Hoon; Yoo, Jong Shin; Kim, Seung Il

2006-02-01

Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jiménez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli.

PubMed

Sayari, Adel; Mosbah, Habib; Gargouri, Youssef

2007-05-01

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.

Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites.

PubMed

Peterson, Eric C; Celikel, Reha; Gokulan, Kuppan; Varughese, Kottayil I

2013-01-01

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K(D) = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy"). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni(2+). Two of the histidine residues of each C-terminal His-tag interact with Ni(2+) in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.

Expression and purification of mouse peptide ESP4 in Escherichia coli.

PubMed

Hirakane, Makoto; Taniguchi, Masahiro; Yoshinaga, Sosuke; Misumi, Shogo; Terasawa, Hiroaki

2014-04-01

Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviors in many animals. In mice, the male-specific peptide ESP1 (exocrine gland-secreting peptide 1) is secreted into tear fluids and enhances female sexual receptive behavior. ESP1 belongs to the ESP family, a multigene family with 38 genes in mice. ESP1 shares the highest homology with ESP4. ESP1 is expressed in the extraorbital lacrimal gland, whereas ESP4 is expressed in some exocrine glands. Thus, ESP4 is expected to have a function that has not been elucidated yet. Large amounts of the purified ESP4 protein are required for structural and biochemical studies. Here we present an expression and purification scheme for the recombinant ESP4 protein. The N-terminally histidine-tagged ESP4 fusion protein was expressed in Escherichia coli as inclusion bodies, which were solubilized and purified by nickel affinity chromatography. The histidine tag was cleaved with thrombin and removed by a second nickel affinity chromatography step. The ESP4 protein was isolated with high purity by reversed-phase chromatography. For NMR analyses, we prepared a stable isotope-labeled ESP4 protein. Three repeated freeze-drying steps after the reversed-phase chromatography were required, to remove a volatile contaminating compound and to obtain an NMR spectrum with a homogeneous line shape. AMS-modification and far-UV CD spectroscopic analyses suggested that ESP4 has an intramolecular disulfide bridge and a helical structure, respectively. The present study provides a powerful tool for structural and biochemical studies of ESP4, leading toward the elucidation of the roles of the ESP family members. Copyright © 2014 Elsevier Inc. All rights reserved.

Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.

PubMed

Schilling, Birgit; Rardin, Matthew J; MacLean, Brendan X; Zawadzka, Anna M; Frewen, Barbara E; Cusack, Michael P; Sorensen, Dylan J; Bereman, Michael S; Jing, Enxuan; Wu, Christine C; Verdin, Eric; Kahn, C Ronald; Maccoss, Michael J; Gibson, Bradford W

2012-05-01

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.

Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

PubMed Central

Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.; Zawadzka, Anna M.; Frewen, Barbara E.; Cusack, Michael P.; Sorensen, Dylan J.; Bereman, Michael S.; Jing, Enxuan; Wu, Christine C.; Verdin, Eric; Kahn, C. Ronald; MacCoss, Michael J.; Gibson, Bradford W.

2012-01-01

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models. PMID:22454539

Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

PubMed Central

Kaur, Jasvir; Reinhardt, Dieter P.

2012-01-01

Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

PubMed

Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

2009-01-01

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

Comparative analysis of viral RNA signatures on different RIG-I-like receptors

PubMed Central

Sanchez David, Raul Y; Combredet, Chantal; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Coppée, Jean-Yves; Mura, Marie; Guerbois Galla, Mathilde; Despres, Philippe; Tangy, Frédéric; Komarova, Anastassia V

2016-01-01

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection. DOI: http://dx.doi.org/10.7554/eLife.11275.001 PMID:27011352

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

PubMed Central

Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

2014-01-01

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

PubMed

Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M; Climent, Víctor; Sanz, Jesús M

2014-01-01

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein–protein interactions directly from DNA

PubMed Central

Layton, Curtis J; Hellinga, Homme W

2011-01-01

Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein–protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody–affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed. PMID:21674663

Tumor-targeting domains for chimeric antigen receptor T cells.

PubMed

Bezverbnaya, Ksenia; Mathews, Ashish; Sidhu, Jesse; Helsen, Christopher W; Bramson, Jonathan L

2017-01-01

Immunotherapy with chimeric antigen receptor (CAR) T cells has been advancing steadily in clinical trials. Since the ability of engineered T cells to recognize intended tumor-associated targets is crucial for the therapeutic success, antigen-binding domains play an important role in shaping T-cell responses. Single-chain antibody and T-cell receptor fragments, natural ligands, repeat proteins, combinations of the above and universal tag-specific domains have all been used in the antigen-binding moiety of chimeric receptors. Here we outline the advantages and disadvantages of different domains, discuss the concepts of affinity and specificity, and highlight the recent progress of each targeting strategy.

Melatonin attenuates postharvest physiological deterioration of cassava storage roots.

PubMed

Ma, Qiuxiang; Zhang, Ting; Zhang, Peng; Wang, Zhen-Yu

2016-05-01

Melatonin reportedly increases abiotic and biotic stress tolerance in plants, but information on its in vivo effects during postharvest physiological deterioration (PPD) in cassava is limited. In this study, we investigated the effect of melatonin in regulating cassava PPD. Treatment with 500 mg/L melatonin significantly delayed cassava PPD and reduced the accumulation of hydrogen peroxide (H2O2) while increasing the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), but not ascorbate peroxidase (APX). Transcript analysis further showed that expression of copper/zinc SOD (MeCu/ZnSOD), MeCAT1, glutathione peroxidase (MeGPX), peroxidase 3 (MePX3), and glutathione S-transferases (MeGST) was higher in cassava roots sliced treated with 500 mg/L melatonin than in those not exposed to exogenous melatonin. These data demonstrate that melatonin delays cassava PPD by directly or indirectly maintaining homoeostasis of cellular reactive oxygen species (ROS). We also found that accumulation of endogenous melatonin and the transcript levels of melatonin biosynthesis genes changed dynamically during the PPD process. This finding suggested that endogenous melatonin acts as a signal modulator for maintaining cassava PPD progression and that manipulation of melatonin biosynthesis genes through genetic engineering might prevent cassava root deterioration. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

PubMed

Muro, Eleonora; Hoang, Thang Q; Jobart-Malfait, Aude; Hernandez-Verdun, Danièle

2008-05-01

The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.

Frameshifting in the p6 cDNA phage display system.

PubMed

Govarts, Cindy; Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2010-12-20

Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.

Heterofunctional Magnetic Metal-Chelate-Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity.

PubMed

Tural, Servet; Tural, Bilsen; Demir, Ayhan S

2015-09-01

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor-made magnetic chelate-epoxy supports. In order to selectively adsorb and then covalently immobilize the poly-His-tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co(2+)-chelate groups (38 µmol Co(2+)/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine-tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free-enzyme-catalyzed reaction. The enantiomeric excess (ee) of (R)-benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles. © 2015 Wiley Periodicals, Inc.

Affinity purification–mass spectrometry and network analysis to understand protein-protein interactions

PubMed Central

Morris, John H; Knudsen, Giselle M; Verschueren, Erik; Johnson, Jeffrey R; Cimermancic, Peter; Greninger, Alexander L; Pico, Alexander R

2015-01-01

By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification–mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography–MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one’s familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2–4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one’s data and find the right questions. PMID:25275790

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

PubMed

Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

2016-12-01

The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

Direct and selective immobilization of proteins by means of an inorganic material-binding peptide: discussion on functionalization in the elongation to material-binding peptide.

PubMed

Yokoo, Nozomi; Togashi, Takanari; Umetsu, Mitsuo; Tsumoto, Kouhei; Hattori, Takamitsu; Nakanishi, Takeshi; Ohara, Satoshi; Takami, Seiichi; Naka, Takashi; Abe, Hiroya; Kumagai, Izumi; Adschiri, Tadafumi

2010-01-14

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.

Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

PubMed

Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

2016-03-01

Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Watermelon profilin: characterization of a major allergen as a model for plant-derived food profilins.

PubMed

Cases, Bárbara; Pastor-Vargas, Carlos; Dones, Félix Gil; Perez-Gordo, Marina; Maroto, Aroa S; de las Heras, Manuel; Vivanco, Fernando; Cuesta-Herranz, Javier

2010-01-01

Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus).The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZαA vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelon-allergic patients. Biological activity was tested by the basophil activation test. Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis. Copyright © 2010 S. Karger AG, Basel.

RNA aptamers that functionally interact with green fluorescent protein and its derivatives

PubMed Central

Shui, Bo; Ozer, Abdullah; Zipfel, Warren; Sahu, Nevedita; Singh, Avtar; Lis, John T.; Shi, Hua; Kotlikoff, Michael I.

2012-01-01

Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways. PMID:22189104

VisEL: Visualisation of Expertise Level in a Special Interest Group Knowledge Portal

NASA Astrophysics Data System (ADS)

Zulhafizsyam Wan Ahmad, Wan Muhammad; Sulaiman, Shahida; Yusof, Umi Kalsom

A variety of portals are available nowadays to support diverse purposes such as commercial, publishing, personal, affinity and corporate portals. Affinity portals promote electronic communities who share common interest such as a special interest group (SIG). Knowledge portal is an emerging trend that benefits the existing portal technology by designing such portals with proper representation of the members' shared knowledge. Besides textual representation for diverse expertise levels, graphical visualisation will be able to support the requirements in searching and representing expertise level among e-community. There is a number of existing SIG portals available. However, they do not visualise effectively and accurately the expertise level of members and make it difficult for users to search their targeted experts for instance searching the highest expertise level to have a discussion and to solve their problems related to a project. The goal of this paper is to propose a graphical visualisation of expertise level method (VisEL) using an interactive tag cloud technique that represents expertise level of each member based on their knowledge in a software engineering SIG portal.

Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

PubMed

Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

2017-01-01

Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between aromatase expression and aromatase inhibitors are warranted.

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

PubMed

Salehi, Nasrin; Peng, Ching-An

2016-07-08

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.

Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

NASA Astrophysics Data System (ADS)

Gober, Isaiah Nathaniel

This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the possible ways for detecting PTMs and may find use in the development of new assays for enzymes that lack robust methods for measuring their activity. The third section explores the development of new small molecule receptors capable of selectively binding hydrophilic guests in water, such as the lower methylation states of lysine. We identified a receptor, A2I, that has improved binding affinity and selectivity for dimethyllysine (Kme2). The receptor was discovered and synthesized by using dynamic combinatorial chemistry (DCC) to redesign a small molecule receptor (A2B ) that preferentially binds trimethyllysine (Kme3). Incorporating a biphenyl monomer with ortho-di-substituted carboxylates into the receptor lead to the formation of a salt bridge interaction with Kme2. These favorable electrostatic and hydrogen bonding interactions produced a receptor with 32-fold tighter binding to Kme2, which is the highest affinity synthetic receptor for Kme2 in the context of a peptide that has been reported. This work provides insight into effective strategies for binding hydrophilic, cationic guests in water and is an encouraging result toward a synthetic receptor that selectively binds Kme2 over other methylation states of lysine. In the final section, a small molecule receptor for Kme3 (A 2B) was redesigned using DCC to incorporate either aromatic or acidic amino acids into the receptor. We proposed that the incorporation of amino acids could introduce additional non-covalent interactions (such as cation-pi, electrostatic, and hydrogen bonding) with a guest bound inside the pocket of the receptor. However, selective non-covalent interactions between the amino acid side chain on the modified receptor and the bound methylated lysine guest could not be achieved. This is most likely due to the conformational flexibility of the amino acid-functionalized receptors. Furthermore, attaching amino acids to the receptor seemed to increase non-specific electrostatic interactions, resulting in tighter binding to the unmethylated lysine peptide (compared to A2B). Ultimately, this highlights the importance of incorporating monomers with less conformational flexibility that can rigidly place functional groups into the binding pocket.

Differential Temperature Dependence of Tobacco Etch Virus and Rhinovirus 3C Proteases

PubMed Central

Raran-Kurussi, Sreejith; Tözsér, József; Cherry, Scott; Tropea, Joseph E.; Waugh, David S.

2014-01-01

Because of their stringent sequence specificity, the 3C-like proteases from tobacco etch virus (TEV3) and human rhinovirus are often used for the removal of affinity tags. The latter enzyme is rumored to have greater catalytic activity at 4°C, the temperature at which fusion protein substrates are usually digested. Here, we report that experiments with fusion protein and peptide substrates confirm this conjecture. Whereas the catalytic efficiency of rhinovirus 3C protease is approximately the same at its optimum temperature (30°C) and at 4°C, TEV protease is 10-fold less active at the latter temperature, due primarily to a reduction in kcat. PMID:23395976

Using Haloarcula marismortui Bacteriorhodopsin as a Fusion Tag for Enhancing and Visible Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Hsu, Min-Feng; Yu, Tsung-Fu; Chou, Chia-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen; Wang, Andrew H. J.

2013-01-01

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system. PMID:23457558

A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein ligase.

PubMed

Bond, Thomas E H; Sorenson, Alanna E; Schaeffer, Patrick M

2017-12-01

Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins. The data obtained in this study reveal how biotin and ATP significantly increase the thermal stability (ΔT m =+16.5°C) of M. tuberculosis BirA and lead to formation of a high affinity holoenzyme complex (K obs =7.7nM). The new findings and mycobacterial BirA high-throughput assay presented in this work could provide an efficient platform for future anti-tubercular drug discovery campaigns. Copyright © 2017 Elsevier GmbH. All rights reserved.

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology.

PubMed

Gurung, Ishwori; Berry, Jamie-Lee; Hall, Alexander M J; Pelicic, Vladimir

2017-04-07

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functionalization of paramagnetic nanoparticles for protein immobilization and purification.

PubMed

Carneiro, Lara A B C; Ward, Richard J

2018-01-01

A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe 3 O 4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged β-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni 2+ , nanoparticles demonstrated a binding capacity of 250 μg of a polyhistidine-tagged β-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the β-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity. Copyright © 2017. Published by Elsevier Inc.

In vivo system for analyzing the function of the PsbP protein using Chlamydomonas reinhardtii.

PubMed

Nishimura, Taishi; Sato, Fumihiko; Ifuku, Kentaro

2017-09-01

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein-protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-∆15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII-LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII-LHCII supercomplex from green algae.

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

PubMed

Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

2017-03-17

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN

PubMed Central

Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

2014-01-01

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

Structural and biochemical basis for the difference in the helicase activity of two different constructs of SARS-CoV helicase.

PubMed

Adedeji, A O; Singh, K; Sarafianos, S G

2012-12-22

The non—structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS—CoV) is a helicase that separates double—stranded RNA or DNA with a 5'—3' polarity, using the energy of nucleotide hydrolysis. We have previously determined the minimal mechanism of helicase function by nsp13 where we demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base—pairs each with a catalytic rate of 30 steps per second. In that study we used different constructs of nsp13 (GST and H6 constructs). GST—nsp13 showed much more efficient nucleic acid unwinding than the H6—tagged counterpart. At 0.1 second, more than 50% of the ATP is hydrolyzed by GST—nsp13 compared to less than 5% ATP hydrolysis by H6—nsp13. Interestingly, the two constructs have the same binding affinity for nucleic acids. We, therefore propose that the difference in the catalytic efficiency of these two constructs is due to the interference of ATP binding by the histidine tag at the amino—terminus of nsp13.

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology

PubMed Central

Gurung, Ishwori; Berry, Jamie-Lee; Hall, Alexander M. J.

2017-01-01

Abstract Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)—exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable. PMID:27903891

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.

PubMed

Rowland, Meng M; Bostic, Heidi E; Gong, Denghuang; Speers, Anna E; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F; Best, Michael D

2011-12-27

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirimura, Kohtaro, E-mail: kkohtaro@waseda.jp; Watanabe, Shotaro; Kobayashi, Keiichi

Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity ofmore » the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.« less

Phosphatidylinositol (3,4,5)-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

PubMed Central

Rowland, Meng M.; Bostic, Heidi E.; Gong, Denghuang; Speers, Anna E.; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F.; Best, Michael D.

2013-01-01

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins. PMID:22074223

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

PubMed

Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

2014-01-01

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

GFP Facilitates Native Purification of Recombinant Perlucin Derivatives and Delays the Precipitation of Calcium Carbonate

PubMed Central

Weber, Eva; Guth, Christina; Weiss, Ingrid M.

2012-01-01

Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO3 − as the first ionic interaction partner, but not necessarily for Ca2+ . The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals. PMID:23056388

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

The high-affinity phosphodiesterase PdeH regulates development and aflatoxin biosynthesis in Aspergillus flavus.

PubMed

Yang, Kunlong; Liu, Yinghang; Liang, Linlin; Li, Zhenguo; Qin, Qiuping; Nie, Xinyi; Wang, Shihua

2017-04-01

Cyclic AMP signaling controls a range of physiological processes in response to extracellular stimuli in organisms. Among the signaling cascades, cAMP, as a second messenger, is orchestrated by adenylate cyclase (biosynthesis) and cAMP phosphodiesterases (PDEs) (hydrolysis). In this study, we investigated the function of the high-affinity (PdeH) and low-affinity (PdeL) cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus Aspergillus flavus, and found that instead of PdeL, inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation. However, the ΔpdeL/ΔpdeH mutant exhibited an enhanced phenotype defects, a similar phenotype defects to wild-type strain treated with exogenous cAMP. The activation of PKA activity was inhibited in the ΔpdeH or ΔpdeL/ΔpdeH mutant, both of whom exhibited increasing AF production. Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain, and affected pdeL transcription. Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol, while PdeL-GFP localized mainly to the nucleus. Overall, our results indicated that PdeH plays a major role, but has overlapping function with PdeL, in vegetative growth, development and AF biosynthesis in A. flavus. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Ultrasensitive Electrochemical Detection of Glycoprotein Based on Boronate Affinity Sandwich Assay and Signal Amplification with Functionalized SiO2@Au Nanocomposites.

PubMed

You, Min; Yang, Shuai; Tang, Wanxin; Zhang, Fan; He, Pin-Gang

2017-04-26

Herein we propose a multiple signal amplification strategy designed for ultrasensitive electrochemical detection of glycoproteins. This approach introduces a new type of boronate-affinity sandwich assay (BASA), which was fabricated by using gold nanoparticles combined with reduced graphene oxide (AuNPs-GO) to modify sensing surface for accelerating electron transfer, the composite of molecularly imprinted polymer (MIP) including 4-vinylphenylboronic acid (VPBA) for specific capturing glycoproteins, and SiO 2 nanoparticles carried gold nanoparticles (SiO 2 @Au) labeled with 6-ferrocenylhexanethiol (FcHT) and 4-mercaptophenylboronic acid (MPBA) (SiO 2 @Au/FcHT/MPBA) as tracing tag for binding glycoprotein and generating electrochemical signal. As a sandwich-type sensing, the SiO 2 @Au/FcHT/MPBA was captured by glycoprotein on the surface of imprinting film for further electrochemical detection in 0.1 M PBS (pH 7.4). Using horseradish peroxidase (HRP) as a model glycoprotein, the proposed approach exhibited a wide linear range from 1 pg/mL to 100 ng/mL, with a low detection limit of 0.57 pg/mL. To the best of our knowledge, this is first report of a multiple signal amplification approach based on boronate-affinity molecularly imprinted polymer and SiO 2 @Au/FcHT/MPBA, exhibiting greatly enhanced sensitivity for glycoprotein detection. Furthermore, the newly constructed BASA based glycoprotein sensor demonstrated HRP detection in real sample, such as human serum, suggesting its promising prospects in clinical diagnostics.

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

PubMed Central

O’Malley, Michelle A.; Lazarova, Tzvetana; Britton, Zachary T.; Robinson, Anne S.

2007-01-01

The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A2a receptor fused to a green fluorescent protein (A2aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A2aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A2aR and A2aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A2aR-GFP-His10. One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A2aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A2a-His10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A2aR yet achieved from any heterologous expression system. PMID:17591446

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

PubMed Central

Kaplan, W.; Hüsler, P.; Klump, H.; Erhardt, J.; Sluis-Cremer, N.; Dirr, H.

1997-01-01

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol. PMID:9041642

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

PubMed

Kaplan, W; Hüsler, P; Klump, H; Erhardt, J; Sluis-Cremer, N; Dirr, H

1997-02-01

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

Identification of Protein Targets of 4-Hydroxynonenal Using Click Chemistry for Ex Vivo Biotinylation of Azido and Alkynyl Derivatives

PubMed Central

Vila, Andrew; Tallman, Keri A.; Jacobs, Aaron T.; Liebler, Daniel C.; Porter, Ned A.; Marnett, Lawrence J.

2009-01-01

Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic α, β-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g. IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates but click chemistry was found to be superior for recovery of biotinylated proteins from streptavidin-coated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 μM. These proteins were affinity purified with streptavidin beads and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90, and the 78-kDa glucose-regulated protein) were selectively adducted by azido- and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE. PMID:18232660

Engineering single-molecule, nanoscale, and microscale bio-functional materials via click chemistry

NASA Astrophysics Data System (ADS)

Daniele, Michael Angelo-Anthony

To expand the design envelope and supplement the materials library available to biomaterials scientists, the copper(I)-catalyzed azide-alkyne cycloaddition (CuCAAC) was explored as a route to design, synthesize and characterize bio-functional small-molecules, nanoparticles, and microfibers. In each engineered system, the use of click chemistry provided facile, bio-orthogonal control for materials synthesis; moreover, the results provided a methodology and more complete, fundamental understanding of the use of click chemistry as a tool for the synergy of biotechnology, polymer and materials science. Fluorophores with well-defined photophysical characteristics (ranging from UV to NIR fluorescence) were used as building blocks for small-molecule, fluorescent biosensors. Fluorophores were paired to exhibit fluorescence resonant energy transfer (FRET) and used to probe the metabolic activity of carbazole 1,9a-dioxygenase (CARDO). The FRET pair exhibited a significant variation in PL response with exposure to the lysate of Pseudomonas resinovorans CA10, an organism which can degrade variants of both the donor and acceptor fluorophores. Nanoparticle systems were modified via CuCAAC chemistry to carry affinity tags for CARDO and were subsequently utilized for affinity based bioseparation of CARDO from crude cell lysate. The enzymes were baited with an azide-modified carbazolyl-moiety attached to a poly(propargyl acrylate) nanoparticle. Magnetic nanocluster systems were also modified via CuCAAC chemistry to carry fluorescent imaging tags. The iron-oxide nanoclusters were coated with poly(acrylic acid-co-propargyl acrylate) to provide a clickable surface. Ultimately, alternate Cu-free click chemistries were utilized to produce biohybrid microfibers. The biohybrid microfibers were synthesized under benign photopolymerization conditions inside a microchannel, allowing the encapsulation of viable bacteria. By adjusting pre-polymer solutions and laminar flow rates within the microchannel, the morphology, hydration, and thermal properties of the fibers were easily tuned. The methodology produced hydrogel fibers that sustained viable cells as demonstrated by the encapsulation and subsequent proliferation of Bacillus cereus and Escherichia coli communities.

Overproduction of superoxide dismutase and catalase confers cassava resistance to Tetranychus cinnabarinus

PubMed Central

Lu, Fuping; Liang, Xiao; Lu, Hui; Li, Qian; Chen, Qing; Zhang, Peng; Li, kaimian; Liu, Guanghua; Yan, Wei; Song, Jiming; Duan, Chunfang; Zhang, Linhui

2017-01-01

To explore the role of protective enzymes in cassava (Manihot esculenta Crantz) resistance to mites, transgenic cassava lines overproducing copper/zinc superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) were used to evaluate and molecularly confirm cassava resistance to Tetranychus cinnabarinus. Laboratory evaluation demonstrated that, compared with the control cultivar TMS60444 (wild type, WT), the survival, reproduction, development and activities of SOD and CAT in T. cinnabarinus feeding on transgenic cassava lines SC2, SC4, and SC11 significantly inhibited. Furthermore, the activities of SOD and CAT in transgenic cassava lines SC2, SC4, and SC11 damaged by T. cinnabarinus significantly increased. These findings were similar to the results in the mite-resistant cassava cultivars. Besides, field evaluation indicated that the transgenic cassava lines SC2, SC4, and SC11 were slightly damaged as the highly mite-resistant control C1115, while the highly mite-susceptible WT was severely damaged by T. cinnabarinus. Laboratory and field evaluation demonstrated that transgenic cassava lines were resistant to T. cinnabarinus, which directly confirmed that the increase in SOD and CAT activities was positively related to cassava resistance to T. cinnabarinus. These results will help in understanding the antioxidant defense responses in the cassava–mite interaction and molecular breeding of mite-resistant cassava for effective pest control. PMID:28054665

Architecture and dynamics of overlapped RNA regulatory networks.

PubMed

Lapointe, Christopher P; Preston, Melanie A; Wilinski, Daniel; Saunders, Harriet A J; Campbell, Zachary T; Wickens, Marvin

2017-11-01

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities-Puf3p, Puf4p, and Puf5p of S. cerevisiae Using RNA Tagging, we identified a "super-network" comprised of four subnetworks: Puf3p, Puf4p, and Puf5p subnetworks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, is determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: The remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution. © 2017 Lapointe et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Folate Receptor-targeted Bioflavonoid Genistein-loaded Chitosan Nanoparticles for Enhanced Anticancer Effect in Cervical Cancers

NASA Astrophysics Data System (ADS)

Cai, Limei; Yu, Rufen; Hao, Xi; Ding, Xiangcui

2017-08-01

In this study, novel folic acid-conjugated chitosan nanoparticle was formulated for specific delivery of bioflavonoid, Genistein (GEN), to the cervical cancer cells. The prepared GEN-loaded chitosan nanoparticles (GCN) and folic acid-conjugated GCN (FGCN) showed smaller size with a controlled drug release profile. FGCN exhibited enhanced internalization potential in HeLa cells than that of GCN. The specific internalization of FGCN was mainly due to the affinity of folic acid (FA) with FRs-α which is present in large numbers in HeLa cells. The results revealed that FGCN has a specific affinity towards HeLa cells that will contribute to the better treatment. Folic acid-tagged nanoformulations exhibited a superior cytotoxic effect compared to that of non-targeted formulations. Consistently, IC50 value of GEN decreased from 33.8 to 14.6 μg/ml when treated with FGCN after 24 h incubation. The apoptosis studies indicated that the FGCN nanoparticles were then either GCN or free GEN in terms of anticancer activity. Overall, results revealed that folate conjugation to the delivery system might have great effect on the survival of cervical cancers that will be beneficial for overall cancer treatment.

Oligolysine-based saccharide clusters: synthesis and specificity.

PubMed Central

Frison, Natacha; Marceau, Philippe; Roche, Annie-Claude; Monsigny, Michel; Mayer, Roger

2002-01-01

In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-beta-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the alpha and epsilon amino groups of the core peptide (Lys( n )-Ala-Cys-NH2) with n =1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells: a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity. PMID:12119048

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

PubMed Central

Tian, Xiaolin; Zhu, Mingwei; Li, Long; Wu, Chunlai

2013-01-01

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond. PMID:24335807

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

The Extrachromosomal EAST Protein of Drosophila Can Associate with Polytene Chromosomes and Regulate Gene Expression

PubMed Central

Wasser, Martin; Chia, William

2007-01-01

The EAST protein of Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations. PMID:17476334

An affinity-directed protein missile system for targeted proteolysis

PubMed Central

Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

2016-01-01

The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

Isolation of Plant Photosystem II Complexes by Fractional Solubilization

PubMed Central

Haniewicz, Patrycja; Floris, Davide; Farci, Domenica; Kirkpatrick, Joanna; Loi, Maria C.; Büchel, Claudia; Bochtler, Matthias; Piano, Dario

2015-01-01

Photosystem II (PSII) occurs in different forms and supercomplexes in thylakoid membranes. Using a transplastomic strain of Nicotiana tabacum histidine tagged on the subunit PsbE, we have previously shown that a mild extraction protocol with β-dodecylmaltoside enriches PSII characteristic of lamellae and grana margins. Here, we characterize residual granal PSII that is not extracted by this first solubilization step. Using affinity purification, we demonstrate that this PSII fraction consists of PSII-LHCII mega- and supercomplexes, PSII dimers, and PSII monomers, which were separated by gel filtration and functionally characterized. Our findings represent an alternative demonstration of different PSII populations in thylakoid membranes, and they make it possible to prepare PSII-LHCII supercomplexes in high yield. PMID:26697050

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

PubMed

Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

2009-01-01

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Crystallization and preliminary crystallographic analysis of an Enterococcus faecalis repressor protein, CylR2, involved in regulating cytolysin production through quorum-sensing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ni, Shuisong; McAteer, Kathleen; Bussiere, Dirksen E.

2004-06-01

CylR2 is one of the two regulatory proteins associated with the quorum-sensing-dependent synthesis of cytolysin for the common pathogen Enterococcus faecalis. The protein was expressed with a C-terminal 6-histidine tag and purified to homogeneity with a cobalt affinity column followed by another size exclusion column. Both native and SeMet proteins were crystallized. A complete X-ray diffraction data set from the native crystal was collected to 2.3 resolution. The crystal was tetragonal, belonging to space group P41/43, with unit-cell dimensions a=b=66.2 , c=40.9 and a=b=g=90. The asymmetric unit contained two molecules of CylR2.

Dissection and engineering of the Escherichia coli formate hydrogenlyase complex.

PubMed

McDowall, Jennifer S; Hjersing, M Charlotte; Palmer, Tracy; Sargent, Frank

2015-10-07

The Escherichia coli formate hydrogenlyase (FHL) complex is produced under fermentative conditions and couples formate oxidation to hydrogen production. In this work, the architecture of FHL has been probed by analysing affinity-tagged complexes from various genetic backgrounds. In a successful attempt to stabilize the complex, a strain encoding a fusion between FdhF and HycB has been engineered and characterised. Finally, site-directed mutagenesis of the hycG gene was performed, which is predicted to encode a hydrogenase subunit important for regulating sensitivity to oxygen. This work helps to define the core components of FHL and provides solutions to improving the stability of the enzyme. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Biotin-Tagged Polysaccharide Vesicular Nanocarriers for Receptor-Mediated Anticancer Drug Delivery in Cancer Cells.

PubMed

Deshpande, Nilesh Umakant; Jayakannan, Manickam

2018-06-15

Biotin-conjugated multi-stimuli-responsive polysaccharide vesicular nanocarriers are designed and developed, for the first time, to accomplish receptor-mediated endocytosis in cancer cells and to deliver anticancer drugs at the intracellular compartments. For this purpose, a new renewable hydrophobic unit was custom designed with redox-degradable disulphide and enzyme-biodegradable aliphatic ester chemical linkages and it was conjugated along with biotin on the dextran backbone. The dextran derivative self-assembled into nanovesicles of < 200 nm in size which were characterized by dynamic and static light scattering, electron and atomic force microscopes. Avidin-HABA assay established the high affinity of biotin-tagged dextran vesicles towards membrane-receptors up to 25 nM concentration. Doxorubicin-hydrochloride (DOX.HCl) loaded dextran vesicles exhibited stable formulation in PBS and FBS. Redox-degradation by glutathione (GSH) showed 60 % drug release whereas lysosomal esterase enzyme enabled > 98 % drug release in 12 h. Confocal microscope and flow cytometry assisted time-dependent cellular uptake studies revealed that the biotin-receptor over expressed cervical cancer cells (HeLa) exhibited larger drug accumulation through receptor-assisted endocytosis process. This process enabled the delivery of higher amount of DOX and significantly enhanced the killing in cancer cells (HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells). Control experiments such as biotin pre-treatment in cancer cells and energy-suppressed cellular uptake at 4 C further supported the occurrence of receptor-mediated endocytosis by the biotin-tagged polymer vesicles. This report provides first insights into the targeted polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in biotin receptors over expressed cervical cancer cells.

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

PubMed

Zhang, Pengpeng; Battchikova, Natalia; Paakkarinen, Virpi; Katoh, Hirokazu; Iwai, Masako; Ikeuchi, Masahiko; Pakrasi, Himadri B; Ogawa, Teruo; Aro, Eva-Mari

2005-09-01

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Identification of novel proteins associated with yeast snR30 small nucleolar RNA

PubMed Central

Lemay, Vincent; Hossain, Ahmed; Osheim, Yvonne N.; Beyer, Ann L.; Dragon, François

2011-01-01

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly. PMID:21893585

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Mahavir; Wang, Zhonghua; Cascio, Duilio

Shq1 is an essential protein involved in the early steps of biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). Shq1 binds to dyskerin (Cbf5 in yeast) at an early step of H/ACA RNP assembly and is subsequently displaced by the H/ACA RNA. Shq1 contains an N-terminal CS and a C-terminal Shq1-specific domain (SSD). Dyskerin harbors many mutations associated with dyskeratosis congenita. Structures of yeast Shq1 SSD bound to Cbf5 revealed that only a subset of these mutations is in the SSD binding site, implicating another subset in the putative CS binding site. Here in this paper, we present the crystalmore » structure of human Shq1 CS (hCS) and the nuclear magnetic resonance (NMR) and crystal structures of hCS containing a serine substitution for proline 22 that is associated with some prostate cancers. The structure of hCS is similar to yeast Shq1 CS domain (yCS) and consists of two β-sheets that form an immunoglobulin-like β-sandwich fold. The N-terminal affinity tag sequence AHHHHHH associates with a neighboring protein in the crystal lattice to form an extra β-strand. Deletion of this tag was required to get spectra suitable for NMR structure determination, while the tag was required for crystallization. NMR chemical shift perturbation (CSP) experiments with peptides derived from putative CS binding sites on dyskerin and Cbf5 revealed a conserved surface on CS important for Cbf5/dyskerin binding. A HADDOCK (high-ambiguity-driven protein-protein docking) model of a Shq1-Cbf5 complex that defines the position of CS domain in the pre-H/ACA RNP was calculated using the CSP data.« less

Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans

PubMed Central

Ramya, T N C; Weerapana, Eranthie; Cravatt, Benjamin F; Paulson, James C

2013-01-01

In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of “robust enrichment” afforded by covalent-labeling techniques and “specificity for glycoproteins” typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status. PMID:23070960

Structure and Interactions of the CS Domain of Human H/ACA RNP Assembly Protein Shq1

DOE PAGES

Singh, Mahavir; Wang, Zhonghua; Cascio, Duilio; ...

2014-12-29

Shq1 is an essential protein involved in the early steps of biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). Shq1 binds to dyskerin (Cbf5 in yeast) at an early step of H/ACA RNP assembly and is subsequently displaced by the H/ACA RNA. Shq1 contains an N-terminal CS and a C-terminal Shq1-specific domain (SSD). Dyskerin harbors many mutations associated with dyskeratosis congenita. Structures of yeast Shq1 SSD bound to Cbf5 revealed that only a subset of these mutations is in the SSD binding site, implicating another subset in the putative CS binding site. Here in this paper, we present the crystalmore » structure of human Shq1 CS (hCS) and the nuclear magnetic resonance (NMR) and crystal structures of hCS containing a serine substitution for proline 22 that is associated with some prostate cancers. The structure of hCS is similar to yeast Shq1 CS domain (yCS) and consists of two β-sheets that form an immunoglobulin-like β-sandwich fold. The N-terminal affinity tag sequence AHHHHHH associates with a neighboring protein in the crystal lattice to form an extra β-strand. Deletion of this tag was required to get spectra suitable for NMR structure determination, while the tag was required for crystallization. NMR chemical shift perturbation (CSP) experiments with peptides derived from putative CS binding sites on dyskerin and Cbf5 revealed a conserved surface on CS important for Cbf5/dyskerin binding. A HADDOCK (high-ambiguity-driven protein-protein docking) model of a Shq1-Cbf5 complex that defines the position of CS domain in the pre-H/ACA RNP was calculated using the CSP data.« less

Studying Protein-Protein Interactions by Biotin AP-Tagged Pulldown and LTQ-Orbitrap Mass Spectrometry.

PubMed

Xie, Zhongqiu; Jia, Yuemeng; Li, Hui

2017-01-01

The study of protein-protein interactions represents a key aspect of biological research. Identifying unknown protein binding partners using mass spectrometry (MS)-based proteomics has evolved into an indispensable strategy in drug discovery. The classic approach of immunoprecipitation with specific antibodies against the proteins of interest has limitations, such as the need for immunoprecipitation-qualified antibody. The biotin AP-tag pull-down system has the advantage of high specificity, ease of use, and no requirement for antibody. It is based on the high specificity, high affinity interaction between biotin and streptavidin. After pulldown, in-gel tryptic digestion and tandem mass spectrometry (MS/MS) analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein bands can be performed. In this work, we provide protocols that can be used for the identification of proteins that interact with FOXM1, a protein that has recently emerged as a potential biomarker and drug target in oncotherapy, as an example. We focus on the pull-down procedure and assess the efficacy of the pulldown with known FOXM1 interactors such as β-catenin. We use a high performance LTQ Orbitrap MSn system that combines rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap analysis to identify the interacting proteins.

Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

PubMed

Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

2017-10-01

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant production of a chimeric antimicrobial peptide in E. coli and assessment of its activity against some avian clinically isolated pathogens.

PubMed

Tanhaiean, Abass; Azghandi, Marjan; Razmyar, Jamshid; Mohammadi, Elyas; Sekhavati, Mohammad Hadi

2018-06-08

Over the last decades, poultry industry faced to the rapid emergence of multidrug-resistant bacteria as a global concern. Antimicrobial peptide (AMPs) known as potential antibiotic alternative and were considered as a new antimicrobial agent. Current methods of production and purification of AMPs have several limitations such as: costly, time-consuming and killing the producing host cells in recombinant form. In the present study, a chimeric peptide derived from camel lactoferrin was produced in Escherichia coli periplasmic space using a pET-based expression system and its antibacterial activity was determined on some avian pathogens in vitro. A carboxy-terminal polyhistidine tag was used for purification by Ni 2+ affinity chromatography with an average yield of 0.42 g/L. The His-tagged chimeric peptide showed different range of antimicrobial activity against clinically isolated avian pathogens with low chicken blood hemolysis activity and high serum stability. Overall, the results of this investigation showed the recombinant chimeric peptide was successfully expressed in pET-based expression system and could be considered as a proper alternative for some currently used antibiotics in poultry industry and drugs veterinary medicine. Copyright © 2018 Elsevier Ltd. All rights reserved.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

PubMed Central

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

2015-01-01

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner. PMID:26709825

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

PubMed

de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

2017-12-06

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brizio, Carmen; Galluccio, Michele; Wait, Robin

2006-06-09

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-stepmore » affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.« less

Dispersal, mortality, and predation on recently-stocked rainbow trout in Dale Hollow Lake, Tennessee

USGS Publications Warehouse

Ivasauskas, Tomas J.; Bettoli, Phillip William

2011-01-01

Forty-four hatchery-raised rainbow trout (Oncorhynchus mykiss) were implanted with ultrasonic tags and stocked into Dale Hollow Lake, Tennessee, and tracked at least once per week for eight weeks to describe post-stocking dispersal rates, movements, and habitat use. Dispersal followed a three-stage pattern characterized by rapid movement away from each stocking site during the first week, relatively little dispersal during the next three weeks, and further dispersion during the final four weeks that fish were tracked. Rainbow trout exhibited a strong affinity for coves and were rarely encountered in the main channel. Tagged fish stocked in March exhibited lower mortality (Zweekly = 0.027) than those stocked in January (Zweekly = 0.062) during the first eight weeks post-stocking. Diets of potential predators in Dale Hollow Lake were examined. Walleye (Sander vitreus), smallmouth bass (Micropterus dolomieu), largemouth bass (M. salmoides), and holdover rainbow trout all preyed on recently stocked trout. Larger walleye were more likely to prey on stocked rainbow trout, and walleye of all sizes tended to prey on the smaller trout in each stocked cohort. Walleye were more likely to feed on rainbow trout during January than March. Effective stocking strategies should focus on reducing predation by stocking larger rainbow trout or by stocking when predation risk is minimized (i.e., March).

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

PubMed

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

2015-12-19

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer.

PubMed

Lee, Young-Tae; Gibbons, Garrett; Lee, Shirley Y; Nikolovska-Coleska, Zaneta; Dou, Yali

2015-06-01

We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted using the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirms that the reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

PubMed Central

Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2011-01-01

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

PubMed

Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

2008-05-30

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

Protein unfolding with a steric trap.

PubMed

Blois, Tracy M; Hong, Heedeok; Kim, Tae H; Bowie, James U

2009-10-07

The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

PubMed

Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

2017-09-01

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

PubMed

Mathieu, Christoph; Macêdo, Juan P; Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

PubMed Central

Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C.; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S.; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei. PMID:28045943

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xinxin; Zhang, Min; Takano, Tetsuo

Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membranemore » and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.« less

Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

PubMed Central

Peng, Tao; Free, Paul; Fernig, David G.; Lim, Sierin; Tomczak, Nikodem

2016-01-01

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles. PMID:27622533

Specific In Vivo Labeling of Tyrosinated α-Tubulin and Measurement of Microtubule Dynamics Using a GFP Tagged, Cytoplasmically Expressed Recombinant Antibody

PubMed Central

Cassimeris, Lynne; Guglielmi, Laurence; Denis, Vincent; Larroque, Christian; Martineau, Pierre

2013-01-01

GFP-tagged proteins are used extensively as biosensors for protein localization and function, but the GFP moiety can interfere with protein properties. An alternative is to indirectly label proteins using intracellular recombinant antibodies (scFvs), but most antibody fragments are insoluble in the reducing environment of the cytosol. From a synthetic hyperstable human scFv library we isolated an anti-tubulin scFv, 2G4, which is soluble in mammalian cells when expressed as a GFP-fusion protein. Here we report the use of this GFP-tagged scFv to label microtubules in fixed and living cells. We found that 2G4-GFP localized uniformly along microtubules and did not disrupt binding of EB1, a protein that binds microtubule ends and serves as a platform for binding by a complex of proteins regulating MT polymerization. TOGp and CLIP-170 also bound microtubule ends in cells expressing 2G4-GFP. Microtubule dynamic instability, measured by tracking 2G4-GFP labeled microtubules, was nearly identical to that measured in cells expressing GFP-α-tubulin. Fluorescence recovery after photobleaching demonstrated that 2G4-GFP turns over rapidly on microtubules, similar to the turnover rates of fluorescently tagged microtubule-associated proteins. These data indicate that 2G4-GFP binds relatively weakly to microtubules, and this conclusion was confirmed in vitro. Purified 2G4 partially co-pelleted with microtubules, but a significant fraction remained in the soluble fraction, while a second anti-tubulin scFv, 2F12, was almost completely co-pelleted with microtubules. In cells, 2G4-GFP localized to most microtubules, but did not co-localize with those composed of detyrosinated α-tubulin, a post-translational modification associated with non-dynamic, more stable microtubules. Immunoblots probing bacterially expressed tubulins confirmed that 2G4 recognized α-tubulin and required tubulin’s C-terminal tyrosine residue for binding. Thus, a recombinant antibody with weak affinity for its substrate can be used as a specific intracellular biosensor that can differentiate between unmodified and post-translationally modified forms of a protein. PMID:23555790

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.

PubMed

Lee, Augustine S; Finkielman, Javier D; Peikert, Tobias; Hummel, Amber M; Viss, Margaret A; Specks, Ulrich

2005-12-20

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.

Nanoantenna harmonic sensor: theoretical analysis of contactless detection of molecules with light

NASA Astrophysics Data System (ADS)

Farhat, Mohamed; Cheng, Mark M. C.; Le, Khai Q.; Chen, Pai-Yen

2015-10-01

The nonlinear harmonic sensor is a popular wireless sensor and radiofrequency identification (RFID) technique, which allows high-performance sensing in a severe interference/clutter background by transmitting a radio wave and detecting its modulated higher-order harmonics. Here we introduce the concept and design of optical harmonic tags based on nonlinear nanoantennas that can contactlessly detect electronic (e.g. electron affinity) and optical (e.g. relative permittivity) characteristics of molecules. By using a dual-resonance gold-molecule-silver nanodipole antenna within the quantum mechanical realm, the spectral form of the second-harmonic scattering can sensitively reveal the physical properties of molecules, paving a new route towards optical molecular sensors and optical identification (OPID) of biological, genetic, and medical events for the ‘Internet of Nano-Things’.

Super-Chelators for Advanced Protein Labeling in Living Cells.

PubMed

Gatterdam, Karl; Joest, Eike F; Dietz, Marina S; Heilemann, Mike; Tampé, Robert

2018-05-14

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His 6 - and His 12 -tagged proteins as well as single-molecule-based super-resolution imaging. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis.

PubMed

Chakrabarti, Rajarshi; Bhowmick, Debajit; Bhargava, Varsha; Bhar, Kaushik; Siddhanta, Anirban

2013-09-20

Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis. Copyright © 2013 Elsevier Inc. All rights reserved.

Towards memory-aware services and browsing through lifelogging sensing.

PubMed

Arcega, Lorena; Font, Jaime; Cetina, Carlos

2013-11-05

Every day we receive lots of information through our senses that is lost forever, because it lacked the strength or the repetition needed to generate a lasting memory. Combining the emerging Internet of Things and lifelogging sensors, we believe it is possible to build up a Digital Memory (Dig-Mem) in order to complement the fallible memory of people. This work shows how to realize the Dig-Mem in terms of interactions, affinities, activities, goals and protocols. We also complement this Dig-Mem with memory-aware services and a Dig-Mem browser. Furthermore, we propose a RFID Tag-Sharing technique to speed up the adoption of Dig-Mem. Experimentation reveals an improvement of the user understanding of Dig-Mem as time passes, compared to natural memories where the level of detail decreases over time.

Nanoantenna harmonic sensor: theoretical analysis of contactless detection of molecules with light.

PubMed

Farhat, Mohamed; Cheng, Mark M C; Le, Khai Q; Chen, Pai-Yen

2015-10-16

The nonlinear harmonic sensor is a popular wireless sensor and radiofrequency identification (RFID) technique, which allows high-performance sensing in a severe interference/clutter background by transmitting a radio wave and detecting its modulated higher-order harmonics. Here we introduce the concept and design of optical harmonic tags based on nonlinear nanoantennas that can contactlessly detect electronic (e.g. electron affinity) and optical (e.g. relative permittivity) characteristics of molecules. By using a dual-resonance gold-molecule-silver nanodipole antenna within the quantum mechanical realm, the spectral form of the second-harmonic scattering can sensitively reveal the physical properties of molecules, paving a new route towards optical molecular sensors and optical identification (OPID) of biological, genetic, and medical events for the 'Internet of Nano-Things'.

Peptide-based biosensors: From self-assembled interfaces to molecular probes in electrochemical assays.

PubMed

Puiu, Mihaela; Bala, Camelia

2018-04-01

Redox-tagged peptides have emerged as functional materials with multiple applications in the area of sensing and biosensing applications due to their high stability, excellent redox properties and versatility of biomolecular interactions. They allow direct observation of molecular interactions in a wide range of affinity and enzymatic assays and act as electron mediators. Short helical peptides possess the ability to self-assemble in specific configurations with the possibility to develop in highly-ordered, stable 1D, 2D and 3D architectures in a hierarchical controlled manner. We provide here a brief overview of the electrochemical techniques available to study the electron transfer in peptide films with particular interest in developing biosensors with immobilized peptide motifs, for biological and clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

PubMed

He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

2012-07-01

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.

PubMed

Book, Adam J; Gladman, Nicholas P; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2010-08-13

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes*

PubMed Central

Book, Adam J.; Gladman, Nicholas P.; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2010-01-01

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis. PMID:20516081

Targeted thermal therapy with genetically engineered magnetite magnetosomes@RGD: Photothermia is far more efficient than magnetic hyperthermia.

PubMed

Plan Sangnier, Anouchka; Preveral, Sandra; Curcio, Alberto; K A Silva, Amanda; Lefèvre, Chistopher T; Pignol, David; Lalatonne, Yoann; Wilhelm, Claire

2018-06-10

Providing appropriate means for heat generation by low intratumoral nanoparticle concentrations is a major challenge for cancer nanotherapy. Here we propose RGD-tagged magnetosomes (magnetosomes@RGD) as a biogenic, genetically engineered, inorganic platform for multivalent thermal cancer treatment. Magnetosomes@RGD are biomagnetite nanoparticles synthesized by genetically modified magnetotactic bacteria thanks to a translational fusion of the RGD peptide with the magnetosomal protein MamC. Magnetosomes@RGD thus combine the high crystallinity of their magnetite core with efficient surface functionalization. The specific affinity of RGD was first quantified by single-cell magnetophoresis with a variety of cell types, including immune, muscle, endothelial, stem and cancer cells. The highest affinity and cellular uptake was observed with PC3 prostatic and HeLa uterine cancer cells. The efficiency of photothermia and magnetic hyperthermia was then compared on PC3 cells. Unexpectedly, photothermia was far more efficient than magnetic hyperthermia, which was almost totally inhibited by the cellular environment. RGD targeting was then assessed in vivo at tumor site, in mice bearing PC3 tumors. As a result, we demonstrate that targeted magnetic nanoparticles could generate heat on a therapeutic level after systemic administration, but only under laser excitation, and successfully inhibit tumor progression. Copyright © 2018 Elsevier B.V. All rights reserved.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

PubMed

Gallo, Eugenio; Jarvik, Jonathan W

2017-08-01

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications. © 2017. Published by The Company of Biologists Ltd.

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

PubMed

Barnidge, David R; Jelinek, Diane F; Muddiman, David C; Kay, Neil E

2005-01-01

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments

PubMed Central

Nesvizhskii, Alexey I.

2013-01-01

Analysis of protein interaction networks and protein complexes using affinity purification and mass spectrometry (AP/MS) is among most commonly used and successful applications of proteomics technologies. One of the foremost challenges of AP/MS data is a large number of false positive protein interactions present in unfiltered datasets. Here we review computational and informatics strategies for detecting specific protein interaction partners in AP/MS experiments, with a focus on incomplete (as opposite to genome-wide) interactome mapping studies. These strategies range from standard statistical approaches, to empirical scoring schemes optimized for a particular type of data, to advanced computational frameworks. The common denominator among these methods is the use of label-free quantitative information such as spectral counts or integrated peptide intensities that can be extracted from AP/MS data. We also discuss related issues such as combining multiple biological or technical replicates, and dealing with data generated using different tagging strategies. Computational approaches for benchmarking of scoring methods are discussed, and the need for generation of reference AP/MS datasets is highlighted. Finally, we discuss the possibility of more extended modeling of experimental AP/MS data, including integration with external information such as protein interaction predictions based on functional genomics data. PMID:22611043

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells.

PubMed

Nakamura, Yuki; Umeki, Nobuhisa; Abe, Mitsuhiro; Sako, Yasushi

2017-10-26

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

An affinity-directed protein missile system for targeted proteolysis.

PubMed

Fulcher, Luke J; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal P

2016-10-01

The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. © 2016 The Authors.

AtCHX13 is a plasma membrane K+ transporter.

PubMed

Zhao, Jian; Cheng, Ning-Hui; Motes, Christy M; Blancaflor, Elison B; Moore, Miranda; Gonzales, Naomi; Padmanaban, Senthilkumar; Sze, Heven; Ward, John M; Hirschi, Kendal D

2008-10-01

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using (86)Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a K(m) of 136 and 196 microm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connelly, Heather M; Pelletier, Dale A; Lu, Tse-Yuan

The GlnK and GlnB proteins are members of the pII signal transduction protein family, which is essential in nitrogen regulation due to this protein family's ability to sense internal cellular ammonium levels and control cellular response. The role of GlnK in nitrogen regulation has been studied in a variety of bacteria but previously has been uncharacterized in the purple nonsulfur anoxygenic phototropic bacterium Rhodopseudomonas palustris. R. palustris has tremendous metabolic versatility in its modes of energy generation and carbon metabolism, and it employs a sensitive nitrogen-ammonium regulation system that may vary from that of other commonly studied bacteria. In R.more » palustris, there are three annotated forms of pII proteins: GlnK1, GlnK2, and GlnB. Here we describe, for the first time, the characterization of GlnK1, GlnK2, and GlnB modifications as a response to nitrogen availability, thereby providing information about how this bacterium regulates the AmtB ammonium transporter and glutamine synthetase, which controls the rate of glutamate to glutamine conversion. Using a strategy of creating C-terminally tagged GlnK and GlnB proteins followed by tandem affinity purification in combination with top-down mass spectrometry, four isoforms of the GlnK2 and GlnB proteins and two isoforms of the GlnK1 protein were characterized at high resolution and mass accuracy. Wild-type or endogenous expression of all three proteins was also examined under normal ammonium conditions and ammonium starvation to ensure that the tagging and affinity purification methods employed did not alter the natural state of the proteins. All three proteins were found to undergo uridylylation under ammonium starvation conditions, presumably to regulate the AmtB ammonium transporter and glutamine synthetase. Under high-ammonium conditions, the GlnK1, GlnK2, and GlnB proteins are unmodified. This experimental protocol involving high-resolution mass spectrometry measurements of intact proteins provides a powerful method of examining the posttranslational modifications that play a crucial role in both the regulation of the AmtB ammonium transporter and glutamine synthetase within R. palustris.« less

A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems.

PubMed

Bras, Eduardo J S; Soares, Ruben R G; Azevedo, Ana M; Fernandes, Pedro; Arévalo-Rodríguez, Miguel; Chu, Virginia; Conde, João P; Aires-Barros, M Raquel

2017-09-15

Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of the GPI anchor of human Thy-1 on antibody recognition and function

PubMed Central

Bradley, John E.; Chan, Joy M.; Hagood, James S.

2012-01-01

Thymocyte differentiation antigen 1 (Thy-1) is a glycosylphosphatidyl inositol (GPI)-linked cell surface glycoprotein expressed on numerous cell types, which regulates signals affecting cell adhesion, migration, differentiation, and survival. In addition, Thy-1 has been detected in serum, cerebral spinal fluid, wound fluid from venous ulcers, synovial fluid from joints in rheumatoid arthritis and more recently urine. We previously detected Thy-1 in the conditioned media of cytokine-stimulated lung fibroblasts, suggesting that Thy-1 shedding may be a response to cellular stress. Soluble and membrane bound forms of Thy-1 from in vivo sources have been shown to be identical in size when deglycosylated, suggesting that soluble Thy-1 is separated from the diacyl glycerol portion of its GPI-anchor by hydrolysis within the GPI moiety. For Thy-1 and other GPI-anchored proteins, delipidation induces a stable change in conformation that manifests itself in a change in antibody affinity for soluble forms. Using epitope tagged recombinant soluble Thy-1, we report that widely available monoclonal antibodies to human Thy-1 are unable to detect soluble Thy-1 by immunoblotting. We reevaluated the Thy-1 that we previously reported in the conditioned media of normal human lung fibroblasts and found it to be entirely insoluble. These findings suggest that most Thy-1 reported in body fluids retains its GPI anchor and may be associated with membrane fragments or vesicles. This phenomenon should be considered in the generation of antibodies and controls for Thy-1 bioassays. Furthermore, the changes in Thy-1 conformation with delipidation, beyond affecting antibody affinity, likely affect the ligand affinity and biological function of soluble vs. released membrane-associated forms. PMID:23358110

Introducing a fluorescence-based standard to quantify protein partitioning into membranes.

PubMed

Thomas, Franziska A; Visco, Ilaria; Petrášek, Zdeněk; Heinemann, Fabian; Schwille, Petra

2015-11-01

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface. Copyright © 2015. Published by Elsevier B.V.

FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

PubMed

Zhang, Shizhu; Zheng, Hailin; Long, Nanbiao; Carbó, Natalia; Chen, Peiying; Aguilar, Pablo S; Lu, Ling

2014-02-01

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.

PubMed

Gädke, Johannes; Kleinfeldt, Lennart; Schubert, Chris; Rohde, Manfred; Biedendieck, Rebekka; Garnweitner, Georg; Krull, Rainer

2017-01-20

This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass. Copyright © 2016 Elsevier B.V. All rights reserved.

Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans.

PubMed

Anu, Prasannan V; Madanan, Madathiparambil G; Nair, Ananthakrishnan J; Nair, Gangaprasad A; Nair, Govinda Pillai M; Sudhakaran, Perumana R; Satheeshkumar, Padikara K

2018-04-01

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn 2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

PubMed

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle

PubMed Central

Stallmach, Robert; Kavishwar, Manoli; Withers-Martinez, Chrislaine; Hackett, Fiona; Collins, Christine R; Howell, Steven A; Yeoh, Sharon; Knuepfer, Ellen; Atid, Avshalom J; Holder, Anthony A; Blackman, Michael J

2015-01-01

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle. PMID:25599609

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

PubMed

Chikamatsu, Satomi; Furuno, Tadahide; Kinoshita, Yosuke; Inoh, Yoshikazu; Hirashima, Naohide; Teshima, Reiko; Nakanishi, Mamoru

2007-03-01

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Hitoshi; Akazawa, Daisuke; Toray Industries, Inc., Kanagawa

2010-05-14

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K.more » Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.« less

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6

PubMed Central

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-01-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P212121, with unit-cell parameters a = 88.35, b = 128.73, c = 131.03 Å. PMID:24100567

SCO5745, a Bifunctional RNase J Ortholog, Affects Antibiotic Production in Streptomyces coelicolor

PubMed Central

Bralley, Patricia; Aseem, Madiha

2014-01-01

The bacterial RNases J are considered bifunctional RNases possessing both endo- and exonucleolytic activities. We have isolated an RNase J ortholog from Streptomyces coelicolor encoded by the gene sco5745. We overexpressed a decahistidine-tagged version of SCO5745 and purified the overexpressed protein by immobilized metal ion affinity chromatography. We demonstrated the presence of both 5′-to-3′ exonucleolytic and endonucleolytic activities on the Bacillus subtilis thrS transcript. Exonucleoytic activity predominated with 5′ monophosphorylated thrS, while endonucleolytic activity predominated with 5′ triphosphorylated thrS. While sco5745 is the only RNase J allele in S. coelicolor, the gene is not essential. Its disruption resulted in delayed production of the antibiotic actinorhodin, overproduction of undecylprodigiosin, and diminished production of the calcium-dependent antibiotic, in comparison with the parental strain. PMID:24415725

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

PubMed

Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

2007-10-01

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

Towards Memory-Aware Services and Browsing through Lifelogging Sensing

PubMed Central

Arcega, Lorena; Font, Jaime; Cetina, Carlos

2013-01-01

Every day we receive lots of information through our senses that is lost forever, because it lacked the strength or the repetition needed to generate a lasting memory. Combining the emerging Internet of Things and lifelogging sensors, we believe it is possible to build up a Digital Memory (Dig-Mem) in order to complement the fallible memory of people. This work shows how to realize the Dig-Mem in terms of interactions, affinities, activities, goals and protocols. We also complement this Dig-Mem with memory-aware services and a Dig-Mem browser. Furthermore, we propose a RFID Tag-Sharing technique to speed up the adoption of Dig-Mem. Experimentation reveals an improvement of the user understanding of Dig-Mem as time passes, compared to natural memories where the level of detail decreases over time. PMID:24196436

Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

PubMed Central

Jiang, D W; Englund, P T

2001-01-01

As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6.

PubMed

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-10-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=88.35, b=128.73, c=131.03 Å.

Analysis of In Vivo Chromatin and Protein Interactions of Arabidopsis Transcript Elongation Factors.

PubMed

Pfab, Alexander; Antosz, Wojciech; Holzinger, Philipp; Bruckmann, Astrid; Griesenbeck, Joachim; Grasser, Klaus D

2017-01-01

A central step to elucidate the function of proteins commonly comprises the analysis of their molecular interactions in vivo. For nuclear regulatory proteins this involves determining protein-protein interactions as well as mapping of chromatin binding sites. Here, we present two protocols to identify protein-protein and chromatin interactions of transcript elongation factors (TEFs) in Arabidopsis. The first protocol (Subheading 3.1) describes protein affinity-purification coupled to mass spectrometry (AP-MS) that utilizes suspension cultured cells as experimental system. This approach provides an unbiased view of proteins interacting with epitope-tagged TEFs. The second protocol (Subheading 3.2) depicts details about a chromatin immunoprecipitation (ChIP) procedure to characterize genomic binding sites of TEFs. These methods should be valuable tools for the analysis of a broad variety of nuclear proteins.

Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

PubMed Central

2010-01-01

Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

PubMed Central

Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott

2013-01-01

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.

Participation of the extracellular domain in (pro)renin receptor dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki-Nakagawa, Chiharu; Nishimura, Misa; Tsukamoto, Tomoko

Highlights: • The (pro)renin receptor [(P)RR] is a regulator of the renin–angiotensin system. • The region responsible for (P)RR dimerization was investigated. • (P)RR extracellular domain constructs were retained intracellularly. • The extracellular domain of (P)RR is responsible for its dimerization. • Novel insight into the regulatory mechanism of soluble (P)RR secretion is provided. - Abstract: The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but themore » region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.« less

Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses*

PubMed Central

Komarova, Anastassia V.; Combredet, Chantal; Meyniel-Schicklin, Laurène; Chapelle, Manuel; Caignard, Grégory; Camadro, Jean-Michel; Lotteau, Vincent; Vidalain, Pierre-Olivier; Tangy, Frédéric

2011-01-01

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells. PMID:21911578

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana

PubMed Central

Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John; Mittler, Ron; Harmon, Alice; Harper, Jeffrey

2014-01-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, a 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” (TAP) tag and expressed in transgenic plants. Purified complexes were analyzed by tandem mass spectrometry. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (1) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (GLRs 1.2, 2.1, 2.9, 3.4, 3.7); (2) hormone signaling, such as ACC synthase (isoforms ACS-6, 7 and 8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (3) transcription, such as 7 WRKY family transcription factors; (4) metabolism, such as phosphoenol pyruvate (PEP) carboxylase; and (5) lipid signaling, such as phospholipase D (β, and γ). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300. PMID:19452453

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

PubMed

Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

2009-06-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein.

PubMed

Balogh, Ria K; Gyurcsik, Béla; Hunyadi-Gulyás, Éva; Christensen, Hans E M; Jancsó, Attila

2016-07-01

Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure. Copyright © 2016 Elsevier Inc. All rights reserved.

Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

PubMed

Guo, Chuanliang; Xue, Yan; Yang, Guanheng; Yin, Shang; Shi, Wansheng; Cheng, Yan; Yan, Xiaoshuang; Fan, Shuyue; Zhang, Huijun; Zeng, Fanyi

2016-08-01

Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs. © 2015 International Federation for Cell Biology.

The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

PubMed Central

Shimoi, Hitoshi; Sakamoto, Kazutoshi; Okuda, Masaki; Atthi, Ratchanee; Iwashita, Kazuhiro; Ito, Kiyoshi

2002-01-01

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast. PMID:11916725

Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies

PubMed Central

Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol

2012-01-01

Summary VP22, encoded by the UL49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. PMID:22569534

Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

PubMed Central

Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

2012-01-01

We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

PubMed

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Occurrence State and Molecular Structure Analysis of Extracellular Proteins with Implications on the Dewaterability of Waste-Activated Sludge.

PubMed

Wu, Boran; Ni, Bing-Jie; Horvat, Kristine; Song, Liyan; Chai, Xiaoli; Dai, Xiaohu; Mahajan, Devinder

2017-08-15

The occurrence state and molecular structure of extracellular proteins were analyzed to reveal the influencing factors on the water-holding capacities of protein-like substances in waste-activated sludge (WAS). The gelation process of extracellular proteins verified that advanced oxidation processes (AOPs) for WAS dewaterability improvement eliminated the water affinity of extracellular proteins and prevented these macromolecules from forming stable colloidal aggregates. Isobaric tags for relative and absolute quantitation proteomics identified that most of the extracellular proteins were originally derived from the intracellular part and the proteins originally located in the extracellular part were mainly membrane-associated. The main mechanism of extracellular protein transformation during AOPs could be represented by the damage of the membrane or related external encapsulating structure and the release of intracellular substances. For the selected representative extracellular proteins, the strong correlation (R 2 > 0.97, p < 0.03) between the surface hydrophilicity index and α-helix percentages in the secondary structure indicated that the water affinity relied more on the spatial distribution of hydrophilic functional groups rather than the content. Destructing the secondary structure represented by the α-helix and stretching the polypeptide aggregation in the water phase through disulfide bond removal might be the key to eliminating the inhibitory effects of extracellular proteins on the interstitial water removal from WAS.

AtCHX13 Is a Plasma Membrane K+ Transporter1[C][W][OA

PubMed Central

Zhao, Jian; Cheng, Ning-Hui; Motes, Christy M.; Blancaflor, Elison B.; Moore, Miranda; Gonzales, Naomi; Padmanaban, Senthilkumar; Sze, Heven; Ward, John M.; Hirschi, Kendal D.

2008-01-01

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using 86Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a Km of 136 and 196 μm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment. PMID:18676662

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

PubMed Central

Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

2015-01-01

Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

Identification of Proteolytic Cleavage Sites of EphA2 by Membrane Type 1 Matrix Metalloproteinase on the Surface of Cancer Cells.

PubMed

Kikuchi, Keiji; Kozuka-Hata, Hiroko; Oyama, Masaaki; Seiki, Motoharu; Koshikawa, Naohiko

2018-01-01

Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification . The purified fragment was digested with trypsin to generate proteolytic peptides , and the amino acid sequences of these peptides were determined by nano-LC-mass spectrometry to identify the MT1-MMP-mediated cleavage site(s) of EphA2.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

PubMed

Das, Samir; Nag, Arundhati; Liang, JingXin; Bunck, David N; Umeda, Aiko; Farrow, Blake; Coppock, Matthew B; Sarkes, Deborah A; Finch, Amethist S; Agnew, Heather D; Pitram, Suresh; Lai, Bert; Yu, Mary Beth; Museth, A Katrine; Deyle, Kaycie M; Lepe, Bianca; Rodriguez-Rivera, Frances P; McCarthy, Amy; Alvarez-Villalonga, Belen; Chen, Ann; Heath, John; Stratis-Cullum, Dimitra N; Heath, James R

2015-11-02

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

PubMed Central

2018-01-01

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie

2007-08-10

We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand ({alpha}/{beta}-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) nomore » specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.« less

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

PubMed

Golovko, A O; Koroleva, O N; Drutsa, V L

2017-12-01

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His) 6 -tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

Recombinant expression and characterization of L-asparaginase II from a moderately thermotolerant bacterial isolate.

PubMed

Vidya, Jalaja; Pandey, Ashok

2012-07-01

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; L-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver-Burk plot, and K(m) and V(max) were 0.89 mM and 0.18 U/mg, respectively.

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

PubMed

Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

1995-07-01

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei.

PubMed

Nguyen, Tu N; Schimanski, Bernd; Zahn, André; Klumpp, Birgit; Günzl, Arthur

2006-09-01

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

PubMed Central

Lukinavičius, Gražvydas; Lapinaitė, Audronė; Urbanavičiūtė, Giedrė; Gerasimaitė, Rūta; Klimašauskas, Saulius

2012-01-01

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA. PMID:23042683

Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions - identification of a new nonpeptide inhibitor using Biacore™ T200.

PubMed

Wear, Martin A; Nowicki, Matthew W; Blackburn, Elizabeth A; McNae, Iain W; Walkinshaw, Malcolm D

2017-04-01

We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his-tagged human cyclophilin-A. Our orientation-specific stabilisation approach captures his-tagged protein under 'physiological conditions' (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni 2+ -nitrilotriacetic acid surfaces, very briefly activated for primary amine-coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin-A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo-kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin-A-cyclosporin-A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin-A, from a screen of a fragment library. This fragment, 2,3-diaminopyridine, bound specifically with a mean affinity of 248 ± 60 μm. The X-ray structure of this 109-Da fragment bound in the active site of cyclophilin-A was solved to a resolution of 1.25 Å (PDB: 5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin-A inhibitors.

Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin*

PubMed Central

Vance, David J.; Tremblay, Jacqueline M.; Mantis, Nicholas J.; Shoemaker, Charles B.

2013-01-01

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics. PMID:24202178

Stepwise engineering of heterodimeric single domain camelid VHH antibodies that passively protect mice from ricin toxin.

PubMed

Vance, David J; Tremblay, Jacqueline M; Mantis, Nicholas J; Shoemaker, Charles B

2013-12-20

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH "heterodimers." As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.

Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

PubMed

Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

2018-04-01

Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

Expression, purification, and kinetic characterization of full-length human fibroblast activation protein.

PubMed

Sun, Shaoxian; Albright, Charles F; Fish, Barbara H; George, Henry J; Selling, Bernard H; Hollis, Gregory F; Wynn, Richard

2002-03-01

Human fibroblast activation protein (FAP), an integral membrane serine protease, was produced in insect cells as a hexa-His-tagged protein using a recombinant baculovirus expression system. Two isoforms of FAP, glycosylated and nonglycosylated, were identified by Western blotting using an anti-His-tag antibody and separated by lectin chromatography. The glycosylated FAP was purified to near homogeneity using immobilized metal affinity chromatography and was shown to have both postprolyl dipeptidyl peptidase and postgelatinase activities. In contrast, the nonglycosylated isoform demonstrated no detectable gelatinase activity by either zymography or a fluorescence-based gelatinase activity assay. The kinetic parameters of the dipeptidyl peptidase activity for glycosylated FAP were determined using dipeptide Ala-Pro-7-amino-trifluoromethyl-coumarin as the substrate. The k(cat) is 2.0 s(-1) and k(cat)/K(m) is 1.0 x 10(4) M(-1) s(-1) at pH 8.5. The pH dependence of k(cat) reveals two ionization groups with pK(a1) of 7.0 and pK(a2) of 11.0. The pH profile of k(cat)/K(m) yields similar results with pK(a1) 6.2 and pK(a2) 11.0. The neutral pK(a1) is associated with His at the active site. The basic pK(a2) might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure. Copyright 2002 Elsevier Science (USA).

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.

PubMed

Chen, Ying-Hui; Wang, Gao-Yuan; Hao, Hao-Chao; Chao, Chun-Jiang; Wang, Yamei; Jin, Quan-Wen

2017-03-01

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe , we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale. © 2017. Published by The Company of Biologists Ltd.

Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

PubMed

Singh, Ram Sarup; Yadav, Mukesh

2012-07-01

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

Comparison of three nonlinear models to describe long-term tag shedding by lake trout

USGS Publications Warehouse

Fabrizio, Mary C.; Swanson, Bruce L.; Schram, Stephen T.; Hoff, Michael H.

1996-01-01

We estimated long-term tag-shedding rates for lake trout Salvelinus namaycush using two existing models and a model we developed to account for the observed permanence of some tags. Because tag design changed over the course of the study, we examined tag-shedding rates for three types of numbered anchor tags (Floy tags FD-67, FD-67C, and FD-68BC) and an unprinted anchor tag (FD-67F). Lake trout from the Gull Island Shoal region, Lake Superior, were double-tagged, and subsequent recaptures were monitored in annual surveys conducted from 1974 to 1992. We modeled tag-shedding rates, using time at liberty and probabilities of tag shedding estimated from fish released in 1974 and 1978–1983 and later recaptured. Long-term shedding of numbered anchor tags in lake trout was best described by a nonlinear model with two parameters: an instantaneous tag-shedding rate and a constant representing the proportion of tags that were never shed. Although our estimates of annual shedding rates varied with tag type (0.300 for FD-67, 0.441 for FD-67C, and 0.656 for FD-68BC), differences were not significant. About 36% of tags remained permanently affixed to the fish. Of the numbered tags that were shed (about 64%), two mechanisms contributed to tag loss: disintegration and dislodgment. Tags from about 11% of recaptured fish had disintegrated, but most tags were dislodged. Unprinted tags were shed at a significant but low rate immediately after release, but the long-term, annual shedding rate of these tags was only 0.013. Compared with unprinted tags, numbered tags dislodged at higher annual rates; we hypothesized that this was due to the greater frictional drag associated with the larger cross-sectional area of numbered tags.

Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

PubMed Central

Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

2009-01-01

The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

PubMed

Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

2016-01-01

Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

System and method for acquisition management of subject position information

DOEpatents

Carrender, Curt

2005-12-13

A system and method for acquisition management of subject position information that utilizes radio frequency identification (RF ID) to store position information in position tags. Tag programmers receive position information from external positioning systems, such as the Global Positioning System (GPS), from manual inputs, such as keypads, or other tag programmers. The tag programmers program each position tag with the received position information. Both the tag programmers and the position tags can be portable or fixed. Implementations include portable tag programmers and fixed position tags for subject position guidance, and portable tag programmers for collection sample labeling. Other implementations include fixed tag programmers and portable position tags for subject route recordation. Position tags can contain other associated information such as destination address of an affixed subject for subject routing.

System and method for acquisition management of subject position information

DOEpatents

Carrender, Curt [Morgan Hill, CA

2007-01-23

A system and method for acquisition management of subject position information that utilizes radio frequency identification (RF ID) to store position information in position tags. Tag programmers receive position information from external positioning systems, such as the Global Positioning System (GPS), from manual inputs, such as keypads, or other tag programmers. The tag programmers program each position tag with the received position information. Both the tag programmers and the position tags can be portable or fixed. Implementations include portable tag programmers and fixed position tags for subject position guidance, and portable tag programmers for collection sample labeling. Other implementations include fixed tag programmers and portable position tags for subject route recordation. Position tags can contain other associated information such as destination address of an affixed subject for subject routing.

A laboratory evaluation of tagging-related mortality and tag loss in juvenile humpback chub

USGS Publications Warehouse

Ward, David L.; Persons, William R.; Young, Kirk; Stone, Dennis M.; Van Haverbeke, Randy; Knight, William R.

2015-01-01

We quantified tag retention, survival, and growth in juvenile, captive-reared Humpback Chub Gila cypha marked with three different tag types: (1) Biomark 12.5-mm, 134.2-kHz, full duplex PIT tags injected into the body cavity with a 12-gauge needle; (2) Biomark 8.4-mm, 134.2-kHz, full duplex PIT tags injected with a 16-gauge needle; and (3) Northwest Marine Technology visible implant elastomer (VIE) tags injected under the skin with a 29-gauge needle. Estimates of tag loss, tagging-induced mortality, and growth were evaluated for 60 d with each tag type for four different size-groups of fish: 40–49 mm, 50–59 mm, 60–69 mm, and 70–79 mm TL. Total length was a significant predictor of the probability of PIT tag retention and mortality for both 8-mm and 12-mm PIT tags, and the smallest fish had the highest rates of tag loss (12.5–30.0%) and mortality (7.5–20.0%). Humpback Chub of sizes 40–49 mm TL and tagged with VIE tags had no mortality but did have a 17.5% tag loss. Growth rates of all tagged fish were similar to controls. Our data indicate Humpback Chub can be effectively tagged using either 8-mm or 12-mm PIT tags with little tag loss or mortality at sizes as low as 65 mm TL.

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

PubMed

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak-Reppel, Katrin; Fernández-Montalván, Amaury E; IJzerman, Adriaan P; Heitman, Laura H

2016-01-01

Drug-target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin-releasing hormone (GnRH) receptor for the treatment of hormone-dependent diseases. Surprisingly, the kinetic receptor-binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor-binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. A novel radioligand-binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [(125) I]-triptorelin. In addition to radioligand-binding studies, a homogeneous time-resolved FRET Tag-lite™ method was developed as an alternative assay for the same purpose. Two novel competition association assays were successfully developed and applied to determine the kinetic receptor-binding characteristics of 12 high-affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. © 2015 The British Pharmacological Society.

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

PubMed Central

Ahn, K; Meyer, T H; Uebel, S; Sempé, P; Djaballah, H; Yang, Y; Peterson, P A; Früh, K; Tampé, R

1996-01-01

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP. Images PMID:8670825

Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

NASA Astrophysics Data System (ADS)

Sukhanova, Alyona; Even-Desrumeaux, Klervi; Millot, Jean-Marc; Chames, Patrick; Baty, Daniel; Artemyev, Mikhail; Oleinikov, Vladimir; Cohen, Jacques H. M.; Nabiev, Igor

2012-03-01

Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes <12 nm in diameter comprising four copies of sdAbs linked to the same QD in an oriented manner. sdAb-QD conjugates against carcinoembryonic antigen (CEA) and HER2 exhibited an extremely high specificity in flow cytometry; the quality of immunohistochemical labeling of biopsy samples was found to be superior to that of labeling according to the current "gold standard" protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.

PubMed

Santos, João H P M; Costa, Iris M; Molino, João V D; Leite, Mariana S M; Pimenta, Marcela V; Coutinho, João A P; Pessoa, Adalberto; Ventura, Sónia P M; Lopes, André M; Monteiro, Gisele

2017-03-01

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His) 6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni 2+ -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg -1 ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017. © 2016 American Institute of Chemical Engineers.

Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin

PubMed Central

Moreno-Habel, Daniela A.; Biglang-awa, Ivan M.; Dulce, Angelica; DeeLuu, Dee; Garcia, Peter; Weers, Paul M. M.; Haas-Stapleton, Eric J.

2012-01-01

Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released. PMID:22401766

System and method for inventorying multiple remote objects

DOEpatents

Carrender, Curtis L.; Gilbert, Ronald W.

2007-10-23

A system and method of inventorying multiple objects utilizing a multi-level or a chained radio frequency identification system. The system includes a master tag and a plurality of upper level tags and lower level tags associated with respective objects. The upper and lower level tags communicate with each other and the master tag so that reading of the master tag reveals the presence and absence of upper and lower level tags. In the chained RF system, the upper and lower level tags communicate locally with each other in a manner so that more remote tags that are out of range of some of the upper and lower level tags have their information relayed through adjacent tags to the master tag and thence to a controller.

System and method for inventorying multiple remote objects

DOEpatents

Carrender, Curtis L [Morgan Hill, CA; Gilbert, Ronald W [Morgan Hill, CA

2009-12-29

A system and method of inventorying multiple objects utilizing a multi-level or a chained radio frequency identification system. The system includes a master tag and a plurality of upper level tags and lower level tags associated with respective objects. The upper and lower level tags communicate with each other and the master tag so that reading of the master tag reveals the presence and absence of upper and lower level tags. In the chained RF system, the upper and lower level tags communicate locally with each other in a manner so that more remote tags that are out of range of some of the upper and lower level tags have their information relayed through adjacent tags to the master tag and thence to a controller.

Assessment of PIT tag retention and post-tagging survival in metamorphosing juvenile Sea Lamprey

USGS Publications Warehouse

Simard, Lee G.; Sotola, V. Alex; Marsden, J. Ellen; Miehls, Scott M.

2017-01-01

Background: Passive integrated transponder (PIT) tags have been used to document and monitor the movement or behavior of numerous species of fishes. Data on short-term and long-term survival and tag retention are needed before initiating studies using PIT tags on a new species or life stage. We evaluated the survival and tag retention of 153 metamorphosing juvenile Sea Lamprey Petromyzon marinus tagged with 12 mm PIT tags on three occasions using a simple surgical procedure. Results: Tag retention was 100% and 98.6% at 24 h and 28-105 d post-tagging. Of the lamprey that retained their tags, 87.3% had incisions sufficiently healed to prevent further loss. Survival was 100% and 92.7% at 24 h and 41-118 d post-tagging with no significant difference in survival between tagged and untagged control lamprey. Of the 11 lamprey that died, four had symptoms that indicated their death was directly related to tagging. Survival was positively correlated with Sea Lamprey length. Conclusions: Given the overall high level of survival and tag retention in this study, future studies can utilize 12 mm PIT tags to monitor metamorphosing juvenile Sea Lamprey movement and migration patterns.

Characterization of the Igf-II Binding Site of the IGF-II/MAN-6-P Receptor Extracellular Domain.

NASA Astrophysics Data System (ADS)

Garmroudi, Farideh

1995-01-01

In mammals, insulin-like growth factor II (IGF -II) and glycoproteins bearing the mannose 6-phosphate (Man -6-P) recognition marker bind with high affinity to the same receptor. The functional consequences of IGF-II binding to the receptor at the cell surface are not clear. In these studies, we sought to broaden our understanding of the functional regions of the receptor regarding its IGF -II binding site. The IGF-II binding/cross-linking domain of the IGF-II/Man-6-P receptor was mapped by sequencing receptor fragments covalently attached to IGF-II. Purified rat placental or bovine liver receptors were affinity-labeled, with ^{125}I-IGF-II and digested with endoproteinase Glu-C. Analysis of digests by gel electrophoresis revealed a major radiolabeled band of 18 kDa, which was purified by gel filtration chromatography followed by reverse-phase HPLC and electroblotting. Sequence analysis revealed that, the peptide S(H)VNSXPMF, located within extracellular repeat 10 and beginning with serine 1488 of the bovine receptor, was the best candidate for the IGF-II cross-linked peptide. These data indicated that residues within repeats 10-11 were important for IGF -II binding. To define the location of the IGF-II binding site further, a nested set of six human receptor cDNA constructs was designed to produce epitope-tagged fusion proteins encompassing the region between repeats 8 and 11 of the human IGF-II/Man-6-P receptor extracellular domain. These truncated receptors were transiently expressed in COS-7 cells, immunoprecipitated and analyzed for their abilities to bind and cross-link to IGF-II. All of the constructs were capable of binding/cross-linking to IGF-II, except for the 9.0-11 construct. Displacement curve analysis indicated that the truncated receptors were approximately equivalent in IGF-II binding affinity, but were of 5- to 10-fold lower affinity than full-length receptors. Sequencing of the 9.0-11 construct indicated the presence of a point mutation substituting threonine for isoleucine at position 1621, which is located in the N-terminal half of repeat 11, and was found to abrogate IGF-II binding. Collectively, our work indicates that repeat 11 of the IGF-II/Man-6-P receptor's extracellular domain encompasses the elements both for binding and cross-linking to IGF-II.

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

PubMed

Arruda, Maria Augusta; Stoddart, Leigh A; Gherbi, Karolina; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J

2017-01-01

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A 3 AR. These were K114 (pK i 6.43), retinoic acid p -hydroxyanilide (pK i 6.13) and SU 6556 (pK i 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A 3 AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

Tag-mediated cooperation with non-deterministic genotype-phenotype mapping

NASA Astrophysics Data System (ADS)

Zhang, Hong; Chen, Shu

2016-01-01

Tag-mediated cooperation provides a helpful framework for resolving evolutionary social dilemmas. However, most of the previous studies have not taken into account genotype-phenotype distinction in tags, which may play an important role in the process of evolution. To take this into consideration, we introduce non-deterministic genotype-phenotype mapping into a tag-based model with spatial prisoner's dilemma. By our definition, the similarity between genotypic tags does not directly imply the similarity between phenotypic tags. We find that the non-deterministic mapping from genotypic tag to phenotypic tag has non-trivial effects on tag-mediated cooperation. Although we observe that high levels of cooperation can be established under a wide variety of conditions especially when the decisiveness is moderate, the uncertainty in the determination of phenotypic tags may have a detrimental effect on the tag mechanism by disturbing the homophilic interaction structure which can explain the promotion of cooperation in tag systems. Furthermore, the non-deterministic mapping may undermine the robustness of the tag mechanism with respect to various factors such as the structure of the tag space and the tag flexibility. This observation warns us about the danger of applying the classical tag-based models to the analysis of empirical phenomena if genotype-phenotype distinction is significant in real world. Non-deterministic genotype-phenotype mapping thus provides a new perspective to the understanding of tag-mediated cooperation.

Anchor and visible implant elastomer tag retention by hatchery rainbow trout stocked into an Ozark stream

USGS Publications Warehouse

Walsh, M.G.; Winkelman, D.L.

2004-01-01

As part of a study to evaluate the stocking of rainbow trout Oncorhynchus mykiss in an Oklahoma Ozark stream, we tagged 2,542 hatchery-reared rainbow trout (123-366 mm total length) with individually numbered Floy FD-68B anchor tags and visible implant fluorescent elastomer (VIE) tags. We experimentally stocked double-marked rainbow trout into a small northeastern Oklahoma stream from November 2001 to March 2002 and resampled them monthly from December 2001 to October 2002 by electrofishing. Anchor tag retention was 91% through 6 months, and VIE tag retention was 96% through 6 months despite extensive handling of fish within 24 h of tagging. Based on the ease of application, high visibility, and high retention observed in this study, we recommend the use of VIE tags as a batch mark in similarly sized, similarly pigmented fish. The retention of VIE tags was slightly higher than that of anchor tags, and cost per fish was less for VIE than for anchor tags. However, VIE tags would have limited utility if numerous individual tags are necessary; therefore, we recommend anchor tags as individual marks in similarly sized salmonids. Retention for both tag types was relatively high and could be corrected for when estimating population parameters from tagging data.

Comparison of migration rate and survival between radio-tagged and PIT-tagged migrant yearling chinook salmon in the Snake and Columbia rivers

USGS Publications Warehouse

Hockersmith, E.E.; Muir, W.D.; Smith, S.G.; Sandford, B.P.; Perry, R.W.; Adams, N.S.; Rondorf, D.W.

2003-01-01

A study was conducted to compare the travel times, detection probabilities, and survival of migrant hatchery-reared yearling chinook salmon Oncorhynchus tshawytscha tagged with either gastrically or surgically implanted sham radio tags (with an imbedded passive integrated transponder [PIT] tag) with those of their cohorts tagged only with PIT tags in the Snake and Columbia rivers. Juvenile chinook salmon with gastrically implanted radio tags migrated significantly faster than either surgically radio-tagged or PIT-tagged fish, while migration rates were similar among surgically radio-tagged and PIT-tagged fish. The probabilities of PIT tag detection at downstream dams varied by less than 5% and were not significantly different among the three groups. Survival was similar among treatments for median travel times of less than approximately 6 d (migration distance of 106 km). However, for both gastrically and surgically radio-tagged fish, survival was significantly less than for PIT-tagged fish, for which median travel times exceeded approximately 10 d (migration distance of 225 km). The results of this study support the use of radio tags to estimate the survival of juvenile chinook salmon having a median fork length of approximately 150 mm (range, 127-285 mm) and a median travel time of migration of less than approximately 6 d.

A Hybrid Probabilistic Model for Unified Collaborative and Content-Based Image Tagging.

PubMed

Zhou, Ning; Cheung, William K; Qiu, Guoping; Xue, Xiangyang

2011-07-01

The increasing availability of large quantities of user contributed images with labels has provided opportunities to develop automatic tools to tag images to facilitate image search and retrieval. In this paper, we present a novel hybrid probabilistic model (HPM) which integrates low-level image features and high-level user provided tags to automatically tag images. For images without any tags, HPM predicts new tags based solely on the low-level image features. For images with user provided tags, HPM jointly exploits both the image features and the tags in a unified probabilistic framework to recommend additional tags to label the images. The HPM framework makes use of the tag-image association matrix (TIAM). However, since the number of images is usually very large and user-provided tags are diverse, TIAM is very sparse, thus making it difficult to reliably estimate tag-to-tag co-occurrence probabilities. We developed a collaborative filtering method based on nonnegative matrix factorization (NMF) for tackling this data sparsity issue. Also, an L1 norm kernel method is used to estimate the correlations between image features and semantic concepts. The effectiveness of the proposed approach has been evaluated using three databases containing 5,000 images with 371 tags, 31,695 images with 5,587 tags, and 269,648 images with 5,018 tags, respectively.

Understanding why users tag: A survey of tagging motivation literature and results from an empirical study.

PubMed

Strohmaier, Markus; Körner, Christian; Kern, Roman

2012-12-01

While recent progress has been achieved in understanding the structure and dynamics of social tagging systems, we know little about the underlying user motivations for tagging, and how they influence resulting folksonomies and tags. This paper addresses three issues related to this question. (1) What distinctions of user motivations are identified by previous research, and in what ways are the motivations of users amenable to quantitative analysis? (2) To what extent does tagging motivation vary across different social tagging systems? (3) How does variability in user motivation influence resulting tags and folksonomies? In this paper, we present measures to detect whether a tagger is primarily motivated by categorizing or describing resources, and apply these measures to datasets from seven different tagging systems. Our results show that (a) users' motivation for tagging varies not only across, but also within tagging systems, and that (b) tag agreement among users who are motivated by categorizing resources is significantly lower than among users who are motivated by describing resources . Our findings are relevant for (1) the development of tag-based user interfaces, (2) the analysis of tag semantics and (3) the design of search algorithms for social tagging systems.

Understanding why users tag: A survey of tagging motivation literature and results from an empirical study

PubMed Central

Strohmaier, Markus; Körner, Christian; Kern, Roman

2012-01-01

While recent progress has been achieved in understanding the structure and dynamics of social tagging systems, we know little about the underlying user motivations for tagging, and how they influence resulting folksonomies and tags. This paper addresses three issues related to this question. (1) What distinctions of user motivations are identified by previous research, and in what ways are the motivations of users amenable to quantitative analysis? (2) To what extent does tagging motivation vary across different social tagging systems? (3) How does variability in user motivation influence resulting tags and folksonomies? In this paper, we present measures to detect whether a tagger is primarily motivated by categorizing or describing resources, and apply these measures to datasets from seven different tagging systems. Our results show that (a) users’ motivation for tagging varies not only across, but also within tagging systems, and that (b) tag agreement among users who are motivated by categorizing resources is significantly lower than among users who are motivated by describing resources. Our findings are relevant for (1) the development of tag-based user interfaces, (2) the analysis of tag semantics and (3) the design of search algorithms for social tagging systems. PMID:23471473

29 CFR 1915.89 - Control of hazardous energy (lockout/tags-plus).

Code of Federal Regulations, 2012 CFR

2012-07-01

... employee: (A) Sign a group tag (or a group tag equivalent), attach a personal identification device to a... group tag (or the group tag equivalent), remove the personal identification device, or perform a... safe exposure status of each authorized employee, and (b) signs a group tag (or a group tag equivalent...

29 CFR 1915.89 - Control of hazardous energy (lockout/tags-plus).

Code of Federal Regulations, 2013 CFR

2013-07-01

... employee: (A) Sign a group tag (or a group tag equivalent), attach a personal identification device to a... group tag (or the group tag equivalent), remove the personal identification device, or perform a... safe exposure status of each authorized employee, and (b) signs a group tag (or a group tag equivalent...

29 CFR 1915.89 - Control of hazardous energy (lockout/tags-plus).

Code of Federal Regulations, 2014 CFR

2014-07-01

... employee: (A) Sign a group tag (or a group tag equivalent), attach a personal identification device to a... group tag (or the group tag equivalent), remove the personal identification device, or perform a... safe exposure status of each authorized employee, and (b) signs a group tag (or a group tag equivalent...

Tag loss and short-term mortality associated with passive integrated transponder tagging of juvenile Lost River suckers

USGS Publications Warehouse

Burdick, Summer M.

2011-01-01

Passive integrated transponder (PIT) tags are commonly used to mark small catostomids, but tag loss and the effect of tagging on mortality have not been assessed for juveniles of the endangered Lost River sucker Deltistes luxatus. I evaluated tag loss and short-term (34-d) mortality associated with the PIT tagging of juvenile Lost River suckers in the laboratory by using a completely randomized design and three treatment groups (PIT tagged, positive control, and control). An empty needle was inserted into each positive control fish, whereas control fish were handled but not tagged. Only one fish expelled its PIT tag. Mortality rate averaged 9.8 ± 3.4% (mean ± SD) for tagged fish; mortality was 0% for control and positive control fish. All tagging mortalities occurred in fish with standard lengths of 71 mm or less, and most of the mortalities occurred within 48 h of tagging. My results indicate that 12.45- × 2.02-mm PIT tags provide a viable method of marking juvenile Lost River suckers that are 72 mm or larger.

Roles for stress-inducible lambda glutathione transferases in flavonoid metabolism in plants as identified by ligand fishing.

PubMed

Dixon, David P; Edwards, Robert

2010-11-19

The glutathione transferases (GSTs) of plants are a superfamily of abundant enzymes whose roles in endogenous metabolism are largely unknown. For example, the lambda class of GSTs (GSTLs) have members that are selectively induced by chemical stress treatments and based on their enzyme chemistry are predicted to have roles in redox homeostasis. However, using conventional approaches these functions have yet to be determined. To address this, recombinant GSTLs from wheat and Arabidopsis were tagged with a Strep tag and after affinity-immobilization, incubated with extracts from Arabidopsis, tobacco, and wheat. Bound ligands were then recovered by solvent extraction and identified by mass spectrometry (MS). With the wheat enzyme TaGSTL1, the ligand profiles obtained with in vitro extracts from tobacco closely matched those observed after the protein had been expressed in planta, demonstrating that these associations were physiologically representative. The stress-inducible TaGSTL1 was found to selectively recognize flavonols (e.g. taxifolin; K(d) = 25 nM), with this binding being dependent upon S-glutathionylation of an active site cysteine. In the case of the wheat extracts, this selectivity in ligand recognitions lead to the detection of flavonols that had not been previously described in this cereal. Subsequent in vitro assays showed that the co-binding of flavonols, such as quercetin, to the thiolated TaGSTL1 represented an intermediate step in the reduction of the respective S-glutathionylated quinone derivatives to yield free flavonols. These results suggest a novel role for GSTLs in maintaining the flavonoid pool under stress conditions.

Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration.

PubMed

McCormick, Aleesha M; Jarmusik, Natalie A; Endrizzi, Elizabeth J; Leipzig, Nic D

2014-01-22

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

PubMed

Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

PubMed Central

Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

PubMed Central

McCormick, Aleesha M.; Jarmusik, Natalie A.; Endrizzi, Elizabeth J.; Leipzig, Nic D.

2014-01-01

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification. PMID:24513608

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

PubMed

Chandrayan, Sanjeev K; McTernan, Patrick M; Hopkins, R Christopher; Sun, Junsong; Jenney, Francis E; Adams, Michael W W

2012-01-27

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

PubMed Central

Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

2012-01-01

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

PubMed

Nietzel, Thomas; Mostertz, Jörg; Hochgräfe, Falko; Schwarzländer, Markus

2017-03-01

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Method for designing gas tag compositions

DOEpatents

Gross, Kenny C.

1995-01-01

For use in the manufacture of gas tags such as employed in a nuclear reactor gas tagging failure detection system, a method for designing gas tagging compositions utilizes an analytical approach wherein the final composition of a first canister of tag gas as measured by a mass spectrometer is designated as node #1. Lattice locations of tag nodes in multi-dimensional space are then used in calculating the compositions of a node #2 and each subsequent node so as to maximize the distance of each node from any combination of tag components which might be indistinguishable from another tag composition in a reactor fuel assembly. Alternatively, the measured compositions of tag gas numbers 1 and 2 may be used to fix the locations of nodes 1 and 2, with the locations of nodes 3-N then calculated for optimum tag gas composition. A single sphere defining the lattice locations of the tag nodes may be used to define approximately 20 tag nodes, while concentric spheres can extend the number of tag nodes to several hundred.

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding.

PubMed

Hutchinson, Matthew H; Chase, Howard A

2006-01-01

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

PubMed

Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

2016-05-15

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX. Copyright © 2016 Elsevier B.V. All rights reserved.

Cetacean Tag Design Workshop Held in Arlington, Virginia on 16-17 March 2009

DTIC Science & Technology

2009-03-01

tagging to assess tag performance and animal health . • Examine performance of current tag attachment designs by assessing holding power and anchoring...tag attachment performance, and potentially adversely affect animal health . The second recommended step was to test and collect baseline data on...the tag attachment site to assess animal health and tag performance. Best to test tags on populations that are not listed as threatened or

Ontologies and tag-statistics

NASA Astrophysics Data System (ADS)

Tibély, Gergely; Pollner, Péter; Vicsek, Tamás; Palla, Gergely

2012-05-01

Due to the increasing popularity of collaborative tagging systems, the research on tagged networks, hypergraphs, ontologies, folksonomies and other related concepts is becoming an important interdisciplinary area with great potential and relevance for practical applications. In most collaborative tagging systems the tagging by the users is completely ‘flat’, while in some cases they are allowed to define a shallow hierarchy for their own tags. However, usually no overall hierarchical organization of the tags is given, and one of the interesting challenges of this area is to provide an algorithm generating the ontology of the tags from the available data. In contrast, there are also other types of tagged networks available for research, where the tags are already organized into a directed acyclic graph (DAG), encapsulating the ‘is a sub-category of’ type of hierarchy between each other. In this paper, we study how this DAG affects the statistical distribution of tags on the nodes marked by the tags in various real networks. The motivation for this research was the fact that understanding the tagging based on a known hierarchy can help in revealing the hidden hierarchy of tags in collaborative tagging systems. We analyse the relation between the tag-frequency and the position of the tag in the DAG in two large sub-networks of the English Wikipedia and a protein-protein interaction network. We also study the tag co-occurrence statistics by introducing a two-dimensional (2D) tag-distance distribution preserving both the difference in the levels and the absolute distance in the DAG for the co-occurring pairs of tags. Our most interesting finding is that the local relevance of tags in the DAG (i.e. their rank or significance as characterized by, e.g., the length of the branches starting from them) is much more important than their global distance from the root. Furthermore, we also introduce a simple tagging model based on random walks on the DAG, capable of reproducing the main statistical features of tag co-occurrence. This model has high potential for further practical applications, e.g., it can provide the starting point for a benchmark system in ontology retrieval or it may help pinpoint unusual correlations in the co-occurrence of tags.

Communication methods, systems, apparatus, and devices involving RF tag registration

DOEpatents

Burghard, Brion J [W. Richland, WA; Skorpik, James R [Kennewick, WA

2008-04-22

One technique of the present invention includes a number of Radio Frequency (RF) tags that each have a different identifier. Information is broadcast to the tags from an RF tag interrogator. This information corresponds to a maximum quantity of tag response time slots that are available. This maximum quantity may be less than the total number of tags. The tags each select one of the time slots as a function of the information and a random number provided by each respective tag. The different identifiers are transmitted to the interrogator from at least a subset of the RF tags.

75 FR 53271 - Marine Mammals; File No. 15271

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-31

... suction-cup tagged, 10 would be dart-tagged, and 10 would be tagged with small implantable tags; 30 fin whales would be approached for photo-ID and biopsy, of which 10 would be suction-cup tagged, 5 would be... approached for photo-ID and biopsy, of which 30 would be suction-cup tagged, 10 would be dart-tagged, and 10...

Some Fundamental Limits on SAW RFID Tag Information Capacity and Collision Resolution

NASA Technical Reports Server (NTRS)

Barton, Richard J.

2013-01-01

In this paper, we apply results from multi-user information theory to study the limits of information capacity and collision resolution for SAW RFID tags. In particular, we derive bounds on the achievable data rate per tag as a function of fundamental parameters such as tag time-bandwidth product, tag signal-to-noise ratio (SNR), and number of tags in the environment. We also discuss the implications of these bounds for tag waveform design and tag interrogation efficiency

Effects of coded-wire-tagging on stream-dwelling Sea Lamprey larvae

USGS Publications Warehouse

Johnson, Nicholas; Swink, William D.; Dawson, Heather A.; Jones, Michael L.

2016-01-01

The effects of coded wire tagging Sea Lamprey Petromyzon marinus larvae from a known-aged stream-dwelling population were assessed. Tagged larvae were significantly shorter on average than untagged larvae from 3 to 18 months after tagging. However, 30 months after tagging, the length distribution of tagged and untagged larvae did not differ and tagged Sea Lampreys were in better condition (i.e., higher condition factor) and more likely to have undergone metamorphosis than the untagged population. The reason why tagged larvae were more likely to metamorphose is not clear, but the increased likelihood of metamorphosis could have been a compensatory response to the period of slower growth after tagging. Slower growth after tagging was consistent across larval size-classes, so handling and displacement from quality habitat during the early part of the growing season was likely the cause rather than the tag burden. The tag effects observed in this study, if caused by displacement and handling, may be minimized in future studies if tagging is conducted during autumn after growth has concluded for the year.

Method for designing gas tag compositions

DOEpatents

Gross, K.C.

1995-04-11

For use in the manufacture of gas tags such as employed in a nuclear reactor gas tagging failure detection system, a method for designing gas tagging compositions utilizes an analytical approach wherein the final composition of a first canister of tag gas as measured by a mass spectrometer is designated as node No. 1. Lattice locations of tag nodes in multi-dimensional space are then used in calculating the compositions of a node No. 2 and each subsequent node so as to maximize the distance of each node from any combination of tag components which might be indistinguishable from another tag composition in a reactor fuel assembly. Alternatively, the measured compositions of tag gas numbers 1 and 2 may be used to fix the locations of nodes 1 and 2, with the locations of nodes 3-N then calculated for optimum tag gas composition. A single sphere defining the lattice locations of the tag nodes may be used to define approximately 20 tag nodes, while concentric spheres can extend the number of tag nodes to several hundred. 5 figures.

Survival, growth, and tag retention in age-0 Chinook Salmon implanted with 8-, 9-, and 12-mm PIT tags

USGS Publications Warehouse

Tiffan, Kenneth F.; Perry, Russell W.; Connor, William P.; Mullins, Frank L.; Rabe, Craig; Nelson, Doug D

2015-01-01

The ability to represent a population of migratory juvenile fish with PIT tags becomes difficult when the minimum tagging size is larger than the average size at which fish begin to move downstream. Tags that are smaller (e.g., 8 and 9 mm) than the commonly used 12-mm PIT tags are currently available, but their effects on survival, growth, and tag retention in small salmonid juveniles have received little study. We evaluated growth, survival, and tag retention in age-0 Chinook Salmon Oncorhynchus tshawytscha of three size-groups: 40–49-mm fish were implanted with 8- and 9-mm tags, and 50– 59-mm and 60–69-mm fish were implanted with 8-, 9-, and 12-mm tags. Survival 28 d after tagging ranged from 97.8% to 100% across all trials, providing no strong evidence for a fish-size-related tagging effect or a tag size effect. No biologically significant effects of tagging on growth in FL (mm/d) or weight (g/d) were observed. Although FL growth in tagged fish was significantly reduced for the 40–49-mm and 50–59-mm groups over the first 7 d, growth rates were not different thereafter, and all fish were similar in size by the end of the trials (day 28). Tag retention across all tests ranged from 93% to 99%. We acknowledge that actual implantation of 8- or 9-mm tags into small fish in the field will pose additional challenges (e.g., capture and handling stress) beyond those observed in our laboratory. However, we conclude that experimental use of the smaller tags for small fish in the field is supported by our findings.

Uncertainty of exploitation estimates made from tag returns

USGS Publications Warehouse

Miranda, L.E.; Brock, R.E.; Dorr, B.S.

2002-01-01

Over 6,000 crappies Pomoxis spp. were tagged in five water bodies to estimate exploitation rates by anglers. Exploitation rates were computed as the percentage of tags returned after adjustment for three sources of uncertainty: postrelease mortality due to the tagging process, tag loss, and the reporting rate of tagged fish. Confidence intervals around exploitation rates were estimated by resampling from the probability distributions of tagging mortality, tag loss, and reporting rate. Estimates of exploitation rates ranged from 17% to 54% among the five study systems. Uncertainty around estimates of tagging mortality, tag loss, and reporting resulted in 90% confidence intervals around the median exploitation rate as narrow as 15 percentage points and as broad as 46 percentage points. The greatest source of estimation error was uncertainty about tag reporting. Because the large investments required by tagging and reward operations produce imprecise estimates of the exploitation rate, it may be worth considering other approaches to estimating it or simply circumventing the exploitation question altogether.

Radio tag retention and tag-related mortality among adult sockeye salmon

USGS Publications Warehouse

Ramstad, Kristina M.; Woody, Carol Ann

2003-01-01

Tag retention and tag-related mortality are concerns for any tagging study but are rarely estimated. We assessed retention and mortality rates for esophageal radio tag implants in adult sockeye salmon Oncorhynchus nerka. Migrating sockeye salmon captured at the outlet of Lake Clark, Alaska, were implanted with one of four different radio tags (14.5 × 43 mm (diameter × length), 14.5 × 49 mm, 16 × 46 mm, and 19 × 51 mm). Fish were observed for 15 to 35 d after tagging to determine retention and mortality rates. The overall tag retention rate was high (0.98; 95% confidence interval (CI), 0.92-1.00; minimum, 33 d), with one loss of a 19-mm × 51- mm tag. Mortality of tagged sockeye salmon (0.02; 95% CI, 0-0.08) was similar to that of untagged controls (0.03 (0-0.15)). Sockeye salmon with body lengths (mid-eye to tail fork) of 585-649 mm retained tags as large as 19 × 51 mm and those with body lengths of 499-628 mm retained tags as small as 14.5 × 43 mm for a minimum of 33 d with no increase in mortality. The tags used in this study represent a suite of radio tags that vary in size, operational life, and cost but that are effective in tracking adult anadromous salmon with little tag loss or increase in fish mortality.

SparkClouds: visualizing trends in tag clouds.

PubMed

Lee, Bongshin; Riche, Nathalie Henry; Karlson, Amy K; Carpendale, Sheelash

2010-01-01

Tag clouds have proliferated over the web over the last decade. They provide a visual summary of a collection of texts by visually depicting the tag frequency by font size. In use, tag clouds can evolve as the associated data source changes over time. Interesting discussions around tag clouds often include a series of tag clouds and consider how they evolve over time. However, since tag clouds do not explicitly represent trends or support comparisons, the cognitive demands placed on the person for perceiving trends in multiple tag clouds are high. In this paper, we introduce SparkClouds, which integrate sparklines into a tag cloud to convey trends between multiple tag clouds. We present results from a controlled study that compares SparkClouds with two traditional trend visualizations—multiple line graphs and stacked bar charts—as well as Parallel Tag Clouds. Results show that SparkClouds ability to show trends compares favourably to the alternative visualizations.

Tag retention, growth, and survival of red swamp crayfish marked with a visible implant tag

USGS Publications Warehouse

Isely, J.J.; Stockett, P.E.

2001-01-01

Eighty juvenile (means: 42.4 mm total length, 1.6 g) red swamp crayfish Procambarus clarkii were implanted with sequentially numbered visible implant tags and held in the laboratory. Tags were injected transversely into the musculature just beneath the exoskeleton of the third abdominal segment from the cephalothorax; tags were visible upon inspection. An additional 20 crayfish were left untagged and served as controls. After 150 d, tag retention was 80% and all tags were readable. No tagged crayfish died during the study, and no differences in total length or weight were detected between tagged and control crayfish. All individuals molted at least three times during the 150-d study, and some individuals molted up to six times, suggesting that most tags would be permanently retained. The readability in the field without specialized equipment makes the visible implant tag ideal for studies of crayfish ecology, management, and culture.

77 FR 51761 - Proposed Information Collection; Comment Request; Groundfish Tagging Program

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-27

... required by the Paperwork Reduction Act of 1995. DATES: Written comments must be submitted on or before... are two general categories of tags. Simple plastic tags (spaghetti tags) are external tags... fish. Archival tags are microchips with sensors encased in plastic cylinders that record the depth...

Secure Integration of Radio Frequency Identification (RFID) Technology into a Supply Chain

DTIC Science & Technology

2005-09-01

serves as the rough equivalent of a license plate on an automobile . Figure 1 (below) illustrates the typical construction of an RFID tag. An antenna...writable passive tags (RW) Reprogrammable Class 3 Semi-active tags Reprogrammable Class 4 Active tags Reprogrammable Class 5 Readers... Reprogrammable Table 1. EPC Tag Classes[3]. Table 2 summarizes the advantages, disadvantages and applications of each type of tag. Tag Type Advantages

Measurement of tag confidence in user generated contents retrieval

NASA Astrophysics Data System (ADS)

Lee, Sihyoung; Min, Hyun-Seok; Lee, Young Bok; Ro, Yong Man

2009-01-01

As online image sharing services are becoming popular, the importance of correctly annotated tags is being emphasized for precise search and retrieval. Tags created by user along with user-generated contents (UGC) are often ambiguous due to the fact that some tags are highly subjective and visually unrelated to the image. They cause unwanted results to users when image search engines rely on tags. In this paper, we propose a method of measuring tag confidence so that one can differentiate confidence tags from noisy tags. The proposed tag confidence is measured from visual semantics of the image. To verify the usefulness of the proposed method, experiments were performed with UGC database from social network sites. Experimental results showed that the image retrieval performance with confidence tags was increased.

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

PubMed Central

Bodhinathan, Karthik; Slesinger, Paul A.

2013-01-01

Alcohol (ethanol) produces a wide range of pharmacological effects on the nervous system through its actions on ion channels. The molecular mechanism underlying ethanol modulation of ion channels is poorly understood. Here we used a unique method of alcohol-tagging to demonstrate that alcohol activation of a G-protein–gated inwardly rectifying potassium (GIRK or Kir3) channel is mediated by a defined alcohol pocket through changes in affinity for the membrane phospholipid signaling molecule phosphatidylinositol 4,5-bisphosphate. Surprisingly, hydrophobicity and size, but not the canonical hydroxyl, were important determinants of alcohol-dependent activation. Altering levels of G protein Gβγ subunits, conversely, did not affect alcohol-dependent activation, suggesting a fundamental distinction between receptor and alcohol gating of GIRK channels. The chemical properties of the alcohol pocket revealed here might extend to other alcohol-sensitive proteins, revealing a unique protein microdomain for targeting alcohol-selective therapeutics in the treatment of alcoholism and addiction. PMID:24145411

A young root-specific gene (ArMY2) from horseradish encoding a MYR II myrosinase with kinetic preference for the root-specific glucosinolate gluconasturtiin.

PubMed

Loebers, Andreas; Müller-Uri, Frieder; Kreis, Wolfgang

2014-03-01

The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Jerry; Tomlinson, Ian; Warnement, Michael

2011-01-01

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependentmore » decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.« less

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

PubMed

Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

2016-10-20

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

PubMed Central

Menger, Katja E.; James, Andrew M.; Cochemé, Helena M.; Harbour, Michael E.; Chouchani, Edward T.; Ding, Shujing; Fearnley, Ian M.; Partridge, Linda; Murphy, Michael P.

2015-01-01

Summary Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster. PMID:26095360