Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens.

PubMed

Chen, Jing; Toghi Eshghi, Shadi; Bova, George Steven; Li, Qing Kay; Li, Xingde; Zhang, Hui

2013-12-01

The rapid advancement of high-throughput tools for quantitative measurement of proteins has demonstrated the potential for the identification of proteins associated with cancer. However, the quantitative results on cancer tissue specimens are usually confounded by tissue heterogeneity, e.g. regions with cancer usually have significantly higher epithelium content yet lower stromal content. It is therefore necessary to develop a tool to facilitate the interpretation of the results of protein measurements in tissue specimens. Epithelial cell adhesion molecule (EpCAM) and cathepsin L (CTSL) are two epithelial proteins whose expressions in normal and tumorous prostate tissues were confirmed by measuring staining intensity with immunohistochemical staining (IHC). The expressions of these proteins were measured by ELISA in protein extracts from OCT embedded frozen prostate tissues. To eliminate the influence of tissue heterogeneity on epithelial protein quantification measured by ELISA, a color-based segmentation method was developed in-house for estimation of epithelium content using H&E histology slides from the same prostate tissues and the estimated epithelium percentage was used to normalize the ELISA results. The epithelium contents of the same slides were also estimated by a pathologist and used to normalize the ELISA results. The computer based results were compared with the pathologist's reading. We found that both EpCAM and CTSL levels, measured by ELISA assays itself, were greatly affected by epithelium content in the tissue specimens. Without adjusting for epithelium percentage, both EpCAM and CTSL levels appeared significantly higher in tumor tissues than normal tissues with a p value less than 0.001. However, after normalization by the epithelium percentage, ELISA measurements of both EpCAM and CTSL were in agreement with IHC staining results, showing a significant increase only in EpCAM with no difference in CTSL expression in cancer tissues. These results

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

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

Raymond, Amy; Lovell, Scott; Lorimer, Don

2009-12-01

With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. colimore » and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.« less

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

PubMed

Shih, Ying-Chun; Chen, Chao-Ling; Zhang, Yan; Mellor, Rebecca L; Kanter, Evelyn M; Fang, Yun; Wang, Hua-Chi; Hung, Chen-Ting; Nong, Jing-Yi; Chen, Hui-Ju; Lee, Tzu-Han; Tseng, Yi-Shuan; Chen, Chiung-Nien; Wu, Chau-Chung; Lin, Shuei-Liong; Yamada, Kathryn A; Nerbonne, Jeanne M; Yang, Kai-Chien

2018-04-13

Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4

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

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.

Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells.

PubMed

Zhao, Hong-Bing; Zhang, Xi-Feng; Jia, Xue-Lin; Wang, Hao-Bin

2017-09-01

Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling. The objective of this study was to investigate the expression of Grb7 and its clinicopathological significance in cervical cancer. Utilising immunohistochemical staining, we examined the expression of Grb7 in 120 cases of human cervical cancer tissue and 10 cases of adjacent non-cancerous cervical tissue. The positive rate of Grb7 protein expression was 34.2%, which was significantly higher than that in adjacent non-cancerous cervical tissue (0%, p<0.05). The expression of Grb7 was found to be correlated with age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis. Kaplan-Meier survival analysis showed that patients with positive Grb7 protein expression had a lower overall survival rate than patients without Grb7 expression. In addition, Grb7 plays an important role in promoting tumor progression, including invasion and anti-apoptosis, in cervical cancer cell line. Down-regulation of Grb7 repressed the expression of MMP-9 and Bcl-2, and increased the expression of Bax in Grb7 knockdown Hela cells. Cell invasion assay showed decreased number of Grb7 knockdown Hela cells (18.7±2.1) compared to Hela cells (65.3±2.5, P<0.05). Our results indicated that Grb7 over-expression may facilitate invasion and inhibit apoptosis in cervical cancer and Grb7 is a potentially molecular target of cervical cancer chemotherapy. Copyright © 2017 Elsevier GmbH. All rights reserved.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

PubMed

Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

2017-07-01

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

Facilitating protein solubility by use of peptide extensions

DOEpatents

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Facilitated release of substrate protein from prefoldin by chaperonin.

PubMed

Zako, Tamotsu; Iizuka, Ryo; Okochi, Mina; Nomura, Tomoko; Ueno, Taro; Tadakuma, Hisashi; Yohda, Masafumi; Funatsu, Takashi

2005-07-04

Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly measured using fluorescence microscopy. Our results suggest that binding and release of prefoldin from hyperthermophilic archaea with substrate proteins were in a dynamic equilibrium. Interestingly, the release of substrate proteins from prefoldin was facilitated when chaperonin was present, supporting a handoff mechanism of substrate proteins from prefoldin to the chaperonin.

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

PubMed Central

Godovikova, Valentina; Goetting-Minesky, M. Paula; Shin, Jae M.; Kapila, Yvonne L.; Rickard, Alexander H.

2015-01-01

Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism. PMID:26162875

Expression of the oncoprotein gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumor.

PubMed

Ando, Satoshi; Matsuoka, Taeko; Kawai, Koji; Sugita, Shintaro; Joraku, Akira; Kojima, Takahiro; Suetomi, Takahiro; Miyazaki, Jun; Fujita, Jun; Nishiyama, Hiroyuki

2014-10-01

The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors. © 2014 The Japanese Urological Association.

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

-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

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.

The Drosophila SH2-SH3 adapter protein Dock is expressed in embryonic axons and facilitates synapse formation by the RP3 motoneuron.

PubMed

Desai, C J; Garrity, P A; Keshishian, H; Zipursky, S L; Zinn, K

1999-04-01

The Dock SH2-SH3 domain adapter protein, a homolog of the mammalian Nck oncoprotein, is required for axon guidance and target recognition by photoreceptor axons in Drosophila larvae. Here we show that Dock is widely expressed in neurons and at muscle attachment sites in the embryo, and that this expression pattern has both maternal and zygotic components. In motoneurons, Dock is concentrated in growth cones. Loss of zygotic dock function causes a selective delay in synapse formation by the RP3 motoneuron at the cleft between muscles 7 and 6. These muscles often completely lack innervation in late stage 16 dock mutant embryos. RP3 does form a synapse later in development, however, because muscles 7 and 6 are normally innervated in third-instar mutant larvae. The absence of zygotically expressed Dock also results in subtle defects in a longitudinal axon pathway in the embryonic central nervous system. Concomitant loss of both maternally and zygotically derived Dock dramatically enhances these central nervous system defects, but does not increase the delay in RP3 synaptogenesis. These results indicate that Dock facilitates synapse formation by the RP3 motoneuron and is also required for guidance of some interneuronal axons The involvement of Dock in the conversion of the RP3 growth cone into a presynaptic terminal may reflect a role for Dock-mediated signaling in remodeling of the growth cone's cytoskeleton.

A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy.

PubMed

Gaupels, Frank; Knauer, Torsten; van Bel, Aart J E

2008-01-01

This study investigated advantages and drawbacks of two sieve-tube sap sampling methods for comparison of phloem proteins in powdery mildew-infested vs. non-infested Hordeum vulgare plants. In one approach, sieve tube sap was collected by stylectomy. Aphid stylets were cut and immediately covered with silicon oil to prevent any contamination or modification of exudates. In this way, a maximum of 1muL pure phloem sap could be obtained per hour. Interestingly, after pathogen infection exudation from microcauterized stylets was reduced to less than 40% of control plants, suggesting that powdery mildew induced sieve tube-occlusion mechanisms. In contrast to the laborious stylectomy, facilitated exudation using EDTA to prevent calcium-mediated callose formation is quick and easy with a large volume yield. After two-dimensional (2D) electrophoresis, a digital overlay of the protein sets extracted from EDTA solutions and stylet exudates showed that some major spots were the same with both sampling techniques. However, EDTA exudates also contained large amounts of contaminative proteins of unknown origin. A combinatory approach may be most favourable for studies in which the protein composition of phloem sap is compared between control and pathogen-infected plants. Facilitated exudation may be applied for subtractive identification of differentially expressed proteins by 2D/mass spectrometry, which requires large amounts of protein. A reference gel loaded with pure phloem sap from stylectomy may be useful for confirmation of phloem origin of candidate spots by digital overlay. The method provides a novel opportunity to study differential expression of phloem proteins in monocotyledonous plant species.

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

PubMed Central

Corridon, Peter R.; Rhodes, George J.; Leonard, Ellen C.; Basile, David P.; Gattone, Vincent H.; Bacallao, Robert L.

2013-01-01

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10–50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys. PMID:23467422

Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

PubMed

Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

2017-01-01

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity

Protein expression profiling in head fragments during planarian regeneration after amputation.

PubMed

Chen, Xiaoguang; Xu, Cunshuan

2015-04-01

Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

PubMed

Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

2016-01-01

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

PSI:Biology-Materials Repository: A Biologist’s Resource for Protein Expression Plasmids

PubMed Central

Cormier, Catherine Y.; Park, Jin G.; Fiacco, Michael; Steel, Jason; Hunter, Preston; Kramer, Jason; Singla, Rajeev; LaBaer, Joshua

2011-01-01

The Protein Structure Initiative:Biology-Materials Repository (PSI:Biology-MR; MR; http://psimr.asu.edu) sequence-verifies, annotates, stores, and distributes the protein expression plasmids and vectors created by the Protein Structure Initiative (PSI). The MR has developed an informatics and sample processing pipeline that manages this process for thousands of samples per month from nearly a dozen PSI centers. DNASU (http://dnasu.asu.edu), a freely searchable database, stores the plasmid annotations, which include the full-length sequence, vector information, and associated publications for over 130,000 plasmids created by our laboratory, by the PSI and other consortia, and by individual laboratories for distribution to researchers worldwide. Each plasmid links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org), which facilitates cross-referencing of a particular plasmid to additional protein annotations and experimental data. To expedite and simplify plasmid requests, the MR uses an expedited material transfer agreement (EP-MTA) network, where researchers from network institutions can order and receive PSI plasmids without institutional delays. Currently over 39,000 protein expression plasmids and 78 empty vectors from the PSI are available upon request from DNASU. Overall, the MR’s repository of expression-ready plasmids, its automated pipeline, and the rapid process for receiving and distributing these plasmids more effectively allows the research community to dissect the biological function of proteins whose structures have been studied by the PSI. PMID:21360289

Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

PubMed

Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

2015-12-01

The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses. © 2015 The Protein Society.

Facilitative glucose transporter gene expression in human lymphocytes, monocytes, and macrophages: a role for GLUT isoforms 1, 3, and 5 in the immune response and foam cell formation.

PubMed

Fu, Yuchang; Maianu, Lidia; Melbert, Barry R; Garvey, W Timothy

2004-01-01

Cellular glucose uptake is mediated by a family of facilitative glucose transporters (GLUT) exhibiting differences in kinetics, substrate specificity, and tissue-specific expression. GLUT isoform expression has not been comprehensively studied in human leukocytes, which participate in immune and inflammatory responses and are critical for host defense. Therefore, we studied the regulated expression of GLUT 1-5 mRNA and protein in isolated human lymphocytes and monocytes and in human THP-1 macrophages and foam cells. Lymphocytes expressed GLUT 1 and GLUT 3 proteins, and cellular levels of both isoforms were augmented 3.5- to 6-fold following activation by phytohemagglutinin (PHA). Monocytes expressed 8.4-fold more GLUT 3 protein and 88% less GLUT 1 than lymphocytes, and activation by lipopolysaccharide (LPS) led to a 1.9-fold increase in GLUT 1. At the level of mRNA expression, GLUT 3 mRNA was the most prevalent GLUT mRNA species in monocytes, while lymphocytes expressed equal numbers of GLUT 1 and GLUT 3 transcripts. Differentiation of THP-1 monocytes into macrophages was associated with marked induction of GLUT 3 and GLUT 5 protein expression, and high levels of GLUT 1, GLUT 3, and GLUT 5 were maintained after transformation to foam cells. GLUT 5 mRNA was expressed in 2-fold greater abundance in macrophages and foam cells than that observed for GLUT 1 mRNA, while the level of GLUT 3 mRNA was intermediate. This facilitative glucose transporters are differentially expressed and regulated in human leukocytes in a pattern that could facilitate cellular functions. Speculatively, high GLUT 1 and GLUT 3 expression could provide cellular fuel for the immune response, and high levels of high-affinity GLUT 3 in macrophages might allow the cell to compete with pathogens for hexoses, even in the presence of low interstitial glucose concentrations. Ample expression of GLUT 1 and GLUT 3 in foam cells could also provide hexose substrates and promote lipid loading. The role for

The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa.

PubMed

Laur, Joan; Hacke, Uwe G

2014-01-01

Gas exchange is constrained by the whole-plant hydraulic conductance (Kplant). Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf.

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.

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

PubMed

Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.

[Prokaryotic expression systems].

PubMed

Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał

2013-03-01

For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

BiP clustering facilitates protein folding in the endoplasmic reticulum.

PubMed

Griesemer, Marc; Young, Carissa; Robinson, Anne S; Petzold, Linda

2014-07-01

The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

Downregulation of RBO-PI4KIIIα Facilitates Aβ42 Secretion and Ameliorates Neural Deficits in Aβ42-Expressing Drosophila.

PubMed

Zhang, Xiao; Wang, Wen-An; Jiang, Li-Xiang; Liu, Hai-Yan; Zhang, Bao-Zhu; Lim, Nastasia; Li, Qing-Yi; Huang, Fu-De

2017-05-10

Phosphoinositides and their metabolizing enzymes are involved in Aβ 42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI 4 P). Here, we report that RBO binds to Drosophila PI4KIIIα, and that in an Aβ 42 -expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aβ 42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aβ 42 release and that PI4P facilitated the assembly or oligomerization of Aβ 42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aβ 42 release and consequently reduces neuronal Aβ 42 accumulation likely via decreasing Aβ 42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment. SIGNIFICANCE STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aβ 42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aβ 42 -expressing Drosophila model, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα-a protein that tightly controls the plasmalemmal level of PI 4 P-against the defects caused by Aβ 42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aβ 42 accumulation, and interestingly increased neuronal Aβ 42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates. Copyright �

Heat Shock Protein 83 (Hsp83) Facilitates Methoprene-tolerant (Met) Nuclear Import to Modulate Juvenile Hormone Signaling*

PubMed Central

He, Qianyu; Wen, Di; Jia, Qiangqiang; Cui, Chunlai; Wang, Jian; Palli, Subba R.; Li, Sheng

2014-01-01

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR. PMID:25122763

Improving membrane protein expression and function using genomic edits

DOE PAGES

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

2017-10-12

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Improving membrane protein expression and function using genomic edits

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

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

PubMed Central

McCarty, Thomas; Martin, Scott E.; Le Nouën, Cyril; Buehler, Eugen; Chen, Yu-Chi; Smelkinson, Margery; Ganesan, Sundar; Fischer, Elizabeth R.; Brock, Linda G.; Liang, Bo; Munir, Shirin; Collins, Peter L.; Buchholz, Ursula J.

2016-01-01

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread. PMID:27926942

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

PubMed Central

Schmied, Stéfanie; Affentranger, Sarah; Parvanova, Iana; Kang'a, Simon; Nene, Vishvanath; Katzer, Frank; McKeever, Declan; Müller, Joachim; Bishop, Richard; Pain, Arnab; Dobbelaere, Dirk A. E.

2009-01-01

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential

A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis.

PubMed

Song, Kai; Xue, Yiqun; Wang, Xiaohua; Wan, Yinglang; Deng, Xin; Lin, Jinxing

2017-06-01

Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFP A206K driven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFP A206K line was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFP A206K can be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana. Copyright © 2017 Elsevier GmbH. All rights reserved.

Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)

PubMed Central

Zhao, Jie

2010-01-01

Arabinogalactan proteins (AGPs) comprise a family of hydroxyproline-rich glycoproteins that are implicated in plant growth and development. In this study, 69 AGPs are identified from the rice genome, including 13 classical AGPs, 15 arabinogalactan (AG) peptides, three non-classical AGPs, three early nodulin-like AGPs (eNod-like AGPs), eight non-specific lipid transfer protein-like AGPs (nsLTP-like AGPs), and 27 fasciclin-like AGPs (FLAs). The results from expressed sequence tags, microarrays, and massively parallel signature sequencing tags are used to analyse the expression of AGP-encoding genes, which is confirmed by real-time PCR. The results reveal that several rice AGP-encoding genes are predominantly expressed in anthers and display differential expression patterns in response to abscisic acid, gibberellic acid, and abiotic stresses. Based on the results obtained from this analysis, an attempt has been made to link the protein structures and expression patterns of rice AGP-encoding genes to their functions. Taken together, the genome-wide identification and expression analysis of the rice AGP gene family might facilitate further functional studies of rice AGPs. PMID:20423940

Phage-Induced Expression of CRISPR-Associated Proteins is Revealed by Shotgun Proteomics in Streptococcus thermophilus

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

Young, Jacque C; Dill, Brian; Pan, Chongle

The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response: infection of S. thermophilus DGCC7710 with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infectionmore » and across various time points using two-dimensional liquid chromatography tandem mass spectroscopy. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance during peak infection, including the Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.« less

The E4 protein; structure, function and patterns of expression

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

Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

2013-10-15

{sup ∧}E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.« less

Facilitated Diffusion of Transcription Factor Proteins with Anomalous Bulk Diffusion.

PubMed

Liu, Lin; Cherstvy, Andrey G; Metzler, Ralf

2017-02-16

What are the physical laws of the diffusive search of proteins for their specific binding sites on DNA in the presence of the macromolecular crowding in cells? We performed extensive computer simulations to elucidate the protein target search on DNA. The novel feature is the viscoelastic non-Brownian protein bulk diffusion recently observed experimentally. We examine the influence of the protein-DNA binding affinity and the anomalous diffusion exponent on the target search time. In all cases an optimal search time is found. The relative contribution of intermittent three-dimensional bulk diffusion and one-dimensional sliding of proteins along the DNA is quantified. Our results are discussed in the light of recent single molecule tracking experiments, aiming at a better understanding of the influence of anomalous kinetics of proteins on the facilitated diffusion mechanism.

Human Cytomegalovirus-Encoded Receptor US28 Is Expressed in Renal Allografts and Facilitates Viral Spreading In Vitro.

PubMed

Lollinga, Wouter T; de Wit, Raymond H; Rahbar, Afsar; Vasse, Gwenda F; Davoudi, Belghis; Diepstra, Arjan; Riezebos-Brilman, Annelies; Harmsen, Martin C; Hillebrands, Jan-Luuk; Söderberg-Naucler, Cecilia; van Son, Willem J; Smit, Martine J; Sanders, Jan-Stephan; van den Born, Jacob

2017-03-01

Renal transplantation is the preferred treatment for patients with end-stage renal disease. Human cytomegalovirus (HCMV) activation is associated with decreased renal graft function and survival. Human cytomegalovirus encodes several immune modulatory proteins, including the G protein-coupled receptor US28, which scavenges human chemokines and modulates intracellular signaling. Our aim was to identify the expression and localization of US28 in renal allograft biopsies by immunohistochemistry and determine its role in viral spreading in vitro. Immunohistochemistry revealed US28 in 31 of 34 renal transplant biopsies from HCMV-seropositive donors. Expression was independent of HCMV viremia or IgG serostatus. US28 was predominantly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs) and tubular epithelial cells, with a median positivity of 20% and 40%, respectively. Also, US28-positive cells were present within arterial neointima. In contrast to US28, HCMV-encoded immediate early antigen was detected in less than 5% of VSMCs, tubular epithelial cells, interstitial endothelium, interstitial inflammatory infiltrates, and glomerular cells.Primary VSMCs were infected with green fluorescent protein-tagged wild type or US28-deficient HCMV. The viral spreading of US28-deficient HCMV, via culture medium or cell-to-cell transmission, was significantly impeded as shown by green fluorescent protein (ie, infected) cell quantification and quantitative real-time polymerase chain reaction. Additionally, the number and size of foci was smaller. In summary, HCMV-encoded US28 was detected in renal allografts from HCMV-positive donors independent of viremia and serostatus. Also, US28 facilitates HCMV spreading in VSMCs in vitro. Because the vasculature is affected in chronic renal transplant dysfunction, US28 may provide a potential target for therapeutic intervention.

Heat shock protein-70 neutralizes apoptosis inducing factor in Bcr/Abl expressing cells.

PubMed

Wang, Fang; Dai, An-Ya; Tao, Kun; Xiao, Qing; Huang, Zheng-Lan; Gao, Miao; Li, Hui; Wang, Xin; Cao, Wei-Xi; Feng, Wen-Li

2015-10-01

Bcr/Abl fusion protein is a hallmark of human chronic myeloid leukemia (CML). The protein can activate various signaling pathways to make normal cells transform malignantly and thus to facilitate tumorigenesis. It has been reported that heat shock protein-70 (HSP-70) can be served as an anti-apoptotic protein that suppresses Bax and Apo-2L/TRAIL. But it is unclear whether HSP-70 affects AIF-initiated apoptosis in Bcr/Abl expressing cells considering that HSP-70 is coincidentally over-regulated in these cells. Our findings supported that abundant HSP-70 in Bcr/Abl cells neutralizes AIF by segregating it from nucleus via direct interaction, leading to the failure of AIF initiating cell death and the silence of caspase-independent apoptotic pathway upon apoptotic induction. Moderate inhibition of HSP-70 expression by siRNA leads to Vp-16 triggered re-distribution of AIF in nucleus. In addition, AIF bears a HSP-70 binding domain allowing association with HSP-70. Therefore, disruption of the association using an AIF mutant lacking this domain can restore the potential of AIF importing into nucleus, and finally triggers cell death in a time dependent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

PubMed Central

Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

2015-01-01

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.

PubMed

Kroemer, Jeremy A; Bonning, Bryony C; Harrison, Robert L

2015-01-21

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

Exocyst Complex Protein Expression in the Human Placenta

PubMed Central

Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

2014-01-01

Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction

PubMed Central

Tugaeva, Kristina V.; Tsvetkov, Philipp O.

2017-01-01

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods. PMID:28575131

Dynamics of Hippocampal Protein Expression During Long-term Spatial Memory Formation*

PubMed Central

Borovok, Natalia; Nesher, Elimelech; Levin, Yishai; Reichenstein, Michal; Pinhasov, Albert

2016-01-01

Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein

Protein expression in Arabidopsis thaliana after chronic clinorotation

NASA Technical Reports Server (NTRS)

Piastuch, W. C.; Brown, C. S.

1995-01-01

Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

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

Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana

2008-02-20

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defectivemore » clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.« less

Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda)

PubMed Central

Cortés, Alba; Sotillo, Javier; Muñoz-Antoli, Carla; Fried, Bernard; Esteban, J. Guillermo; Toledo, Rafael

2015-01-01

Background Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. Methodology/Principal Findings To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. Conclusion/Significance Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection. PMID:26390031

Protein expression in Arabidopsis thaliana after chronic clinorotation

NASA Technical Reports Server (NTRS)

Piastuch, William C.; Brown, Christopher S.

1994-01-01

Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

PubMed Central

2010-01-01

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this

Infection of epithelial cells with dengue virus promotes the expression of proteins favoring the replication of certain viral strains.

PubMed

Martínez-Betancur, Viviana; Marín-Villa, Marcel; Martínez-Gutierrez, Marlén

2014-08-01

Dengue virus (DENV) is the causative agent of dengue and severe dengue. To understand better the dengue virus-host interaction, it is important to determine how the expression of cellular proteins is modified due to infection. Therefore, a comparison of protein expression was conducted in Vero cells infected with two different DENV strains, both serotype 2: DENV-2/NG (associated with dengue) and DENV-2/16681 (associated with severe dengue). The viability of the infected cells was determined, and neither strain induced cell death at 48 hr. In addition, the viral genomes and infectious viral particles were quantified, and the genome of the DENV-2/16681 strain was determined to have a higher replication rate compared with the DENV-2/NG strain. Finally, the proteins from infected and uninfected cultures were separated using two-dimensional gel electrophoresis, and the differentially expressed proteins were identified by mass spectrometry. Compared with the uninfected controls, the DENV-2/NG- and DENV-2/16681-infected cultures had five and six differentially expressed proteins, respectively. The most important results were observed when the infected cultures were compared to each other (DENV-2/NG vs. DENV-2/16681), and 18 differentially expressed proteins were identified. Based on their cellular functions, many of these proteins were linked to the increase in the replication efficiency of DENV. Among the proteins were calreticulin, acetyl coenzyme A, acetyl transferase, and fatty acid-binding protein. It was concluded that the infection of Vero cells with DENV-2/NG or DENV-2/16681 differentially modifies the expression of certain proteins, which can, in turn, facilitate infection. © 2013 Wiley Periodicals, Inc.

Dark proteins: effect of inclusion body formation on quantification of protein expression.

PubMed

Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

2008-09-01

Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

Clofazimine Modulates the Expression of Lipid Metabolism Proteins in Mycobacterium leprae-Infected Macrophages

PubMed Central

Degang, Yang; Akama, Takeshi; Hara, Takeshi; Tanigawa, Kazunari; Ishido, Yuko; Gidoh, Masaichi; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

2012-01-01

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine. PMID:23236531

Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

PubMed

Degang, Yang; Akama, Takeshi; Hara, Takeshi; Tanigawa, Kazunari; Ishido, Yuko; Gidoh, Masaichi; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

2012-01-01

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

An incoherent feedforward loop facilitates adaptive tuning of gene expression.

PubMed

Hong, Jungeui; Brandt, Nathan; Abdul-Rahman, Farah; Yang, Ally; Hughes, Tim; Gresham, David

2018-04-05

We studied adaptive evolution of gene expression using long-term experimental evolution of Saccharomyces cerevisiae in ammonium-limited chemostats. We found repeated selection for non-synonymous variation in the DNA binding domain of the transcriptional activator, GAT1, which functions with the repressor, DAL80 in an incoherent type-1 feedforward loop (I1-FFL) to control expression of the high affinity ammonium transporter gene, MEP2. Missense mutations in the DNA binding domain of GAT1 reduce its binding to the GATAA consensus sequence. However, we show experimentally, and using mathematical modeling, that decreases in GAT1 binding result in increased expression of MEP2 as a consequence of properties of I1-FFLs. Our results show that I1-FFLs, one of the most commonly occurring network motifs in transcriptional networks, can facilitate adaptive tuning of gene expression through modulation of transcription factor binding affinities. Our findings highlight the importance of gene regulatory architectures in the evolution of gene expression. © 2018, Hong et al.

Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

NASA Technical Reports Server (NTRS)

Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

2001-01-01

The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

Effects of immunosuppressive treatment on protein expression in rat kidney

PubMed Central

Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

2014-01-01

The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

PubMed

Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M

2013-01-01

N-MYC down-regulated-like (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

PubMed Central

Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

2013-01-01

Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Methodology/Principal Findings Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. Conclusion/Significance NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients. PMID:24223735

Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile.

PubMed

Wang, Jing; Bie, Jinghua; Ghosh, Shobha

2016-09-01

While HDL-associated unesterified or free cholesterol (FC) is thought to be rapidly secreted into the bile, the fate of HDL-associated cholesteryl esters (HDL-CEs) that represent >80% of HDL-cholesterol, is only beginning to be understood. In the present study, we examined the hypothesis that intracellular cholesterol transport proteins [sterol carrier protein 2 (SCP2) and fatty acid binding protein-1 (FABP1)] not only facilitate CE hydrolase-mediated hydrolysis of HDL-CEs, but also enhance elimination of cholesterol into bile. Adenovirus-mediated overexpression of FABP1 or SCP2 in primary hepatocytes significantly increased hydrolysis of HDL-[(3)H]CE, reduced resecretion of HDL-CE-derived FC as nascent HDL, and increased its secretion as bile acids. Consistently, the flux of [(3)H]cholesterol from HDL-[(3)H]CE to biliary bile acids was increased by overexpression of SCP2 or FABP1 in vivo and reduced in SCP2(-/-) mice. Increased flux of HDL-[(3)H]CE to biliary FC was noted with FABP1 overexpression and in SCP2(-/-) mice that have increased FABP1 expression. Lack of a significant decrease in the flux of HDL-[(3)H]CE to biliary FC or bile acids in FABP1(-/-) mice indicates the likely compensation of its function by an as yet unidentified mechanism. Taken together, these studies demonstrate that FABP1 and SCP2 facilitate the preferential movement of HDL-CEs to bile for final elimination. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile

PubMed Central

Wang, Jing; Bie, Jinghua; Ghosh, Shobha

2016-01-01

While HDL-associated unesterified or free cholesterol (FC) is thought to be rapidly secreted into the bile, the fate of HDL-associated cholesteryl esters (HDL-CEs) that represent >80% of HDL-cholesterol, is only beginning to be understood. In the present study, we examined the hypothesis that intracellular cholesterol transport proteins [sterol carrier protein 2 (SCP2) and fatty acid binding protein-1 (FABP1)] not only facilitate CE hydrolase-mediated hydrolysis of HDL-CEs, but also enhance elimination of cholesterol into bile. Adenovirus-mediated overexpression of FABP1 or SCP2 in primary hepatocytes significantly increased hydrolysis of HDL-[3H]CE, reduced resecretion of HDL-CE-derived FC as nascent HDL, and increased its secretion as bile acids. Consistently, the flux of [3H]cholesterol from HDL-[3H]CE to biliary bile acids was increased by overexpression of SCP2 or FABP1 in vivo and reduced in SCP2−/− mice. Increased flux of HDL-[3H]CE to biliary FC was noted with FABP1 overexpression and in SCP2−/− mice that have increased FABP1 expression. Lack of a significant decrease in the flux of HDL-[3H]CE to biliary FC or bile acids in FABP1−/− mice indicates the likely compensation of its function by an as yet unidentified mechanism. Taken together, these studies demonstrate that FABP1 and SCP2 facilitate the preferential movement of HDL-CEs to bile for final elimination. PMID:27381048

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

PubMed

He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

2017-04-01

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

The Arabidopsis GASA10 gene encodes a cell wall protein strongly expressed in developing anthers and seeds.

PubMed

Trapalis, Menelaos; Li, Song Feng; Parish, Roger W

2017-07-01

The Arabidopsis GASA10 gene encodes a GAST1-like (Gibberellic Acid-Stimulated) protein. Reporter gene analysis identified consistent expression in anthers and seeds. In anthers expression was developmentally regulated, first appearing at stage 7 of anther development and reaching a maximum at stage 11. Strongest expression was in the tapetum and developing microspores. GASA10 expression also occurred throughout the seed and in root vasculature. GASA10 was shown to be transported to the cell wall. Using GASA1 and GASA6 as positive controls, gibberellic acid was found not to induce GASA10 expression in Arabidopsis suspension cells. Overexpression of GASA10 (35S promoter-driven) resulted in a reduction in silique elongation. GASA10 shares structural similarities to the antimicrobial peptide snakin1, however, purified GASA10 failed to influence the growth of a variety of bacterial and fungal species tested. We propose cell wall associated GASA proteins are involved in regulating the hydroxyl radical levels at specific sites in the cell wall to facilitate wall growth (regulating cell wall elongation). Copyright © 2017 Elsevier B.V. All rights reserved.

Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies.

PubMed

Mačinković, Igor S; Abughren, Mohamed; Mrkic, Ivan; Grozdanović, Milica M; Prodanović, Radivoje; Gavrović-Jankulović, Marija

2013-12-01

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies. Copyright © 2013 Elsevier B.V. All rights reserved.

[Expression, purification and activity analysis of GGDEF and EAL domain-containing proteins from Lactobacillus acidophilus].

PubMed

He, Jia-Hui; Sun, Jie-Li; Yan, Wen-Juan; Wang, Fang

2017-05-20

To identify the functions of the proteins containing the GGDEF or EAL domain in Lactobacillus acidophilus for investigation of the regulatory mechanism of c-di-GMP in this strain. The DNA fragments of NH13_07045-GGDEF, NH13_07050 and NH13_07055 from Lactobacillus acidophilus ATCC4356 were amplified by PCR and cloned into the expression vector pMAL-His-c2. After sequencing, the recombinant plasmids were transformed into competent Escherichia coli cells, which were induced by IPTG to express the recombinant proteins fused with maltose binding protein (MBP). The fusion proteins were purified using amylose resin column for diguanylate cyclase (DGC) or phosphodiesterase (PDE) activity assays in vitro followed by analysis with high-performance liquid chromatography (HPLC). The target DNA fragments were obtained by PCR, and their sequences were all identical to that in GenBank. The purified and concentrated fusion proteins, which were identified by SDS-PAGE and Western blotting, had relative molecular masses of 59 kD, 67 kD and 72 kD. HPLC analysis showed no DGC activity in NH13_07045-GGDEF, while PDE activity was found in NH13_07050 but not in NH13_07055. We obtained the protein encoded by NH13_07050 that possesses PDE activity in vitro. This protein may facilitate the evaluation of the regulatory function of c-di-GMP in Lactobacillus acidophilus.

Expression of multidrug resistance proteins in retinoblastoma

PubMed Central

Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

2017-01-01

AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

PubMed

Rubenstein, R C; Lyons, B M

2001-07-01

Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Modulation of N-glycosylation by mesalamine facilitates membranous E-cadherin expression in colon epithelial cells☆

PubMed Central

Khare, Vineeta; Lang, Michaela; Dammann, Kyle; Campregher, Christoph; Lyakhovich, Alex; Gasche, Christoph

2014-01-01

Genome wide association studies have implicated intestinal barrier function genes in the pathogenesis of ulcerative colitis. One of such loci CDH1, encoding E-cadherin, a transmembrane glycoprotein with known tumor suppressor functions, is also linked to the susceptibility to colorectal cancer. Loss of membranous E-cadherin expression is common in both colitis and cancer. We have recently demonstrated that mesalamine (5-ASA); the anti-inflammatory drug used to treat ulcerative colitis, induces membranous expression of E-cadherin and increases intercellular adhesion. Using colorectal cancer epithelial cells with aberrant E-cadherin expression, we investigated the mechanism underlying such an effect of 5-ASA. Post-translational modification of E-cadherin glycosylation was analyzed by biotin/streptavidin detection of sialylated glycoproteins. GnT-III (N-acetylglucosaminyltransferase III) expression was assessed by qRT-PCR, Western blot and immunofluorescence. GnT-III activity was analyzed by reactivity with E-4/L-4-PHA. Expression, localization and interaction of E-cadherin and β-catenin were analyzed by Western blot, immunocytochemistry and RNA interference. 5-ASA activity modulated E-cadherin glycosylation and increased both mRNA and protein levels of GnT-III and its activity as detected by increased E4-lectin reactivity. Intestinal APCMin polyps in mice showed low expression of GnT-III and 5-ASA was effective in increasing its expression. The data demonstrated that remodeling of glycans by GnT-III mediated bisect glycosylation, contributes to the membranous retention of E-cadherin by 5-ASA; facilitating intercellular adhesion. Induction of membranous expression of E-cadherin by 5-ASA is a novel mechanism for mucosal healing in colitis that might impede tumor progression by modulation of GnT-III expression. PMID:24184502

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins ☆

PubMed Central

Donnelly, Mark I.; Zhou, Min; Millard, Cynthia Sanville; Clancy, Shonda; Stols, Lucy; Eschenfeldt, William H.; Collart, Frank R.; Joachimiak, Andrzej

2009-01-01

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his6-tag–maltose-binding protein (MBP), intended to facilitate purification and enhance proteins’ solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his6-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his6-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his6-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his6-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his6-tag. PMID:16497515

Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

PubMed Central

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Raghavendra, Kamaraju; Sharma, Arun

2015-01-01

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins

Annotated differentially expressed salivary proteins of susceptible and insecticide-resistant mosquitoes of Anopheles stephensi.

PubMed

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Raghavendra, Kamaraju; Sharma, Arun

2015-01-01

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito-parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins

Advances in recombinant protein expression for use in pharmaceutical research.

PubMed

Assenberg, Rene; Wan, Paul T; Geisse, Sabine; Mayr, Lorenz M

2013-06-01

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells. Copyright © 2013. Published by Elsevier Ltd.

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

SBDS Protein Expression Patterns in the Bone Marrow

PubMed Central

Wong, Trisha E.; Calicchio, Monica L.; Fleming, Mark D.; Shimamura, Akiko; Harris, Marian H.

2010-01-01

Shwachman Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome caused by biallelic SBDS gene mutations. Here we examined SBDS protein levels in human bone marrow. SBDS protein expression was high in neutrophil progenitors, megakaryocytes, plasma cells and osteoblasts. In contrast, SBDS protein levels were low in all hematopoietic cell lineages from patients harboring the common SBDS mutations. We conclude that SBDS protein levels vary widely between specific marrow lineages. Uniformly low SBDS protein expression levels distinguish the majority of SDS patients from controls or other marrow failure syndromes. PMID:20658628

Vectors for co-expression of an unrestricted number of proteins

PubMed Central

Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

2007-01-01

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

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

Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung

Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1more » plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.« less

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

PubMed

Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

2013-08-01

To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

The growing impact of lyophilized cell-free protein expression systems

PubMed Central

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

2017-01-01

ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

PubMed

Kim, Eugene Y; Sudini, Kuladeep; Singh, Anil K; Haque, Mahamudul; Leaman, Douglas; Khuder, Sadik; Ahmed, Salahuddin

2018-05-25

Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts (RASFs) that cause joint destruction. We have shown earlier that RASFs exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K 48 -linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.-Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Well, slap my thigh: expression of surprise facilitates memory of surprising material.

PubMed

Parzuchowski, Michal; Szymkow-Sudziarska, Aleksandra

2008-06-01

Two studies examined the general prediction that one's emotional expression should facilitate memory for material that matches the expression. The authors focused on specific facial expressions of surprise. In the first study, participants who were mimicking a surprised expression showed better recall for the surprising words and worse recall for neutral words, relative to those who were mimicking a neutral expression. Study 2 replicated the results of Study 1, showing that participants who mimicked a surprised expression recalled more words spoken in a surprising manner compared with those that sounded neutral or sad. Conversely, participants who mimicked sad facial expressions showed greater recall for sad than neutral or surprising words. The results provide evidence of the importance of matching the emotional valence of the recall content to the facial expression of the recaller during the memorization period. (PsycINFO Database Record (c) 2008 APA, all rights reserved). (Copyright) 2008 APA, all rights reserved.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

PubMed

de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

2017-08-10

Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open

The stress-induced heat shock protein 70.3 expression is regulated by a dual-component mechanism involving alternative polyadenylation and HuR.

PubMed

Kraynik, Stephen M; Gabanic, Andrew; Anthony, Sarah R; Kelley, Melissa; Paulding, Waltke R; Roessler, Anne; McGuinness, Michael; Tranter, Michael

2015-06-01

Heat shock protein 70.3 (Hsp70.3) expression increases in response to cellular stress and plays a cytoprotective role. We have previously shown that Hsp70.3 expression is controlled through coordinated post-transcriptional regulation by miRNAs and alternative polyadenylation (APA), and APA-mediated shortening of the Hsp70.3 3'-UTR facilitates increased protein expression. A stress-induced increase in Hsp70.3 mRNA and protein expression is accompanied by alternative polyadenylation (APA)-mediated truncation of the 3'UTR of the Hsp70.3 mRNA transcript. However, the role that APA plays in stress-induced expression of Hsp70.3 remains unclear. Our results show that APA-mediated truncation of the Hsp70.3 3'UTR increases protein expression through enhanced polyribosome loading. Additionally, we demonstrate that the RNA binding protein HuR, which has been previously shown to play a role in mediating APA, is necessary for heat shock mediated increase in Hsp70.3 mRNA and protein. However, it is somewhat surprising to note that HuR does not play a role in APA of the Hsp70.3 mRNA, and these two regulatory events appear to be mutually exclusive regulators of Hsp70.3 expression. These results not only provide important insight to the regulation of stress response genes following heat shock, but also contribute an enhanced understanding of how alternative polyadenylation contributes to gene regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.

PubMed

Kramer, Marianne C; Liang, Dongming; Tatomer, Deirdre C; Gold, Beth; March, Zachary M; Cherry, Sara; Wilusz, Jeremy E

2015-10-15

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes. © 2015 Kramer et al.; Published by Cold Spring Harbor Laboratory Press.

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

PubMed Central

Kramer, Marianne C.; Liang, Dongming; Tatomer, Deirdre C.; Gold, Beth; March, Zachary M.; Cherry, Sara; Wilusz, Jeremy E.

2015-01-01

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3′ end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine–arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes. PMID:26450910

[Expression of c-jun protein after experimental rat brain concussion].

PubMed

Wang, Feng; Li, Yong-hong

2010-02-01

To observe e-jun protein expression after rat brain concussion and explore the forensic pathologic markers following brain concussion. Fifty-five rats were randomly divided into brain concussion group and control group. The expression of c-jun protein was observed by immunohistochemistry. There were weak positive expression of c-jun protein in control group. In brain concussion group, however, some neutrons showed positive expression of c-jun protein at 15 min after brain concussion, and reach to the peak at 3 h after brain concussion. The research results suggest that detection of c-jun protein could be a marker to determine brain concussion and estimate injury time after brain concussion.

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

PubMed Central

Rasala, Beth A; Muto, Machiko; Lee, Philip A; Jager, Michal; Cardoso, Rosa MF; Behnke, Craig A; Kirk, Peter; Hokanson, Craig A; Crea, Roberto; Mendez, Michael; Mayfield, Stephen P

2010-01-01

Summary Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear if this is due to few attempts or to limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, greater than 50% expressed at levels sufficient for commercial production. Three expressed at 2% to 3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivty achieved demonstrate the utility of C. reinhardtii as a robust platform for human therapeutic protein production. PMID:20230484

Single-strand DNA binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs

PubMed Central

Pandita, Raj K.; Chow, Tracy T.; Udayakumar, Durga; Bain, Amanda L.; Cubeddu, Liza; Hunt, Clayton R.; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E.; Khanna, Kum Kum; Shay, Jerry W.; Pandita, Tej K.

2015-01-01

Proliferating mammalian stem and cancer cells express telomerase (TERT) in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA binding protein SSB1, which has a critical role in DNA double-strand break repair. Here we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacted with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduced TERT interaction with telomeres and lead to G-overhang loss. While SSB1 was recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relied upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. PMID:25589350

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

PubMed

Mehlin, Christopher; Boni, Erica; Buckner, Frederick S; Engel, Linnea; Feist, Tiffany; Gelb, Michael H; Haji, Lutfiyah; Kim, David; Liu, Colleen; Mueller, Natascha; Myler, Peter J; Reddy, J T; Sampson, Joshua N; Subramanian, E; Van Voorhis, Wesley C; Worthey, Elizabeth; Zucker, Frank; Hol, Wim G J

2006-08-01

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

[Pichia pastoris as an expression system for recombinant protein production].

PubMed

Ciarkowska, Anna; Jakubowska, Anna

2013-01-01

Pichia pastoris has become increasingly popular as a host for recombinant protein production in recent years. P. pastoris is more cost effective and allows achieving higher expression levels than insect and mammalian cells. It also offers some significant advantages over E. coli expression systems, such as avoiding problems with proper protein folding. Also, P. pastoris as an eukaryotic organism can carry out posttranslational modifications of produced proteins. Additionally, P. pastoris can produce high levels of recombinant proteins in extracellular medium which simplifies protein purification. Having many advantages over other expression systems makes P. pastoris an organism of choice for industrial protein production.

Purification of proteins from baculovirus-infected insect cells.

PubMed

O'Shaughnessy, Luke; Doyle, Sean

2011-01-01

Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

Coating nanoparticles with tunable surfactants facilitates control over the protein corona.

PubMed

Müller, J; Bauer, K N; Prozeller, D; Simon, J; Mailänder, V; Wurm, F R; Winzen, S; Landfester, K

2017-01-01

Nanoparticles with long blood circulation time are a prerequisite for targeted drug delivery. To make the nanoparticles invisible for phagocytizing cells, functional moieties on the particle surface are believed to be necessary to attract specific so-called 'stealth' proteins forming a protein 'corona'. Currently, covalent attachment of those moieties represents the only way to achieve that attraction. However, that approach requires a high synthetic effort and is difficult to control. Therefore, we present the coating of model nanoparticles with biodegradable polymeric surfactants as an alternative method. The thermodynamic parameters of the coating process can be tuned by adjusting the surfactants' block lengths and hydrophilicity. Consequently, the unspecific protein adsorption and aggregation tendency of the particles can be controlled, and stealth proteins inhibiting cell uptake are enriched on their surface. This non-covalent approach could be applied to any particle type and thus facilitates tuning the protein corona and its biological impact. Copyright © 2016 Elsevier Ltd. All rights reserved.

Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER.

PubMed

Kuhl, D; Kennedy, T E; Barzilai, A; Kandel, E R

1992-12-01

Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis. These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation. In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex. We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II. We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins. Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons. The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent. These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.

[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.

Biotechnology Protein Expression and Purification Facility

NASA Technical Reports Server (NTRS)

2003-01-01

The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

Performance benchmarking of four cell-free protein expression systems.

PubMed

Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

2016-02-01

Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.

Toll-like receptor-mediated inhibition of Gas6 and ProS expression facilitates inflammatory cytokine production in mouse macrophages

PubMed Central

Deng, Tingting; Zhang, Yue; Chen, Qiaoyuan; Yan, Keqin; Han, Daishu

2012-01-01

Activation of Toll-like receptors (TLRs) triggers rapid inflammatory cytokine production in various cell types. The exogenous product of growth-arrest-specific gene 6 (Gas6) and Protein S (ProS) inhibit the TLR-triggered inflammatory responses through the activation of Tyro3, Axl and Mer (TAM) receptors. However, regulation of the Gas6/ProS-TAM system remains largely unknown. In the current study, mouse macrophages are shown to constitutively express Gas6 and ProS, which synergistically suppress the basal and TLR-triggered production of inflammatory cytokines, including those of tumour necrosis factor-α, interleukin-6 and interleukin-1β, by the macrophages in an autocrine manner. Notably, TLR signalling markedly decreases Gas6 and ProS expression in macrophages through the activation of the nuclear factor-κB. Further, the down-regulation of Gas6 and ProS by TLR signalling facilitates the TLR-mediated inflammatory cytokine production in mouse macrophages. These results describe a self-regulatory mechanism of TLR signalling through the suppression of Gas6 and ProS expression. PMID:22043818

C-Type Lectin Receptor MCL Facilitates Mincle Expression and Signaling through Complex Formation.

PubMed

Miyake, Yasunobu; Masatsugu, Oh-hora; Yamasaki, Sho

2015-06-01

C-type lectin receptors expressed in APCs are recently defined pattern recognition receptors that play a crucial role in immune responses against pathogen-associated molecular patterns. Among pathogen-associated molecular patterns, cord factor (trehalose-6,6'-dimycolate [TDM]) is the most potent immunostimulatory component of the mycobacterial cell wall. Two C-type lectin receptors, macrophage-inducible C-type lectin (Mincle) and macrophage C-type lectin (MCL), are required for immune responses against TDM. Previous studies indicate that MCL is required for TDM-induced Mincle expression. However, the mechanism by which MCL induces Mincle expression has not been fully understood. In this study, we demonstrate that MCL interacts with Mincle to promote its surface expression. After LPS or zymosan stimulation, MCL-deficient bone marrow-derived dendritic cells (BMDCs) had a lower level of Mincle protein expression, although mRNA expression was comparable with wild-type BMDCs. Meanwhile, BMDCs from MCL transgenic mice showed an enhanced level of Mincle expression on the cell surface. MCL was associated with Mincle through the stalk region and this region was necessary and sufficient for the enhancement of Mincle expression. This interaction appeared to be mediated by the hydrophobic repeat of MCL, as substitution of four hydrophobic residues within the stalk region with serine (MCL(4S)) abolished the function to enhance the surface expression of Mincle. MCL(4S) mutant failed to restore the defective TDM responses in MCL-deficient BMDCs. These results suggest that MCL positively regulates Mincle expression through protein-protein interaction via its stalk region, thereby magnifying Mincle-mediated signaling. Copyright © 2015 by The American Association of Immunologists, Inc.

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.

Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

PubMed

Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

2009-10-01

The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions.

Modeling Protein Expression and Protein Signaling Pathways

PubMed Central

Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

2015-01-01

High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

[Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

PubMed

Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

2014-01-01

To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

AR-v7 protein expression is regulated by protein kinase and phosphatase

PubMed Central

Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

2015-01-01

Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

Differential expression pattern of protein markers for predicting chemosensitivity of dexamethasone-based chemotherapy of B cell acute lymphoblastic leukemia.

PubMed

Dehghan-Nayeri, Nasrin; Eshghi, Peyman; Pour, Kourosh Goudarzi; Rezaei-Tavirani, Mostafa; Omrani, Mir Davood; Gharehbaghian, Ahmad

2017-07-01

Dexamethasone is considered as a direct chemotherapeutic agent in the treatment of pediatric acute lymphoblastic leukemia (ALL). Beside the advantages of the drug, some problems arising from the dose-related side effects are challenging issues during the treatment. Accordingly, the classification of patients to dexamethasone sensitive and resistance groups can help to select optimizing the therapeutic dose with the lowest adverse effects particularly in sensitive cases. For this purpose, we investigated inhibited proliferation and induced cytotoxicity in NALM-6 cells, as sensitive cells, after dexamethasone treatment. In addition, comparative protein expression analysis using the 2DE-MALDI-TOF MS technique was performed to identify the specific altered proteins. In addition, we evaluated mRNA expression levels of the identified proteins in bone-marrow samples from pediatric ALL patients using the real-time q-PCR method. Eventually, proteomic analysis revealed a combination of biomarkers, including capping proteins (CAPZA1 and CAPZB), chloride channel (CLIC1), purine nucleoside phosphorylase (PNP), and proteasome activator (PSME1), in response to the dexamethasone treatment. In addition, our results indicated low expression of identified proteins at both the mRNA and protein expression levels after drug treatment. Moreover, quantitative real-time PCR data analysis indicated that independent of the molecular subtypes of the leukemia, CAPZA1, CAPZB, CLIC1, and PNP expression levels were lower in ALL samples than normal samples, although PSME1 expression level was higher in ALL samples than normal samples. Furthermore, the expression level of all proteins (except PSME1) was different between high-risk and standard-risk patients that suggesting the prognostic value of them. In conclusion, our study suggests a panel of biomarkers comprising CAPZA1, CAPZB, CLIC1, PNP, and PSME1 as early diagnosis and treatment evaluation markers that may differentiate cancer cells which

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

PubMed Central

2012-01-01

Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains. PMID:22569138

Nucleoplasmic Nup98 controls gene expression by regulating a DExH/D-box protein.

PubMed

Capitanio, Juliana S; Montpetit, Ben; Wozniak, Richard W

2018-01-01

The nucleoporin Nup98 has been linked to the regulation of transcription and RNA metabolism, 1-3 but the mechanisms by which Nup98 contributes to these processes remains largely undefined. Recently, we uncovered interactions between Nup98 and several DExH/D-box proteins (DBPs), a protein family well-known for modulating gene expression and RNA metabolism. 4-6 Analysis of Nup98 and one of these DBPs, DHX9, showed that they directly interact, their association is facilitated by RNA, and Nup98 binding stimulates DHX9 ATPase activity. 7 Furthermore, these proteins were dependent on one another for their proper association with a subset of gene loci to control transcription and modulate mRNA splicing. 7 On the basis of these observations, we proposed that Nup98 functions to regulate DHX9 activity within the nucleoplasm. 7 Since Nup98 is associated with several DBPs, regulation of DHX9 by Nup98 may represent a paradigm for understanding how Nup98, and possibly other FG-Nup proteins, could direct the diverse cellular activities of multiple DBPs.

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification

PubMed Central

Kinzer-Ursem, Tamara L.

2018-01-01

As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this “next-generation” calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics. PMID:29864125

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

Protein disorder is positively correlated with gene expression in E. coli

PubMed Central

Paliy, Oleg; Gargac, Shawn M.; Cheng, Yugong; Uversky, Vladimir N.; Dunker, A. Keith

2009-01-01

We considered on a global scale the relationship between the predicted fraction of protein disorder and RNA and protein expression in E. coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins. PMID:18465893

Expression of Clonorchis sinensis GIIIsPLA2 protein in baculovirus-infected insect cells and its overexpression facilitating epithelial-mesenchymal transition in Huh7 cells via AKT pathway.

PubMed

Shang, Mei; Xie, Zhizhi; Tang, Zeli; He, Lei; Wang, Xiaoyun; Wang, Caiqin; Wu, Yinjuan; Li, Ye; Zhao, Lu; Lv, Zhiyue; Wu, Zhongdao; Huang, Yan; Yu, Xinbing; Li, Xuerong

2017-04-01

Although prior studies confirmed that group III secretory phospholipase A 2 of Clonorchis sinensis (CsGIIIsPLA 2 ) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA 2 . The molecular weight of recombinant CsGIIIsPLA 2 protein was about 34 kDa. Further investigation showed that most of the recombinant protein presented intracellular expression in Sf9 insect cell nucleus and could be detected only into cell debris, which made the protein purification and further functional study difficult. Therefore, to study the role of CsGIIIsPLA 2 in hepatocellular carcinoma (HCC) progression, CsGIIIsPLA 2 overexpression Huh7 cell model was applied. Cell proliferation, migration, and the expression level of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, N-cadherin, α-catenin, Vimentin, p300, Snail, and Slug) along with possible mechanism were measured. The results indicated that CsGIIIsPLA 2 overexpression not only inhibited cell proliferation and promoted migration and EMT but also enhanced the phosphorylation of AKT in HCC cells. In conclusion, this study supported that CsGIIIsPLA 2 overexpression suppressed cell proliferation and induced EMT through the AKT pathway.

Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

PubMed

Barz, W P; Walter, P

1999-04-01

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

PubMed Central

Barz, Wolfgang P.; Walter, Peter

1999-01-01

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δ cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins. PMID:10198056

[Tripartite-motif protein 25 and pyruvate kinase M2 protein expression in non-small cell lung cancer].

PubMed

Jing, Huai-Zhi; Qiu, Feng; Chen, Shi-Zhi; Su, Lin; Qu, Can

2015-03-01

To investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC. The expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients. The positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026). TRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

Distinct Protein Expression Profiles of Solid-Pseudopapillary Neoplasms of the Pancreas.

PubMed

Park, Minhee; Lim, Jong-Sun; Lee, Hyoung-Joo; Na, Keun; Lee, Min Jung; Kang, Chang Moo; Paik, Young-Ki; Kim, Hoguen

2015-08-07

Solid-pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumor with mutation in CTNNB1 and distinct clinical and pathological features. We compared the proteomic profiles of SPN to mRNA expression. Pooled SPNs and pooled non-neoplastic pancreatic tissues were examined with high-resolution mass spectrometry. We identified 329 (150 up-regulated and 179 down-regulated) differentially expressed proteins in SPN. We identified 191 proteins (58.1% of the 329 dysregulated proteins) with the same expression tendencies in SPN based on mRNA data. Many overexpressed proteins were related to signaling pathways known to be activated in SPNs. We found that several proteins involved in Wnt signaling, including DKK4 and β-catenin, and proteins that bind β-catenin, such as FUS and NONO, were up-regulated in SPNs. Molecules involved in glycolysis, including PKM2, ENO2, and HK1, were overexpressed in accordance to their mRNA levels. In summary, SPN showed (1) distinct protein expression changes that correlated with mRNA expression, (2) overexpression of Wnt signaling proteins and proteins that bind directly to β-catenin, and (3) overexpression of proteins involved in metabolism. These findings may help develop early diagnostic biomarkers and molecular targets.

Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

DOEpatents

Mayfield, Stephen P.

2010-03-16

Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

DOEpatents

Mayfield, Stephen P

2015-01-13

Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

Expression of multiple proteins in transgenic plants

DOEpatents

Vierstra, Richard D.; Walker, Joseph M.

2002-01-01

A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

Methods and constructs for expression of foreign proteins in photosynthetic organisms

DOEpatents

Laible, Philip D.; Hanson, Deborah K.

2002-01-01

A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

Similar protein expression profiles of ovarian and endometrial high-grade serous carcinomas.

PubMed

Hiramatsu, Kosuke; Yoshino, Kiyoshi; Serada, Satoshi; Yoshihara, Kosuke; Hori, Yumiko; Fujimoto, Minoru; Matsuzaki, Shinya; Egawa-Takata, Tomomi; Kobayashi, Eiji; Ueda, Yutaka; Morii, Eiichi; Enomoto, Takayuki; Naka, Tetsuji; Kimura, Tadashi

2016-03-01

Ovarian and endometrial high-grade serous carcinomas (HGSCs) have similar clinical and pathological characteristics; however, exhaustive protein expression profiling of these cancers has yet to be reported. We performed protein expression profiling on 14 cases of HGSCs (7 ovarian and 7 endometrial) and 18 endometrioid carcinomas (9 ovarian and 9 endometrial) using iTRAQ-based exhaustive and quantitative protein analysis. We identified 828 tumour-expressed proteins and evaluated the statistical similarity of protein expression profiles between ovarian and endometrial HGSCs using unsupervised hierarchical cluster analysis (P<0.01). Using 45 statistically highly expressed proteins in HGSCs, protein ontology analysis detected two enriched terms and proteins composing each term: IMP2 and MCM2. Immunohistochemical analyses confirmed the higher expression of IMP2 and MCM2 in ovarian and endometrial HGSCs as well as in tubal and peritoneal HGSCs than in endometrioid carcinomas (P<0.01). The knockdown of either IMP2 or MCM2 by siRNA interference significantly decreased the proliferation rate of ovarian HGSC cell line (P<0.01). We demonstrated the statistical similarity of the protein expression profiles of ovarian and endometrial HGSC beyond the organs. We suggest that increased IMP2 and MCM2 expression may underlie some of the rapid HGSC growth observed clinically.

The protein expression landscape of the Arabidopsis root

PubMed Central

Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

2012-01-01

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

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

Yamakoshi, Takako; Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp; Ur Rehman, Mati

2013-03-01

Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain andmore » a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.« less

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

PubMed

Zhong, Guitao; Liu, Ronghe; Zhuang, Menglong; Wang, Hao

2017-01-01

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

PubMed Central

Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

2014-01-01

Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of

Positive, but Not Negative, Facial Expressions Facilitate 3-Month-Olds' Recognition of an Individual Face

ERIC Educational Resources Information Center

Brenna, Viola; Proietti, Valentina; Montirosso, Rosario; Turati, Chiara

2013-01-01

The current study examined whether and how the presence of a positive or a negative emotional expression may affect the face recognition process at 3 months of age. Using a familiarization procedure, Experiment 1 demonstrated that positive (i.e., happiness), but not negative (i.e., fear and anger) facial expressions facilitate infants' ability to…

Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

DOE PAGES

Li, Jiying; Hu, Jianping; Bassham, Diane

2015-09-14

Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Usingmore » microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression.

PubMed

Zhu, Shaozhou; Gong, Cuiyu; Ren, Lu; Li, Xingzhou; Song, Dawei; Zheng, Guojun

2013-01-01

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (-)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

PubMed

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessing the utility of gene co-expression stability in combination with correlation in the analysis of protein-protein interaction networks

PubMed Central

2011-01-01

Background Gene co-expression, in the form of a correlation coefficient, has been valuable in the analysis, classification and prediction of protein-protein interactions. However, it is susceptible to bias from a few samples having a large effect on the correlation coefficient. Gene co-expression stability is a means of quantifying this bias, with high stability indicating robust, unbiased co-expression correlation coefficients. We assess the utility of gene co-expression stability as an additional measure to support the co-expression correlation in the analysis of protein-protein interaction networks. Results We studied the patterns of co-expression correlation and stability in interacting proteins with respect to their interaction promiscuity, levels of intrinsic disorder, and essentiality or disease-relatedness. Co-expression stability, along with co-expression correlation, acts as a better classifier of hub proteins in interaction networks, than co-expression correlation alone, enabling the identification of a class of hubs that are functionally distinct from the widely accepted transient (date) and obligate (party) hubs. Proteins with high levels of intrinsic disorder have low co-expression correlation and high stability with their interaction partners suggesting their involvement in transient interactions, except for a small group that have high co-expression correlation and are typically subunits of stable complexes. Similar behavior was seen for disease-related and essential genes. Interacting proteins that are both disordered have higher co-expression stability than ordered protein pairs. Using co-expression correlation and stability, we found that transient interactions are more likely to occur between an ordered and a disordered protein while obligate interactions primarily occur between proteins that are either both ordered, or disordered. Conclusions We observe that co-expression stability shows distinct patterns in structurally and functionally

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

PubMed

Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-Ichi

2018-01-26

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) 14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense . We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M -1 ·cm -1 . The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE

PubMed Central

Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G.; Zheng, Hongjin; Gonen, Tamir

2014-01-01

The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins. PMID:25088546

Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

PubMed

Pandita, Raj K; Chow, Tracy T; Udayakumar, Durga; Bain, Amanda L; Cubeddu, Liza; Hunt, Clayton R; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E; Khanna, Kum Kum; Shay, Jerry W; Pandita, Tej K

2015-03-01

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR. ©2015 American Association for Cancer Research.

Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini

PubMed Central

Suwannatrai, Kulwadee; Suwannatrai, Apiporn; Tabsripair, Pairat; Welbat, Jariya Umka; Tangkawattana, Sirikachorn; Cantacessi, Cinzia; Mulvenna, Jason; Tesana, Smarn; Loukas, Alex

2016-01-01

Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas. PMID:27893749

Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

PubMed

Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

2015-08-01

Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

PubMed

Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

2013-08-20

Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior

Phytomonas: A non-pathogenic trypanosomatid model for functional expression of proteins.

PubMed

Miranda, Mariana R; Sayé, Melisa; Reigada, Chantal; Carrillo, Carolina; Pereira, Claudio A

2015-10-01

Phytomonas are protozoan parasites from the Trypanosomatidae family which infect a wide variety of plants. Herein, Phytomonas Jma was tested as a model for functional expression of heterologous proteins. Green fluorescent protein expression was evaluated in Phytomonas and compared with Trypanosoma cruzi, the etiological agent of Chagas' disease. Phytomonas was able to express GFP at levels similar to T. cruzi although the transgenic selection time was higher. It was possible to establish an efficient transfection and selection protocol for protein expression. These results demonstrate that Phytomonas can be a good model for functional expression of proteins from other trypanosomatids, presenting the advantage of being completely safe for humans. Copyright © 2015 Elsevier Inc. All rights reserved.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Expression of hypoxia-inducible 2 (HIG2) protein in uterine cancer.

PubMed

Nishimura, S; Tsuda, H; Nomura, H; Kataoka, F; Chiyoda, T; Tanaka, H; Tanaka, K; Susumu, N; Aoki, D

2011-01-01

For both cervical cancer (UCC) and endometrial cancer (EMC) there are no effective prognostic markers. In this study, we evaluated HIG2 protein expression in 332 uterine cancers (186 UCCs and 146 EMCs) and examined the relationship between HIG2 protein expression and clinical factors, including prognosis. Totally, HIG2 expression was detected in 58% of UCC and 66% of EMC. However, there was no significant relationship between HIG2 expression and age, clinical stage and histology in either UCC or EMC. In addition, HIG2 protein expression was not related to prognosis of UCC or EMC. The positivity rate of HIG2 protein was 56% and 61% in early-stage UCC and EMC, respectively and 67% in non-squamous cell carcinoma of UCC. The positivity rate of HIG2 protein was high even in early-stage UCC and EMC

The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression

PubMed Central

Crosby, Heidi A.; Schlievert, Patrick M.; Merriman, Joseph A.; King, Jessica M.; Salgado-Pabón, Wilmara; Horswill, Alexander R.

2016-01-01

Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins. PMID:27144398

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

Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase.

PubMed Central

Bergold, P J; Sweatt, J D; Winicov, I; Weiss, K R; Kandel, E R; Schwartz, J H

1990-01-01

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells. Images PMID:1692622

Transient expression of CCL21as recombinant protein in tomato.

PubMed

Beihaghi, Maria; Marashi, Hasan; Bagheri, Abdolreza; Sankian, Mojtaba

2018-03-01

The main goal of this study was to investigate the possibility of expressing recombinant protein of C-C chemokine ligand 21 (CCL21) in Solanum lycopersicum via agroinfiltration. CCL21 is a chemokine can be used for anti-metastatic of cancer cell lines. To examine the expression of CCL21 protein in S. lycopersicum , the construct of ccl21 was synthesized. This construct was cloned into pBI121 and the resulting CCL21 plasmid was agro-infiltrated into S. lycopersicum leaves. Within three days after infiltration, Expression of the foreign gene was confirmed by quantitative Real-time PCR. A recombinant CCL21 protein was immunogenically detected by western blot, dot blot and ELISA assay. And results showed that the foreign gene was expressed in the transformed leaves in high level. Also scratch assay was used to investigate the role of this protein in anti-metastatic function. The results demonstrated anti-metastatic of cancer cells in the presence of this protein.

Particularly interesting Cys-His-rich protein is highly expressed in human intracranial aneurysms and resists aneurysmal rupture

PubMed Central

Peng, Yu-Tao; Shi, Xiang-En; Li, Zhi-Qiang; He, Xin; Sun, Yu-Ming

2016-01-01

Particularly interesting Cys-His-rich protein (PINCH) has several biological functions in cancer development, invasion and metastasis in malignant cells, and the expression of PINCH is upregulated in several cancer types, including breast cancer, gastric adenocarcinoma and rectal cancer. However, the contribution of PINCH to human cerebral aneurysms remains largely unknown. Therefore, the significance of PINCH expression in cerebral aneurysm growth and rupture was examined in the present study. The protein expression levels of alpha-smooth muscle actin, osteopontin (OPN), matrix metalloproteinase (MMP) 9 and PINCH were evaluated using immunohistochemistry and western blot analyses. The results demonstrate that the protein expression levels of OPN, MMP9 and PINCH in the unruptured intracranial aneurysm (UA) and ruptured intracranial aneurysm (RA) groups were markedly higher than those of the control group, whereas OPN and PINCH expression levels were decreased in the RA group compared to those of the UA group. In addition, there was a strong correlation between PINCH and tumor size (r=0.650 and P=0.0026), as well as between PINCH and OPN (r=0.639 and P=0.0033) in the unruptured cerebral aneurysms. However, the correlation between PINCH and tumor size (r=0.450 and P=0.1393) and between PINCH and OPN (r=0.366 and P=0.2426) revealed no obvious difference in the ruptured cerebral aneurysms. In conclusion, PINCH was highly expressed in the UAs, which may be a critical factor for preventing aneurysmal rupture. Moreover, PINCH may facilitate intracranial aneurysm progression, at least partially, through the activation of extracellular signal-regulated kinase signaling and the suppression of c-Jun N-terminal kinase signaling. PMID:28101173

Particularly interesting Cys-His-rich protein is highly expressed in human intracranial aneurysms and resists aneurysmal rupture.

PubMed

Peng, Yu-Tao; Shi, Xiang-En; Li, Zhi-Qiang; He, Xin; Sun, Yu-Ming

2016-12-01

Particularly interesting Cys-His-rich protein (PINCH) has several biological functions in cancer development, invasion and metastasis in malignant cells, and the expression of PINCH is upregulated in several cancer types, including breast cancer, gastric adenocarcinoma and rectal cancer. However, the contribution of PINCH to human cerebral aneurysms remains largely unknown. Therefore, the significance of PINCH expression in cerebral aneurysm growth and rupture was examined in the present study. The protein expression levels of alpha-smooth muscle actin, osteopontin (OPN), matrix metalloproteinase (MMP) 9 and PINCH were evaluated using immunohistochemistry and western blot analyses. The results demonstrate that the protein expression levels of OPN, MMP9 and PINCH in the unruptured intracranial aneurysm (UA) and ruptured intracranial aneurysm (RA) groups were markedly higher than those of the control group, whereas OPN and PINCH expression levels were decreased in the RA group compared to those of the UA group. In addition, there was a strong correlation between PINCH and tumor size ( r =0.650 and P=0.0026), as well as between PINCH and OPN ( r =0.639 and P=0.0033) in the unruptured cerebral aneurysms. However, the correlation between PINCH and tumor size ( r =0.450 and P=0.1393) and between PINCH and OPN ( r =0.366 and P=0.2426) revealed no obvious difference in the ruptured cerebral aneurysms. In conclusion, PINCH was highly expressed in the UAs, which may be a critical factor for preventing aneurysmal rupture. Moreover, PINCH may facilitate intracranial aneurysm progression, at least partially, through the activation of extracellular signal-regulated kinase signaling and the suppression of c-Jun N-terminal kinase signaling.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

PubMed

Fabrick, Jeffrey A; Hull, J Joe

2017-04-20

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

PubMed

Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

2017-10-01

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

PubMed

Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

2014-07-01

Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

PubMed

Yue, Chang-Wu; Zhang, Yi-Zheng

2009-03-01

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

Suppression of polyglutamine protein toxicity by co-expression of a heat-shock protein 40 and a heat-shock protein 110

PubMed Central

Kuo, Y; Ren, S; Lao, U; Edgar, B A; Wang, T

2013-01-01

A network of heat-shock proteins mediates cellular protein homeostasis, and has a fundamental role in preventing aggregation-associated neurodegenerative diseases. In a Drosophila model of polyglutamine (polyQ) disease, the HSP40 family protein, DNAJ-1, is a superior suppressor of toxicity caused by the aggregation of polyQ containing proteins. Here, we demonstrate that one specific HSP110 protein, 70 kDa heat-shock cognate protein cb (HSC70cb), interacts physically and genetically with DNAJ-1 in vivo, and that HSC70cb is necessary for DNAJ-1 to suppress polyglutamine-induced cell death in Drosophila. Expression of HSC70cb together with DNAJ-1 significantly enhanced the suppressive effects of DNAJ-1 on polyQ-induced neurodegeneration, whereas expression of HSC70cb alone did not suppress neurodegeneration in Drosophila models of either general polyQ disease or Huntington's disease. Furthermore, expression of a human HSP40, DNAJB1, together with a human HSP110, APG-1, protected cells from polyQ-induced neural degeneration in flies, whereas expression of either component alone had little effect. Our data provide a functional link between HSP40 and HSP110 in suppressing the cytotoxicity of aggregation-prone proteins, and suggest that HSP40 and HSP110 function together in protein homeostasis control. PMID:24091676

N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

PubMed

Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

2017-05-01

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The N Terminus of Andes Virus L Protein Suppresses mRNA and Protein Expression in Mammalian Cells

PubMed Central

Heinemann, Patrick; Schmidt-Chanasit, Jonas

2013-01-01

Little is known about the structure and function of the 250-kDa L protein of hantaviruses, although it plays a central role in virus genome transcription and replication. When attempting to study Andes virus (ANDV) L protein in mammalian cells, we encountered difficulties. Even in a strong overexpression system, ANDV L protein could not be detected by immunoblotting. Deletion analysis revealed that the 534 N-terminal amino acid residues determine the low-expression phenotype. Inhibition of translation due to RNA secondary structures around the start codon, rapid proteasomal degradation, and reduced half-life time were excluded. However, ANDV L protein expression could be rescued upon mutation of the catalytic PD-E-K motif and further conserved residues of the putative endonuclease at the N terminus of the protein. In addition, wild-type ANDV L rather than expressible L mutants suppressed the level of L mRNA, as well as reporter mRNAs. Wild-type L protein also reduced the synthesis of cellular proteins in the high-molecular-weight range. Using expressible ANDV L mutants as a tool for localization studies, we show that L protein colocalizes with ANDV N and NSs but not Gc protein. A fraction of L protein also colocalized with the cellular processing (P) body component DCP1a. Overall, these data suggest that ANDV L protein possesses a highly active endonuclease at the N terminus suppressing the level of its own as well as heterologous mRNAs upon recombinant expression in mammalian cells. PMID:23576516

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

PubMed

Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

PubMed

Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

2014-01-09

The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems.

PubMed

Douillard, François P; Mahony, Jennifer; Campanacci, Valérie; Cambillau, Christian; van Sinderen, Douwe

2011-09-01

Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses. Copyright © 2011 Elsevier Inc. All rights reserved.

Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

PubMed

Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

2016-11-01

The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P < .0001), and the primary sites of metastatic carcinomas (P < .0001) compared with normal mammary glands. No significant differences in ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

Imbalance in chemical space: How to facilitate the identification of protein-protein interaction inhibitors.

PubMed

Kuenemann, Mélaine A; Labbé, Céline M; Cerdan, Adrien H; Sperandio, Olivier

2016-04-01

Protein-protein interactions (PPIs) play vital roles in life and provide new opportunities for therapeutic interventions. In this large data analysis, 3,300 inhibitors of PPIs (iPPIs) were compared to 17 reference datasets of collectively ~566,000 compounds (including natural compounds, existing drugs, active compounds on conventional targets, etc.) using a chemoinformatics approach. Using this procedure, we showed that comparable classes of PPI targets can be formed using either the similarity of their ligands or the shared properties of their binding cavities, constituting a proof-of-concept that not only can binding pockets be used to group PPI targets, but that these pockets certainly condition the properties of their corresponding ligands. These results demonstrate that matching regions in both chemical space and target space can be found. Such identified classes of targets could lead to the design of PPI-class-specific chemical libraries and therefore facilitate the development of iPPIs to the stage of drug candidates.

Rhythmic expression of DEC2 protein in vitro and in vivo.

PubMed

Sato, Fuyuki; Muragaki, Yasuteru; Kawamoto, Takeshi; Fujimoto, Katsumi; Kato, Yukio; Zhang, Yanping

2016-06-01

Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

Novel isoprenylated proteins identified by an expression library screen.

PubMed

Biermann, B J; Morehead, T A; Tate, S E; Price, J R; Randall, S K; Crowell, D N

1994-10-14

Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

Genome engineering for improved recombinant protein expression in Escherichia coli.

PubMed

Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

2014-12-19

A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

PubMed

Rosmorduc, O; Sirma, H; Soussan, P; Gordien, E; Lebon, P; Horisberger, M; Bréchot, C; Kremsdorf, D

1999-05-01

Chronic hepatitis B treatment has been significantly improved by interferon (IFN) treatment. However, some studies have suggested that hepatitis B virus (HBV) might have a direct effect on the resistance to IFN. Defective particles, generated by spliced HBV RNA and associated with chronic hepatitis B, have been previously characterized; expression of these particles leads to cytoplasmic accumulation of the capsid protein. The aim of this study was to investigate the role of these defective genomes in IFN resistance. The global antiviral activity of IFN was studied by virus yield reduction assays, the expression of three IFN-induced antiviral proteins was analysed by Western blotting and confocal microscopy, and the regulation of MxA gene expression was studied by Northern blotting and the luciferase assay, in Huh7 cells transfected with a complete or the defective HBV genome. Results showed that the expression of the defective genome reduces the antiviral activity of IFN and that this modulation involves a selective inhibition of MxA protein induction by the HBV capsid protein. Our results also show the trans-suppressive effect of the HBV capsid on the MxA promoter, which might participate in this phenomenon. In conclusion, this study shows a direct interplay between the IFN-sensitive pathway and the capsid protein and might implicate this defective HBV genome in virus persistence.

Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

USDA-ARS?s Scientific Manuscript database

The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

PubMed

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

PubMed

Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

2017-01-01

Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

Expression of membrane-associated proteins within single emulsion cell facsimiles.

PubMed

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

Expression and Localization of Plant Protein Disulfide Isomerase.

PubMed Central

Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

1993-01-01

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

PubMed

Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

2013-03-01

The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

PubMed

Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

2014-07-01

Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Insect cells-baculovirus system for the production of difficult to express proteins.

PubMed

Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

2015-01-01

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly

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

Wang, Robert Y.L., E-mail: yuwang@mail.cgu.edu.tw; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan; Kuo, Rei-Lin

2013-09-01

Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted tomore » new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone. - Highlights: • Hsp90β is associated with EV71 virion and is secreted with the release virus. • Hsp90β effects on the correct assembly of viral particles. • Viral titer of cultured medium was reduced in the presence of geldanamycin. • Viral titer was also reduced when Hsp90β was suppressed by siRNA treatment. • The extracellular Hsp90β was also observed in other RNA viruses-infected cells.« less

NDR proteins

PubMed Central

Jones, Alan M

2010-01-01

N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

USDA-ARS?s Scientific Manuscript database

The complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST) data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained an...

Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

PubMed

Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

2010-04-01

"Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

Protein Interactions in T7 DNA Replisome Facilitate DNA Damage Bypass.

PubMed

Zou, Zhenyu; Chen, Ze; Xue, Qizhen; Xu, Ying; Xiong, Jingyuan; Yang, Ping; Le, Shuai; Zhang, Huidong

2018-06-14

DNA replisome inevitably encounters DNA damage during DNA replication. T7 DNA replisome contains DNA polymerase (gp5), the processivity factor thioredoxin (trx), helicase-primase (gp4), and ssDNA binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated the strand-displacement DNA synthesis past 8-oxoG or O6-MeG at the synthetic DNA fork by T7 DNA replisome. DNA damage does not obviously affect the binding affinities among helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6-MeG, as well as GC-rich template sequence clusters, inhibit the strand-displacement DNA synthesis and produce partial extension products. Relative to gp4 ΔC-tail, gp4 promotes the DNA damage bypass. The presence of gp2.5 further promotes this bypass. Thus, the interactions of polymerase with helicase and ssDNA binidng protein faciliate the DNA damage bypass. Similarly, accessory proteins in other complicated DNA replisomes also facilitate the DNA damage bypass. This work provides the novel mechanism information of DNA damage bypass by DNA replisome. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

PubMed Central

Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

2013-01-01

Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

Delta Subunit-Containing Gamma-Aminobutyric Acid A Receptor Disinhibits Lateral Amygdala and Facilitates Fear Expression in Mice.

PubMed

Liu, Zhi-Peng; He, Qing-Hai; Pan, Han-Qing; Xu, Xiao-Bin; Chen, Wen-Bing; He, Ye; Zhou, Jin; Zhang, Wen-Hua; Zhang, Jun-Yu; Ying, Xiao-Ping; Han, Ren-Wen; Li, Bao-Ming; Gao, Tian-Ming; Pan, Bing-Xing

2017-06-15

Maintaining gamma-aminobutyric acidergic (GABAergic) inhibition in the amygdala within a physiological range is critical for the appropriate expression of emotions such as fear and anxiety. The synaptic GABA type A receptor (GABA A R) is generally known to mediate the primary component of amygdala inhibition and prevent inappropriate expression of fear. However, little is known about the contribution of the extrasynaptic GABA A R to amygdala inhibition and fear. By using mice expressing green fluorescent protein in interneurons (INs) and lacking the δ subunit-containing GABA A R (GABA A (δ)R), which is exclusively situated in the extrasynaptic membrane, we systematically investigated the role of GABA A (δ)R in regulating inhibition in the lateral amygdala (LA) and fear learning using the combined approaches of immunohistochemistry, electrophysiology, and behavior. In sharp contrast to the established role of synaptic GABA A R in mediating LA inhibition, we found that either pharmacological or physiological recruitment of GABA A (δ)R resulted in the weakening of GABAergic transmission onto projection neurons in LA while leaving the glutamatergic transmission unaltered, suggesting disinhibition by GABA A (δ)R. The disinhibition arose from IN-specific expression of GABA A (δ)R with its activation decreasing the input resistance of local INs and suppressing their activation. Genetic deletion of GABA A (δ)R attenuated its role in suppressing LA INs and disinhibiting LA. Importantly, the GABA A (δ)R facilitated long-term potentiation in sensory afferents to LA and permitted the expression of learned fear. Our findings suggest that GABA A (δ)R serves as a brake rather than a mediator of GABAergic inhibition in LA. The disinhibition by GABA A (δ)R may help to prevent excessive suppression of amygdala activity and thus ensure the expression of emotion. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

PubMed

Steinmetz, Eric J; Auldridge, Michele E

2017-11-01

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

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.

Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain.

PubMed

Okui, M; Ito, F; Ogita, K; Kuramoto, N; Kudoh, J; Shimizu, N; Ide, T

2000-01-01

APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.

Marker Protein Expression Combined With Expression Heterogeneity is a Powerful Indicator of Malignancy in Acral Lentiginous Melanomas.

PubMed

Cintra Lopes Carapeto, Fernando; Neves Comodo, Andréia; Germano, Andressa; Pereira Guimarães, Daiane; Barcelos, Denise; Fernandes, Mariana; Landman, Gilles

2017-02-01

Samples of acral lentiginous melanomas (ALMs) were obtained from the Department of Pathology at Escola Paulista de Medicina-Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. Demographic, clinical, and follow-up data were obtained from the charts of Hospital São Paulo. From 2 tissue microarrays containing 60 nevi and quadruplicate samples of ≥1.0-mm of 49 ALM, sections were stained to evaluate SCF, KIT, BRAF, CYCLIND1, MYC, and PTEN immunohistochemical protein expression. Nevi and ALM from 2006 to 2010 were reviewed and collected. All specimens were in the vertical growth phase, and histopathological parameters indicated that tumors were at an advanced stage at diagnosis. Average tumor thickness was 6.95 mm, 63% were ulcerated, average mitotic index was 5 mitotic cells per mm, and 43% were at Clark's level V. Compared with nevi, the χ test showed that ALM significantly correlated with SCF protein expression (P = 0.001) and expression heterogeneity (P < 0.000). Similar findings were observed for KIT (P = 0.005, P = 0.003, respectively), MYC (P < 0.000, P < 0.000), and PTEN (P = 0.005, P < 0.000). Malignancy did not correlate with BRAF and CYCLIN D1 expression (P = 0.053 and P = 0.259, respectively), but it did significantly correlate with their heterogeneous expression (P < 0.000, P = 0.024, respectively). Combined protein expression had an odds ratio of greater malignancy when BRAF and MYC were positive and/or heterogeneously expressed (OR of 78 and 95, respectively). We show that marker protein expression, when combined with heterogeneous expression as shown by immunohistochemistry, is a powerful indicator of malignancy in ALMs, especially, when protein pairs are combined.

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

PubMed

Tagliavia, Marcello; Cuttitta, Angela

2016-01-01

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

Sexual experience increases oxytocin receptor gene expression and protein in the medial preoptic area of the male rat.

PubMed

Gil, Mario; Bhatt, Renu; Picotte, Katie B; Hull, Elaine M

2013-09-01

Oxytocin (OT) promotes social and reproductive behaviors in mammals, and OT deficits may be linked to disordered social behaviors like autism and severe anxiety. Male rat sexual behavior is an excellent model for OT regulation of behavior, as its pattern and neural substrates are well characterized. We previously reported that OT microinjected into the medial preoptic area (MPOA), a major integrative site for male sexual behavior, facilitates copulation in sexually experienced male rats, whereas intra-MPOA injection of an OT antagonist (OTA) inhibits copulation. In the present studies, copulation on the day of sacrifice stimulated OTR mRNA expression in the MPOA, irrespective of previous sexual experience, with the highest levels observed in first-time copulators. In addition, sexually experienced males had higher levels of OTR protein in the MPOA than sexually naïve males and first-time copulators. Finally, intra-MPOA injection of OT facilitated mating in sexually naive males. Others have reported a positive correlation between OT mRNA levels and male sexual behavior. Our studies show that OT in the MPOA facilitates mating in both sexually naive and experienced males, some of the behavioral effects of OT are mediated by the OTR, and sexual experience is associated with increased OTR expression in the MPOA. Taken together, these data suggest a reciprocal interaction between central OT and behavior, in which OT facilitates copulation and copulation stimulates the OT/OTR system in the brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prognostic relevance of Centromere protein H expression in esophageal carcinoma.

PubMed

Guo, Xian-Zhi; Zhang, Ge; Wang, Jun-Ye; Liu, Wan-Li; Wang, Fang; Dong, Ju-Qin; Xu, Li-Hua; Cao, Jing-Yan; Song, Li-Bing; Zeng, Mu-Sheng

2008-08-13

Many kinetochore proteins have been shown to be associated with human cancers. The aim of the present study was to clarify the expression of Centromere protein H (CENP-H), one of the fundamental components of the human active kinetochore, in esophageal carcinoma and its correlation with clinicopathological features. We examined the expression of CENP-H in immortalized esophageal epithelial cells as well as in esophageal carcinoma cells, and in 12 cases of esophageal carcinoma tissues and the paired normal esophageal tissues by RT-PCR and Western blot analysis. In addition, we analyzed CENP-H protein expression in 177 clinicopathologically characterized esophageal carcinoma cases by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations. The level of CENP-H mRNA and protein were higher in the immortalized cells, cancer cell lines and most cancer tissues than in normal control tissues. Immunohistochemistry showed that CENP-H was expressed in 127 of 171 ESCC cases (74.3%) and in 3 of 6 esophageal adenocarcinoma cases (50%). Statistical analysis of ESCC cases showed that there was a significant difference of CENP-H expression in patients categorized according to gender (P = 0.013), stage (P = 0.023) and T classification (P = 0.019). Patients with lower CENP-H expression had longer overall survival time than those with higher CENP-H expression. Multivariate analysis suggested that CENP-H expression was an independent prognostic marker for esophageal carcinoma patients. A prognostic value of CENP-H was also found in the subgroup of T3 approximately T4 and N0 tumor classification. Our results suggest that CENP-H protein is a valuable marker of esophageal carcinoma progression. CENP-H might be used as a valuable prognostic marker for esophageal carcinoma patients.

Calcium affecting protein expression in longan under simulated acid rain stress.

PubMed

Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

2015-08-01

Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

PubMed

Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

2009-12-01

The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

Recombinant protein expression in Escherichia coli: advances and challenges

PubMed Central

Rosano, Germán L.; Ceccarelli, Eduardo A.

2014-01-01

Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

Transient expression and cellular localization of recombinant proteins in cultured insect cells

USDA-ARS?s Scientific Manuscript database

Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer.

PubMed

Jung, Yoon Yang; Kim, Hye Min; Koo, Ja Seung

2015-01-01

The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof. Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52). The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054-22.59, p = 0.043). Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.

Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay.

PubMed

Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C J; Wallace, John C; Polyak, Steven W

2008-11-15

Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme's properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

PubMed Central

Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

2013-01-01

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

Differential expression proteins associated with bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

PubMed

Zhang, Y X; Yu, D; Tian, X L; Liu, C Y; Gai, S P; Zheng, G S

2015-01-01

Endo-dormant flower buds of tree peony must have sufficient chilling duration to reinitiate growth, which is a major obstacle to the forcing culture of tree peony in winter. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) to identify the differentially expressed proteins of tree peony after three different chilling treatments: endo-dormancy, endo-dormancy release and eco-dormancy stages. More than 200 highly reproducible protein spots were detected, and 31 differentially expressed spots (P < 0.05) were selected for further analysis. Finally, 20 protein spots were confidently identified from databases, which were annotated and classified into seven functional categories: response to abiotic or biotic stimulus (four), metabolic processes (four), other binding (three), transcription or transcription regulation (two), biological processes (one), cell biogenesis (one) and unclassified (five). The results of qPCR of five genes were mainly consistent with that of the protein accumulation analysis as determined by 2-DE. This indicated that most of these genes were mainly regulated at transcriptional level. The activity of nitrate reductase and pyruvate dehydrogenase E1 was consistent with the 2-DE results. The proteomic profiles indicated activation of citrate cycle, amino acid metabolism, lipid metabolism, energy production, calcium signalling and cell growth processes by chilling fulfilment to facilitate dormancy release in tree peony. Analysis of functions of identified proteins will increase our knowledge of endo-dormancy release in tree peony. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

High-level SLP-2 expression and HER-2/neu protein expression are associated with decreased breast cancer patient survival.

PubMed

Cao, Wenfeng; Zhang, Bin; Liu, Yanxue; Li, Hongtao; Zhang, Shiwu; Fu, Li; Niu, Yun; Ning, Liansheng; Cao, Xuchen; Liu, Zhihua; Sun, Baocun

2007-09-01

There is sufficient evidence that human stomatin-like protein 2 (SLP-2) is a novel cancer-related gene. Its protein is overexpressed in many human cancers. SLP-2 can contribute to the promotion of cell growth, cell adhesion, and tumorigenesis in esophageal squamous cell carcinoma and lymph node metastasis in laryngeal squamous cell carcinoma. Immunohistochemical detection of SLP-2, estrogen and progesterone receptors, and HER-2/neu were performed on 263 cases of primary invasive breast cancer with a tissue microarray. Of 263 cases, 138 (52.5%) showed high expression of SLP-2 protein, and 125 (47.5%) showed low or absent expression. In addition, there were significant positive associations between tumor stage and size (P = .020), lymph node metastasis (P < .001), clinical stage (P < .001), distant metastasis (P = .002), and HER-2/neu protein expression (P = .037) and high-level SLP-2 expression. High-level SLP-2 expression was associated with decreased overall survival (P = .011) and was more often found in patients with tumors larger than 20 mm, lymph node metastasis, advanced clinical stage, distant metastasis, and HER-2/neu protein-positive expression. More important, lymph node metastasis, HER-2/neu-positive expression, and high-level SLP-2 expression were associated with significantly decreased survival.

Expression and purification of RHC-EGFP fusion protein and its application in hyaluronic acid assay.

PubMed

Duan, Ningjun; Lv, Wansheng; Zhu, Lingli; Zheng, Weijuan; Hua, Zichun

2017-03-16

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM-enhanced green fluorescence protein (RHC-EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC-EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.

Expression and crystallization of the plant alternative oxidase.

PubMed

May, Benjamin; Elliott, Catherine; Iwata, Momi; Young, Luke; Shearman, Julia; Albury, Mary S; Moore, Anthony L

2015-01-01

The alternative oxidase (AOX) is an integral monotopic membrane protein located on the inner surface of the inner mitochondrial membrane. Branching from the traditional respiratory chain at the quinone pool, AOX is responsible for cyanide-resistant respiration in plants and fungi, heat generation in thermogenic plants, and survival of parasites, such as Trypanosoma brucei, in the human host. A recently solved AOX structure provides insight into its active site, thereby facilitating rational phytopathogenic and antiparasitic drug design. Here, we describe expression of recombinant AOX using two different expression systems. Purification protocols for the production of highly pure and stable AOX protein in sufficient quantities to facilitate further kinetic, biophysical, and structural analyses are also described.

The Protein Corona around Nanoparticles Facilitates Stem Cell Labeling for Clinical MR Imaging.

PubMed

Nejadnik, Hossein; Taghavi-Garmestani, Seyed-Meghdad; Madsen, Steven J; Li, Kai; Zanganeh, Saeid; Yang, Phillip; Mahmoudi, Morteza; Daldrup-Link, Heike E

2018-03-01

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation.

PubMed

Yan, Jin; Leal, Karina; Magupalli, Venkat G; Nanou, Evanthia; Martinez, Gilbert Q; Scheuer, Todd; Catterall, William A

2014-11-01

Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.

[Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

PubMed

Wang, Ting-Ting; Wang, Xi-Hui; Fan, Zhong-Ling; Chen, Jin-Long; Cao, Bing-Lei; Kong, Na; Hu, Jing-Dong; Zhao, Hong-Kun

2011-02-01

To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

Green fluorescent protein as a reporter of gene expression and protein localization.

PubMed

Kain, S R; Adams, M; Kondepudi, A; Yang, T T; Ward, W W; Kitts, P

1995-10-01

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

PubMed Central

Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

2011-01-01

Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

PubMed Central

Stanley, Rachael L; Fleck, Roland A; Becker, David L; Goodship, Allen E; Ralphs, Jim R; Patterson-Kane, Janet C

2007-01-01

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1–6 months), 5 young adults (2–7 years) and 5 old horses (18–33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury. PMID:17848160

Fe-S Proteins that Regulate Gene Expression

PubMed Central

Mettert, Erin L.; Kiley, Patricia J.

2014-01-01

Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

In vitro protein expression: an emerging alternative to cell-based approaches.

PubMed

He, Mingyue

2011-04-30

Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Subcellular localization of transiently expressed fluorescent fusion proteins.

PubMed

Collings, David A

2013-01-01

The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system.

PubMed

Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W

2001-10-01

The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

Machine learning in computational biology to accelerate high-throughput protein expression.

PubMed

Sastry, Anand; Monk, Jonathan; Tegel, Hanna; Uhlen, Mathias; Palsson, Bernhard O; Rockberg, Johan; Brunk, Elizabeth

2017-08-15

The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

PubMed Central

Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

Interfacial polymerization for colorimetric labeling of protein expression in cells.

PubMed

Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

The Expression and Significance of Neuronal Iconic Proteins in Podocytes

PubMed Central

Sun, Yu; Zhang, Hongxia; Hu, Ruimin; Sun, Jianyong; Mao, Xing; Zhao, Zhonghua; Chen, Qi; Zhang, Zhigang

2014-01-01

Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes. PMID:24699703

p53 AND MDM2 PROTEIN EXPRESSION IN ACTINIC CHEILITIS

PubMed Central

de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

2008-01-01

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia. PMID:19082401

p53 and MDM2 protein expression in actinic cheilitis.

PubMed

de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

2008-01-01

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

PubMed Central

Zhang, Xianghui; Fu, Jianming; Hiromasa, Yasuaki; Pan, Hongyu; Bai, Guihua

2013-01-01

Background Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1. Methods The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins. Results Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification. Conclusions Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance. PMID:24376514

[Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

PubMed

Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

2011-01-01

To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

The TIM Barrel Architecture Facilitated the Early Evolution of Protein-Mediated Metabolism.

PubMed

Goldman, Aaron David; Beatty, Joshua T; Landweber, Laura F

2016-01-01

The triosephosphate isomerase (TIM) barrel protein fold is a structurally repetitive architecture that is present in approximately 10% of all enzymes. It is generally assumed that this ubiquity in modern proteomes reflects an essential historical role in early protein-mediated metabolism. Here, we provide quantitative and comparative analyses to support several hypotheses about the early importance of the TIM barrel architecture. An information theoretical analysis of protein structures supports the hypothesis that the TIM barrel architecture could arise more easily by duplication and recombination compared to other mixed α/β structures. We show that TIM barrel enzymes corresponding to the most taxonomically broad superfamilies also have the broadest range of functions, often aided by metal and nucleotide-derived cofactors that are thought to reflect an earlier stage of metabolic evolution. By comparison to other putatively ancient protein architectures, we find that the functional diversity of TIM barrel proteins cannot be explained simply by their antiquity. Instead, the breadth of TIM barrel functions can be explained, in part, by the incorporation of a broad range of cofactors, a trend that does not appear to be shared by proteins in general. These results support the hypothesis that the simple and functionally general TIM barrel architecture may have arisen early in the evolution of protein biosynthesis and provided an ideal scaffold to facilitate the metabolic transition from ribozymes, peptides, and geochemical catalysts to modern protein enzymes.

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

PubMed Central

Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

2012-01-01

Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

Establishment of stable cell line for inducing KAP1 protein expression.

PubMed

Liu, Xiaoyan; Khan, Md Asaduzzaman; Cheng, Jingliang; Wei, Chunli; Zhang, Lianmei; Fu, Junjiang

2015-06-01

Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells.

PubMed

Albanito, Lidia; Sisci, Diego; Aquila, Saveria; Brunelli, Elvira; Vivacqua, Adele; Madeo, Antonio; Lappano, Rosamaria; Pandey, Deo Prakash; Picard, Didier; Mauro, Loredana; Andò, Sebastiano; Maggiolini, Marcello

2008-08-01

Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

PubMed

Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

2009-09-01

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Variation in Protein Intake Induces Variation in Spider Silk Expression

PubMed Central

Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

2012-01-01

Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells.

PubMed

Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

2016-08-05

The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

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

López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

Hippocampal expression of a virus-derived protein impairs memory in mice.

PubMed

Bétourné, Alexandre; Szelechowski, Marion; Thouard, Anne; Abrial, Erika; Jean, Arnaud; Zaidi, Falek; Foret, Charlotte; Bonnaud, Emilie M; Charlier, Caroline M; Suberbielle, Elsa; Malnou, Cécile E; Granon, Sylvie; Rampon, Claire; Gonzalez-Dunia, Daniel

2018-02-13

The analysis of the biology of neurotropic viruses, notably of their interference with cellular signaling, provides a useful tool to get further insight into the role of specific pathways in the control of behavioral functions. Here, we exploited the natural property of a viral protein identified as a major effector of behavioral disorders during infection. We used the phosphoprotein (P) of Borna disease virus, which acts as a decoy substrate for protein kinase C (PKC) when expressed in neurons and disrupts synaptic plasticity. By a lentiviral-based strategy, we directed the singled-out expression of P in the dentate gyrus of the hippocampus and we examined its impact on mouse behavior. Mice expressing the P protein displayed increased anxiety and impaired long-term memory in contextual and spatial memory tasks. Interestingly, these effects were dependent on P protein phosphorylation by PKC, as expression of a mutant form of P devoid of its PKC phosphorylation sites had no effect on these behaviors. We also revealed features of behavioral impairment induced by P protein expression but that were independent of its phosphorylation by PKC. Altogether, our findings provide insight into the behavioral correlates of viral infection, as well as into the impact of virus-mediated alterations of the PKC pathway on behavioral functions.

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

BAX protein expression and clinical outcome in epithelial ovarian cancer.

PubMed

Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

1998-08-01

Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients

PubMed Central

Bae, Su-Mi; Min, Hyun-Jin; Ding, Guo Hua; Kwak, Sun-Young; Cho, Young-Lae; Nam, Kye-Hyun; Park, Choong Hak; Kim, Yong-Wan; Kim, Chong-Kook; Han, Byoung-Don; Lee, Young-Joo; Kim, Do Kang

2006-01-01

Purpose Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two- dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectrometer. Materials and Methods Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software™. The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. Results A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. Conclusion 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC. PMID:19771267

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

PubMed

Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

2000-02-01

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

PubMed

Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

2014-06-01

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

PubMed

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

ERIC Educational Resources Information Center

Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

2008-01-01

We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

PubMed

Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

2014-03-01

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

Oligomerization state of water channels and glycerol facilitators. Involvement of loop E.

PubMed

Lagrée, V; Froger, A; Deschamps, S; Pellerin, I; Delamarche, C; Bonnec, G; Gouranton, J; Thomas, D; Hubert, J F

1998-12-18

The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast or Xenopus oocytes behaves as an homotetramer when extracted by n-octyl beta-D-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes. The GlpF protein extracted either by SDS or by nondenaturing detergents, OG and Triton X-100, exhibits sedimentation coefficients only compatible with a monomeric form of the protein in micelles. We then substituted in loop E of AQPcic two amino acids predicted to play a role in the functional/structural properties of the MIPs. In two expression systems, yeast and oocytes, the mutant AQPcic-S205D is monomeric in OG and in SDS. The A209K mutation does not modify the tetrameric form of the heterologous protein in OG. This study shows that the serine residue at position 205 is essential for AQPcic tetramerization. Because the serine in this position is highly conserved among aquaporins and systematically replaced by an acid aspartic in GlpFs, we postulate that glycerol facilitators are monomers whereas aquaporins are organized in tetramers. Our data suggest that the role of loop E in MIP properties partly occurs through its ability to allow oligomerization of the proteins.

Protein-disulfide Isomerase Regulates the Thyroid Hormone Receptor-mediated Gene Expression via Redox Factor-1 through Thiol Reduction-Oxidation*

PubMed Central

Hashimoto, Shoko; Imaoka, Susumu

2013-01-01

Protein-disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase that regulates the redox state of proteins. We previously found that overexpression of PDI in rat pituitary tumor (GH3) cells suppresses 3,3′,5-triiodothyronine (T3)-stimulated growth hormone (GH) expression, suggesting the contribution of PDI to the T3-mediated gene expression via thyroid hormone receptor (TR). In the present study, we have clarified the mechanism of regulation by which TR function is regulated by PDI. Overexpression of wild-type but not redox-inactive mutant PDI suppressed the T3-induced GH expression, suggesting that the redox activity of PDI contributes to the suppression of GH. We considered that PDI regulates the redox state of the TR and focused on redox factor-1 (Ref-1) as a mediator of the redox regulation of TR by PDI. Interaction between Ref-1 and TRβ1 was detected. Overexpression of wild-type but not C64S Ref-1 facilitated the GH expression, suggesting that redox activity of Cys-64 in Ref-1 is involved in the TR-mediated gene expression. Moreover, PDI interacted with Ref-1 and changed the redox state of Ref-1, suggesting that PDI controls the redox state of Ref-1. Our studies suggested that Ref-1 contributes to TR-mediated gene expression and that the redox state of Ref-1 is regulated by PDI. Redox regulation of PDI via Ref-1 is a new aspect of PDI function. PMID:23148211

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

PubMed

Schroeter, Christina B; Koehler, Sybille; Kann, Martin; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T; Rinschen, Markus M

2018-04-25

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

PubMed

Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.

PubMed

Kuipers, Grietje; Karyolaimos, Alexandros; Zhang, Zhe; Ismail, Nurzian; Trinco, Gianluca; Vikström, David; Slotboom, Dirk Jan; de Gier, Jan-Willem

2017-12-16

To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector. By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein. Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

PubMed

Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

2005-05-01

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.

PubMed

Sun, Xiaoyan; Wray, Curtis; Tian, Xintian; Hasselgren, Per-Olof; Lu, James

2003-09-01

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.

Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce.

PubMed

Sun, Hyeon-Jin; Cui, Min-Long; Ma, Biao; Ezura, Hiroshi

2006-01-23

Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.

PubMed

Keates, Tracy; Cooper, Christopher D O; Savitsky, Pavel; Allerston, Charles K; Phillips, Claire; Hammarström, Martin; Daga, Neha; Berridge, Georgina; Mahajan, Pravin; Burgess-Brown, Nicola A; Müller, Susanne; Gräslund, Susanne; Gileadi, Opher

2012-06-15

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. Copyright © 2011 Elsevier B.V. All rights reserved.

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

PubMed Central

Keates, Tracy; Cooper, Christopher D.O.; Savitsky, Pavel; Allerston, Charles K.; Phillips, Claire; Hammarström, Martin; Daga, Neha; Berridge, Georgina; Mahajan, Pravin; Burgess-Brown, Nicola A.; Müller, Susanne; Gräslund, Susanne; Gileadi, Opher

2012-01-01

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. PMID:22027370

Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

PubMed

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

PubMed Central

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice

PubMed Central

Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M.; Azizi, Mohammad; Khalaj, Vahid

2018-01-01

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins. PMID

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

PubMed

Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M; Azizi, Mohammad; Khalaj, Vahid

2018-01-01

Saccharomyces boulardii , a subspecies of Saccharomyces cerevisiae , is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi ® ) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3 - S. boulardii . To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group ( P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group ( P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii , as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

Exercise alters myostatin protein expression in sedentary and exercised streptozotocin-diabetic rats.

PubMed

Bassi, Daniela; Bueno, Patricia de Godoy; Nonaka, Keico Okino; Selistre-Araujo, Heloisa Sobreiro; Leal, Angela Merice de Oliveira

2015-04-01

The aim of this study was to analyze the effect of exercise on the pattern of muscle myostatin (MSTN) protein expression in two important metabolic disorders, i.e., obesity and diabetes mellitus. MSTN, is a negative regulator of skeletal muscle mass. We evaluated the effect of exercise on MSTN protein expression in diabetes mellitus and high fat diet-induced obesity. MSTN protein expression in gastrocnemius muscle was analyzed by Western Blot. P < 0.05 was assumed. Exercise induced a significant decrease in glycemia in both diabetic and obese animals. The expression of precursor and processed protein forms of MSTN and the weight of gastrocnemius muscle did not vary in sedentary or exercised obese animals. Diabetes reduced gastrocnemius muscle weight in sedentary animals. However, gastrocnemius muscle weight increased in diabetic exercised animals. Both the precursor and processed forms of muscle MSTN protein were significantly higher in sedentary diabetic rats than in control rats. The precursor form was significantly lower in diabetic exercised animals than in diabetic sedentary animals. However, the processed form did not change. These results demonstrate that exercise can modulate the muscle expression of MSTN protein in diabetic rats and suggest that MSTN may be involved in energy homeostasis.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

PubMed

Liu, Cunbao; Yang, Xu; Yao, Yufeng; Huang, Weiwei; Sun, Wenjia; Ma, Yanbing

2014-05-01

Two versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

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

McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk; Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast BT9 7BL; Patel, Daksha, E-mail: d.patel@qub.ac.uk

2014-01-05

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in themore » cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.« less

Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

PubMed

Houdelet, Marcel; Galinski, Anna; Holland, Tanja; Wenzel, Kathrin; Schillberg, Stefan; Buyel, Johannes Felix

2017-04-01

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD 600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

PubMed Central

Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

2016-01-01

Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

Protein-protein interaction network of gene expression in the hydrocortisone-treated keloid.

PubMed

Chen, Rui; Zhang, Zhiliang; Xue, Zhujia; Wang, Lin; Fu, Mingang; Lu, Yi; Bai, Ling; Zhang, Ping; Fan, Zhihong

2015-01-01

In order to explore the molecular mechanism of hydrocortisone in keloid tissue, the gene expression profiles of keloid samples treated with hydrocortisone were subjected to bioinformatics analysis. Firstly, the gene expression profiles (GSE7890) of five samples of keloid treated with hydrocortisone and five untreated keloid samples were downloaded from the Gene Expression Omnibus (GEO) database. Secondly, data were preprocessed using packages in R language and differentially expressed genes (DEGs) were screened using a significance analysis of microarrays (SAM) protocol. Thirdly, the DEGs were subjected to gene ontology (GO) function and KEGG pathway enrichment analysis. Finally, the interactions of DEGs in samples of keloid treated with hydrocortisone were explored in a human protein-protein interaction (PPI) network, and sub-modules of the DEGs interaction network were analyzed using Cytoscape software. Based on the analysis, 572 DEGs in the hydrocortisone-treated samples were screened; most of these were involved in the signal transduction and cell cycle. Furthermore, three critical genes in the module, including COL1A1, NID1, and PRELP, were screened in the PPI network analysis. These findings enhance understanding of the pathogenesis of the keloid and provide references for keloid therapy. © 2015 The International Society of Dermatology.

Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues.

PubMed

Ohta, K; Iwai, K; Kasahara, Y; Taniguchi, N; Krajewski, S; Reed, J C; Miyawaki, T

1995-11-01

The ability of Bcl-2 to inhibit apoptotic cell death is well established. Several homologues of the bcl-2 gene, such as bax, bcl-x or mcl-1, have recently been identified. Like Bcl-2, both Bcl-XL and Mcl-1 appear to function as repressors of apoptotic cell death, whereas Bax facilitates it, indicating possible interactions among them in the control of cellular survival. To investigate the in vivo role of expression of bcl-2 gene family products, immunoblot analysis using corresponding specific antisera was performed for peripheral blood cells and some lymphoid tissues in humans. We demonstrated that all Bcl-2 family proteins were expressed at various levels in hematolymphoid cell subpopulations isolated from peripheral blood, tonsil, spleen and thymus. Lymphoid expression of Bcl-2 family proteins tended to increase following activation, but declined with time in culture. Loss of Bcl-2 in cultured lymphoid cells was especially marked. Sole expression of Bax, but not other members of the Bcl-2 family, was observed on neutrophils, seemingly reflecting their shortest life-span among blood leukocytes. The results support the notion that a balance of expression of Bcl-2 family proteins may regulate the life and death of hematolymphoid cells at different stages of cell differentiation and activation.

OncoBinder facilitates interpretation of proteomic interaction data by capturing coactivation pairs in cancer.

PubMed

Van Coillie, Samya; Liang, Lunxi; Zhang, Yao; Wang, Huanbin; Fang, Jing-Yuan; Xu, Jie

2016-04-05

High-throughput methods such as co-immunoprecipitationmass spectrometry (coIP-MS) and yeast 2 hybridization (Y2H) have suggested a broad range of unannotated protein-protein interactions (PPIs), and interpretation of these PPIs remains a challenging task. The advancements in cancer genomic researches allow for the inference of "coactivation pairs" in cancer, which may facilitate the identification of PPIs involved in cancer. Here we present OncoBinder as a tool for the assessment of proteomic interaction data based on the functional synergy of oncoproteins in cancer. This decision tree-based method combines gene mutation, copy number and mRNA expression information to infer the functional status of protein-coding genes. We applied OncoBinder to evaluate the potential binders of EGFR and ERK2 proteins based on the gastric cancer dataset of The Cancer Genome Atlas (TCGA). As a result, OncoBinder identified high confidence interactions (annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) or validated by low-throughput assays) more efficiently than co-expression based method. Taken together, our results suggest that evaluation of gene functional synergy in cancer may facilitate the interpretation of proteomic interaction data. The OncoBinder toolbox for Matlab is freely accessible online.

Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

PubMed Central

Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

2016-01-01

The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

PubMed

Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

2016-05-24

The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

PubMed

Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

2015-08-01

To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

PubMed

Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

2016-06-01

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

PubMed Central

Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

2017-01-01

There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

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

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle.

PubMed

Mizuta, Kentaro; Zhang, Yi; Xu, Dingbang; Mizuta, Fumiko; D'Ovidio, Frank; Masaki, Eiji; Emala, Charles W

2013-09-02

Dopamine signaling is mediated by Gs protein-coupled "D1-like" receptors (D1 and D5) and Gi-coupled "D2-like" receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists

Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

PubMed

Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

2011-10-25

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

PubMed Central

McMains, Emily; Krishnan, Vijai; Prasad, Sujitha; Gleason, Evanna

2011-01-01

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue. PMID:21408174

Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

PubMed

Brödel, A K; Raymond, J A; Duman, J G; Bier, F F; Kubick, S

2013-02-10

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications. Copyright © 2012 Elsevier B.V. All rights reserved.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

PubMed

Hu, Jia; Chen, Xiang; Zhang, Xuhua; Yuan, Xiaopeng; Yang, Mingjuan; Dai, Hui; Yang, Wei; Zhou, Qinghua; Wen, Weihong; Wang, Qirui; Qin, Weijun; Zhao, Aizhi

2018-05-01

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (V H ) and light (V L ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development. © 2018 The Protein Society.

Expression of small heat shock proteins from pea seedlings under gravity altered conditions

NASA Astrophysics Data System (ADS)

Talalaev, Alexandr S.

2005-08-01

A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

Repetitive acute intermittent hypoxia increases expression of proteins associated with plasticity in the phrenic motor nucleus

PubMed Central

Satriotomo, Irawan; Dale, Erica A.; Dahlberg, Jenny M.; Mitchell, Gordon S.

2015-01-01

Acute intermittent hypoxia (AIH) initiates plasticity in respiratory motor control, including phrenic long term facilitation (pLTF). Since pLTF is enhanced by preconditioning with repetitive exposure to AIH (rAIH), we hypothesized that a rAIH protocol consisting of 3 AIH exposures per week for 10 weeks (3×wAIH; AIH: 10, 5-min episodes of 10.5% O2; 5-min normoxic intervals) would enhance expression of molecules that play key roles in pLTF within the phrenic motor nucleus. Immunohistochemical analyses revealed that 3×wAIH for 10 weeks increased serotonin terminal density in the C4 phrenic motor nucleus and serotonin 2A (5-HT2A) receptor expression in presumptive phrenic motor neurons. Immunoreactive brain derived neurotrophic factor (BDNF) and its high affinity receptor (TrkB) also increased following 3×wAIH. 3×wAIH also increased expression of another hypoxia-sensitive growth factor known to elicit phrenic motor facilitation, vascular endothelial growth factor (VEGF), and its receptor (VEGFR-2). Kinases “downstream” from TrkB and VEGFR-2 were up-regulated in or near presumptive phrenic motor neurons, including phosphorylated extracellular-signal regulated kinase (p-ERK) and protein kinase B (p-AKT). Thus, 3×wAIH up-regulates neurochemicals known to be associated with phrenic motor plasticity. Since 3×wAIH upregulates pro-plasticity molecules without evidence for CNS pathology, it may be a useful therapeutic tool in treating disorders that cause respiratory insufficiency, such as spinal injury or motor neuron disease. PMID:22704858

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

Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

PubMed Central

Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

2016-01-01

Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

PubMed

Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

2015-10-01

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunogenicity of Newcastle Disease Virus Vectors Expressing Norwalk Virus Capsid Protein in the Presence or Absence of VP2 Protein

PubMed Central

Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y.; Samal, Siba K.

2015-01-01

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirs-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. PMID:26099695

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

PubMed Central

Hudson, Lauren E.; Fasken, Milo B.; McDermott, Courtney D.; McBride, Shonna M.; Kuiper, Emily G.; Guiliano, David B.; Corbett, Anita H.; Lamb, Tracey J.

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders. PMID:25391025

Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

PubMed

Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

Raman microscopy of bladder cancer cells expressing green fluorescent protein

NASA Astrophysics Data System (ADS)

Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

2016-11-01

Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

PubMed Central

Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

2016-01-01

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

The protein expression landscape of mitosis and meiosis in diploid budding yeast.

PubMed

Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

2017-03-06

Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal.

PubMed

Salas-Cortés, L; Jaubert, F; Nihoul-Feketé, C; Brauner, R; Rosemblatt, M; Fellous, M

2000-01-01

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein. Copyright 2001 S. Karger AG, Basel

Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

PubMed

Pushnova, E A; Ostanin, K; Thelen, M P

2001-11-01

Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose. Copyright 2001 Academic Press.

Postsynaptic Regulation of Long-Term Facilitation in Aplysia

PubMed Central

Cai, Diancai; Chen, Shanping; Glanzman, David L.

2009-01-01

Summary Repeated exposure to serotonin (5-HT), an endogenous neurotransmitter that mediates behavioral sensitization in Aplysia [1–3], induces long-term facilitation (LTF) of the Aplysia sensorimotor synapse [4]. LTF, a prominent form of invertebrate synaptic plasticity, is believed to play a major role in long-term learning in Aplysia [5]. Until now, LTF has been thought to be due predominantly to cellular processes activated by 5-HT within the presynaptic sensory neuron [6]. Recent work indicates that LTF depends on the increased expression and release of a sensory neuron-specific neuropeptide, sensorin [7]. Sensorin released during LTF appears to bind to autoreceptors on the sensory neuron, thereby activating critical presynaptic signals, including mitogen-activated protein kinase (MAPK) [8, 9]. Here, we show that LTF depends on elevated postsynaptic Ca2+ and postsynaptic protein synthesis. Furthermore, we find that the increased expression of presynaptic sensorin due to 5-HT stimulation requires elevation of postsynaptic intracellular Ca2+. Our results represent perhaps the strongest evidence to date that the increased expression of a specific presynaptic neuropeptide during LTF is regulated by retrograde signals. PMID:18571411

Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway.

PubMed

Pan, Xingliang; Yang, Yalin; Liu, Xuewei; Li, Dong; Li, Juan; Guo, Xiaoze; Zhou, Zhigang

2016-09-16

The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

PubMed Central

Branco, Luis M; Matschiner, Alex; Fair, Joseph N; Goba, Augustine; Sampey, Darryl B; Ferro, Philip J; Cashman, Kathleen A; Schoepp, Randal J; Tesh, Robert B; Bausch, Daniel G; Garry, Robert F; Guttieri, Mary C

2008-01-01

Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination. PMID:18538016

Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome

PubMed Central

Boqun, Xu; Xiaonan, Dai; YuGui, Cui; Lingling, Gao; Xue, Dai; Gao, Chao; Feiyang, Diao; Jiayin, Liu; Gao, Li; Li, Mei; Zhang, Yuan; Ma, Xiang

2013-01-01

Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS. PMID:23861679

Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome.

PubMed

Boqun, Xu; Xiaonan, Dai; Yugui, Cui; Lingling, Gao; Xue, Dai; Gao, Chao; Feiyang, Diao; Jiayin, Liu; Gao, Li; Li, Mei; Zhang, Yuan; Ma, Xiang

2013-01-01

Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS.

Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy.

PubMed

Liu, Guan-Ting; Kung, Hsiu-Ni; Chen, Chung-Kuan; Huang, Cheng; Wang, Yung-Li; Yu, Cheng-Pu; Lee, Chung-Pei

2018-02-26

Although a vesicular nucleocytoplasmic transport system is believed to exist in eukaryotic cells, the features of this pathway are mostly unknown. Here, we report that the BFRF1 protein of the Epstein-Barr virus improves vesicular transport of nuclear envelope (NE) to facilitate the translocation and clearance of nuclear components. BFRF1 expression induces vesicles that selectively transport nuclear components to the cytoplasm. With the use of aggregation-prone proteins as tools, we found that aggregated nuclear proteins are dispersed when these BFRF1-induced vesicles are formed. BFRF1-containing vesicles engulf the NE-associated aggregates, exit through from the NE, and putatively fuse with autophagic vacuoles. Chemical treatment and genetic ablation of autophagy-related factors indicate that autophagosome formation and autophagy-linked FYVE protein-mediated autophagic proteolysis are involved in this selective clearance of nuclear proteins. Remarkably, vesicular transport, elicited by BFRF1, also attenuated nuclear aggregates accumulated in neuroblastoma cells. Accordingly, induction of NE-derived vesicles by BFRF1 facilitates nuclear protein translocation and clearance, suggesting that autophagy-coupled transport of nucleus-derived vesicles can be elicited for nuclear component catabolism in mammalian cells.-Liu, G.-T., Kung, H.-N., Chen, C.-K., Huang, C., Wang, Y.-L., Yu, C.-P., Lee, C.-P. Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

PubMed

Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

2012-01-01

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

Abiotic stresses modulate expression of major intrinsic proteins in barley (Hordeum vulgare).

PubMed

Ligaba, Ayalew; Katsuhara, Maki; Shibasaka, Mineo; Djira, Gemechis

2011-02-01

In one of the most important crops, barley (Hordeum vulgare L.), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3 and HvTIP4;1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2;1, HvTIP2;3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed. Copyright © 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

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

Inoue-Toyoda, Maki; Kato, Kohsuke; Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575

Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter andmore » enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state. - Highlights: • DEX facilitates the transcription from the HCMV MIE promoter. • GR is involved in DEX-dependent transcription from the HCMV MIE promoter. • A 17 bp repeat is responsible for the HCMV MIE promoter activation by DEX. • An NF-I-like protein is involved in the HCMV MIE promoter activation by DEX.« less

Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

PubMed

Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

2017-09-15

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

Clinicopathologic significance of minichromosome maintenance protein 2 and Tat-interacting protein 30 expression in benign and malignant lesions of the gallbladder.

PubMed

Liu, Dong-cai; Yang, Zhu-lin

2011-11-01

Gallbladder cancers are aggressive tumors with a poor prognosis and high mortality rate. To find specific biological markers for early diagnosis and prognosis and to develop possible alternative treatment strategies, we examined minichromosome maintenance protein 2 (MCM2) and Tat-interacting protein 30 (TIP30) expression in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. Expression of MCM2 was significantly higher in adenocarcinomas than in peritumoral tissues (χ² = 8.41; P < .01), adenomatous polyps (χ² = 6.81; P < .01), and chronic cholecystitis (χ² = 21.00; P < .01). In contrast, Tat-interacting protein 30 expression was significantly less in adenocarcinomas than in peritumoral tissues (χ² = 13.26; P < .01), adenomatous polyps (χ² = 4.76; P < .05), and chronic cholecystitis (χ² = 18.93; P < .01). The benign lesions in gallbladder epithelium with positive MCM2 or negative Tat-interacting protein 30 expression showed moderate to severe atypical hyperplasia. Expression of MCM2 and absence of Tat-interacting protein 30 were significantly associated with poor differentiation, large tumor mass, lymph node metastasis, and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that either elevated MCM2 (P = .006) or lowered Tat-interacting protein 30 (P = .006) expression was closely associated with shorter overall survival. Multivariate Cox regression analysis revealed that expression of MCM2 (P = .007) or nonexpression of Tat-interacting protein 30 (P = .009) was an independent predictor of a poor prognosis in adenocarcinoma. Our results suggest that overexpression of MCM2 or loss of expression of Tat-interacting protein 30 is closely related to carcinogenesis, progression, biological behavior, and prognosis of gallbladder adenocarcinoma. Copyright © 2011 Elsevier Inc. All rights reserved.

Changes in protein expression during honey bee larval development.

PubMed

Chan, Queenie W T; Foster, Leonard J

2008-10-29

The honey bee (Apis mellifera), besides its role in pollination and honey production, serves as a model for studying the biochemistry of development, metabolism, and immunity in a social organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly 800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles. We report that honey bee larval growth is marked by an age-correlated increase of protein transporters and receptors, as well as protein nutrient stores, while opposite trends in protein translation activity and turnover were observed. Levels of the immunity factors prophenoloxidase and apismin are positively correlated with development, while others surprisingly were not significantly age-regulated, suggesting a molecular explanation for why bees are susceptible to major age-associated bee bacterial infections such as American Foulbrood or fungal diseases such as chalkbrood. Previously unreported findings include the reduction of antioxidant and G proteins in aging larvae. These data have allowed us to integrate disparate findings in previous studies to build a model of metabolism and maturity of the immune system during larval development. This publicly accessible resource for protein expression trends will help generate new hypotheses in the increasingly important field of honey bee research.

Heroin use is associated with lower levels of restriction factors and type I interferon expression and facilitates HIV-1 replication.

PubMed

Zhu, Jia-Wu; Liu, Feng-Liang; Mu, Dan; Deng, De-Yao; Zheng, Yong-Tang

Heroin use is associated with increased incidence of infectious diseases such as HIV-1 infection, as a result of immunosuppression to a certain extent. Host restriction factors are recently identified cellular proteins with potent antiviral activities. Whether heroin use impacts on the in vivo expression of restriction factors that result in facilitating HIV-1 replication is poorly understood. Here we recruited 432 intravenous drug users (IDUs) and 164 non-IDUs at high-risk behaviors. Based on serological tests, significantly higher prevalence of HIV-1 infection was observed among IDUs compared with non-IDUs. We included those IDUs and non-IDUs without HIV-1 infection, and found IDUs had significantly lower levels of TRIM5α, TRIM22, APOBEC3G, and IFN-α, -β expression than did non-IDUs. We also directly examined plasma viral load in HIV-1 mono-infected IDUs and non-IDUs and found HIV-1 mono-infected IDUs had significantly higher plasma viral load than did non-IDUs. Moreover, intrinsically positive correlation between type I interferon and TRIM5α or TRIM22 was observed, however, which was dysregulated following heroin use. Collectively, heroin use benefits HIV-1 replication that may be partly due to suppression of host restriction factors and type I interferon expression. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Glucose transporter 3 (GLUT3) protein expression in human placenta across gestation

PubMed Central

Brown, Kelecia; Heller, Debra S.; Zamudio, Stacy; Illsley, Nicholas P.

2012-01-01

Conflicting information regarding expression of GLUT3 protein in the human placenta has been reported and the localization and pattern of expression of GLUT3 protein across gestation has not been clearly defined. The objective of this study was characterization of syncytial GLUT3 protein expression across gestation. We hypothesized that GLUT3 protein is present in the syncytial microvillous membrane and that its expression decreases over gestation. GLUT3 protein was measured in samples from a range of gestational ages (first to third trimester), with human brain and human bowel used as a positive and negative control respectively. As an additional measure of specificity, we transfected BeWo choriocarcinoma cells, a trophoblast cell line expressing GLUT3, with siRNA directed against GLUT3 and analyzed expression by Western blotting. GLUT3 was detected in the syncytiotrophoblast at all gestational ages by immunohistochemistry. Using Western blotting GLUT3 was detected as an integral membrane protein at a molecular weight of ~50kDa in microvillous membranes from all trimesters but not in syncytial basal membranes. The identity of the primary antibody target was confirmed by demonstrating that expression of the immunoblotting signal in GLUT3 siRNA-treated BeWo was decreased to 18 ± 6% (mean ± SEM) of that seen in cells transfected with a non-targeting siRNA. GLUT3 expression in microvillous membranes detected by Western blot decreased through the trimesters such that expression in the second trimester (wks 14–26) was 48 ± 7% of that in the first trimester and by the third trimester (wks 31–40) only 34 ± 10% of first trimester expression. In addition, glucose uptake into BeWo cells treated with GLUT3 siRNA was reduced to 60% of that measured in cells treated with the non-targeting siRNA. This suggests that GLUT3-mediated uptake comprises approximately 50% of glucose uptake into BeWo cells. These results confirm the hypothesis that GLUT3 is present in the

Expression Atlas: gene and protein expression across multiple studies and organisms

PubMed Central

Tang, Y Amy; Bazant, Wojciech; Burke, Melissa; Fuentes, Alfonso Muñoz-Pomer; George, Nancy; Koskinen, Satu; Mohammed, Suhaib; Geniza, Matthew; Preece, Justin; Jarnuczak, Andrew F; Huber, Wolfgang; Stegle, Oliver; Brazma, Alvis; Petryszak, Robert

2018-01-01

Abstract Expression Atlas (http://www.ebi.ac.uk/gxa) is an added value database that provides information about gene and protein expression in different species and contexts, such as tissue, developmental stage, disease or cell type. The available public and controlled access data sets from different sources are curated and re-analysed using standardized, open source pipelines and made available for queries, download and visualization. As of August 2017, Expression Atlas holds data from 3,126 studies across 33 different species, including 731 from plants. Data from large-scale RNA sequencing studies including Blueprint, PCAWG, ENCODE, GTEx and HipSci can be visualized next to each other. In Expression Atlas, users can query genes or gene-sets of interest and explore their expression across or within species, tissues, developmental stages in a constitutive or differential context, representing the effects of diseases, conditions or experimental interventions. All processed data matrices are available for direct download in tab-delimited format or as R-data. In addition to the web interface, data sets can now be searched and downloaded through the Expression Atlas R package. Novel features and visualizations include the on-the-fly analysis of gene set overlaps and the option to view gene co-expression in experiments investigating constitutive gene expression across tissues or other conditions. PMID:29165655

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

PubMed Central

Culver, J N; Lehto, K; Close, S M; Hilf, M E; Dawson, W O

1993-01-01

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8446627

The expression and function of epithelial membrane protein 1 in laryngeal carcinoma.

PubMed

Li, Hong; Zhang, Xiaowen; Jiang, Xuejun; Ji, Xu

2017-01-01

In this study, we compared the expression of epithelial membrane protein 1 (EMP1) on the steady-state mRNA level (by quantitative real-time PCR) and on the protein level (by western immunoblot and immunohistochemistry) in 51 pairs of laryngeal carcinoma tissues and matched cancer-free peritumor tissues, and we analyzed the correlation between EMP1 expression and different clinicopathological factors. Furthermore, we ectopically expressed EMP1 in human laryngeal carcinoma Hep-2 cells and examined the effects on cell viability, apoptosis, colonogenicity, and motility, by MTT assay, flow cytometry, colony formation assay and Transwell migration assay, respectively. EMP1 expression (on both the mRNA and protein levels) was significantly lower in the cancer tissues than in matched peritumor tissues (P<0.05). In laryngeal cancers, the level of EMP1 protein was correlated with histological grade (P<0.05), but not with age, gender, clinical stage, cancer subtype or lymph node metastasis (P>0.05). Functionally, ectopic expression of EMP1 in Hep-2 cells significantly reduced cell viability, colony formation, and migration, but enhanced apoptosis. Therefore, EMP1 is a tumor suppressor in laryngeal carcinoma. Boosting EMP1 expression in laryngeal carcinoma initiates multiple anticancer phenotypes and thus presents a promising therapeutic strategy for laryngeal cancer.

Systematic Analysis of Arabidopsis Organelles and a Protein Localization Database for Facilitating Fluorescent Tagging of Full-Length Arabidopsis Proteins1[W

PubMed Central

Li, Shijun; Ehrhardt, David W.; Rhee, Seung Y.

2006-01-01

Cells are organized into a complex network of subcellular compartments that are specialized for various biological functions. Subcellular location is an important attribute of protein function. To facilitate systematic elucidation of protein subcellular location, we analyzed experimentally verified protein localization data of 1,300 Arabidopsis (Arabidopsis thaliana) proteins. The 1,300 experimentally verified proteins are distributed among 40 different compartments, with most of the proteins localized to four compartments: mitochondria (36%), nucleus (28%), plastid (17%), and cytosol (13.3%). About 19% of the proteins are found in multiple compartments, in which a high proportion (36.4%) is localized to both cytosol and nucleus. Characterization of the overrepresented Gene Ontology molecular functions and biological processes suggests that the Golgi apparatus and peroxisome may play more diverse functions but are involved in more specialized processes than other compartments. To support systematic empirical determination of protein subcellular localization using a technology called fluorescent tagging of full-length proteins, we developed a database and Web application to provide preselected green fluorescent protein insertion position and primer sequences for all Arabidopsis proteins to study their subcellular localization and to store experimentally verified protein localization images, videos, and their annotations of proteins generated using the fluorescent tagging of full-length proteins technology. The database can be searched, browsed, and downloaded using a Web browser at http://aztec.stanford.edu/gfp/. The software can also be downloaded from the same Web site for local installation. PMID:16617091

Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

PubMed

Merchant, Mark; Kinney, Clint; Sanders, Paige

2009-12-01

Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

Dexamethasone Regulates Cochlear Expression of Deafness-associated Proteins Myelin Protein Zero and Heat Shock Protein 70, as Revealed by iTRAQ Proteomics.

PubMed

Maeda, Yukihide; Fukushima, Kunihiro; Kariya, Shin; Orita, Yorihisa; Nishizaki, Kazunori

2015-08-01

Using proteomics, we aimed to identify the proteins differentially regulated by dexamethasone in the mouse cochlea based on mass-spectrometry data. Glucocorticoid therapy is widely used for many forms of sensorineural hearing loss; however, the molecular mechanism of its action in the cochlea remains poorly understood. Dexamethasone or control saline was intratympanically applied to the cochleae of mice. Twelve hours after application, proteins differentially regulated by dexamethasone in the cochlea were analyzed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-mass spectrometry. Next, dexamethasone-dependent regulation of these proteins was verified in the cochleae of mice with noise-induced hearing loss (NIHL) and systemic administration of dexamethasone by western blotting. Immunolocalizations of these proteins were examined in cochleae with NIHL. A total of 247 proteins with a greater than 95% confidence interval of protein identification were found, and 11 differentially expressed proteins by dexamethasone were identified by the iTRAQ-mass spectrometry. One protein, myelin protein zero (Mpz), was upregulated (1.870 ± 0.201-fold change, p < 0.01) at 6 hours post-systemic dexamethasone and noise exposure in a mouse model of NIHL. Heat shock protein 70 (Hsp70) was downregulated (0.511 ± 0.274-fold change, p < 0.05) at 12 hours post-systemic dexamethasone. Immunohistochemistry confirmed Mpz localization to the efferent and afferent processes of the spiral neurons, whereas Hsp70 showed a more ubiquitous expression pattern in the cochlea. Both Mpz and Hsp70 have been reported to be closely associated with sensorineural hearing loss in humans. Dexamethasone significantly modulated the expression levels of these proteins in the cochleae of mice.

Genomics Analysis of Genes Expressed in Maize Endosperm Identifies Novel Seed Proteins and Clarifies Patterns of Zein Gene Expression

PubMed Central