Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.

PubMed

Zhou, Jian-Liang; An, Jing-Jing; Li, Ping; Li, Hui-Jun; Jiang, Yan; Cheng, Jie-Fei

2009-03-20

We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

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

PubMed

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

2015-01-01

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

Virus elimination during the recycling of chromatographic columns used during the manufacture of coagulation factors.

PubMed

Roberts, Peter L

2014-07-01

Various chromatographic procedures are used during the purification and manufacture of plasma products such as coagulation factors. These steps contribute to the overall safety of such products by removing potential virus contamination. Virus removal by two affinity chromatography procedures, i.e. monoclonal antibody chromatography and metal chelate chromatography (immobilised metal ion affinity chromatography), used during the manufacture of the high purity factor VIII (Replenate®) and factor IX (Replenine®-VF), respectively, has been investigated. In addition, as these columns are recycled after use, the effectiveness of the sanitisation procedures for preventing possible cross-contamination, has also been investigated. Both chromatographic steps proved effective for eliminating a range of model enveloped and non-enveloped viruses by 4 to >6 and 5 to >8 log for the monoclonal and metal chelate columns, respectively. The effectiveness of the relatively mild column sanitisation conditions used, i.e. ethanol for factor IX and acetic acid for factor VIII, was confirmed using non-spiked column runs. The chemicals used contributed to virus elimination by inactivation and/or by physical removal of the virus. In summary, these studies demonstrate that potential virus contamination between chromatographic runs can be prevented when an effective column recycling and sanitisation procedure is included. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Development of a 3-step straight-through purification strategy combining membrane adsorbers and resins.

PubMed

Hughson, Michael D; Cruz, Thayana A; Carvalho, Rimenys J; Castilho, Leda R

2017-07-01

The pressures to efficiently produce complex biopharmaceuticals at reduced costs are driving the development of novel techniques, such as in downstream processing with straight-through processing (STP). This method involves directly and sequentially purifying a particular target with minimal holding steps. This work developed and compared six different 3-step STP strategies, combining membrane adsorbers, monoliths, and resins, to purify a large, complex, and labile glycoprotein from Chinese hamster ovary cell culture supernatant. The best performing pathway was cation exchange chromatography to hydrophobic interaction chromatography to affinity chromatography with an overall product recovery of up to 88% across the process and significant clearance of DNA and protein impurities. This work establishes a platform and considerations for the development of STP of biopharmaceutical products and highlights its suitability for integration with single-use technologies and continuous production methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:931-940, 2017. © 2017 American Institute of Chemical Engineers.

Copper(II)-based metal affinity chromatography for the isolation of the anticancer agent bleomycin from Streptomyces verticillus culture.

PubMed

Gu, Jiesi; Codd, Rachel

2012-10-01

The glycopeptide-based bleomycins are structurally complex natural products produced by Streptomyces verticillus used in combination therapy against testicular and other cancers. Bleomycin has a high affinity towards a range of transition metal ions with the 1:1 Fe(II) complex relevant to its mechanism of action in vivo and the 1:1 Cu(II) complex relevant to its production from culture. The affinity between Cu(II) and bleomycin was the underlying principle for using Cu(II)-based metal affinity chromatography in this work to selectively capture bleomycin from crude S. verticillus culture. A solution of standard bleomycin was retained at a binding capacity of 300 nmol mL(-1) on a 1-mL bed volume of Cu(II)-loaded iminodiacetate (IDA) resin at pH 9 via the formation of the heteroleptic immobilized complex [Cu(IDA)(bleomycin)]. Bleomycin was eluted from the resin at pH 5 as the metal-free ligand under conditions where pK(a) (IDA)

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

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

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

PubMed Central

DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.

2016-01-01

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

Isolation and purification of wheat germ agglutinin and analysis of its properties

NASA Astrophysics Data System (ADS)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Inhibition of initial adhesion of oral bacteria through a lectin from Bauhinia variegata L. var. variegata expressed in Escherichia coli.

PubMed

Klafke, G B; Borsuk, S; Gonçales, R A; Arruda, F V S; Carneiro, V A; Teixeira, E H; Coelho da Silva, A L; Cavada, B S; Dellagostin, O A; Pinto, L S

2013-11-01

The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a saliva-coated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation. © 2013 The Society for Applied Microbiology.

Recovery and purification process development for monoclonal antibody production

PubMed Central

Ma, Junfen; Winter, Charles; Bayer, Robert

2010-01-01

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

An affinity chromatography-gel filtration device for preparing thyroid microsomal antigen.

PubMed

Wang, L; Zheng, W F

1987-09-24

On the basis of conventional differential centrifugation for preparing crude thyroid microsomal antigen (TMAg), we have employed Sepharose 4B gel filtration and affinity chromatography separately to study the elution pattern in terms of absorbance and antigenic activity. The result indicates that thyroglobulin (TG) exists in two forms in crude TMAg, i.e., 'free TG' and 'membrane-bound TG'. TMAg is present in two forms in the eluate: (1) the TM fragment or TMAg polymer, which is produced at a higher rate and has greater antigenic activity, but which is less pure; (2) soluble TMAg, which is produced at a lower rate and has less antigenic activity, but which is more pure. We have developed an affinity chromatography-gel filtration (AC-GF) device which is a combination of affinity chromatography and a Sepharose 4B column. Sephadex G-50 is placed between the rubber stopper and Sepharose 4B in the GF column to ensure intactness of the entire system. With such a device, the AC removes the contaminated TG from TM homogenate, and allows the latter to pass directly from AC to GF for rechromatography. This device extracts the full advantages of both methods and each compensates for any deficiency of the other. Using this one-step procedure, one has the greatest chance of removing TG and obtaining TM fragments of TMAg polymers of higher antigenic activity, as well as separating small amounts of more purified soluble TMAg. Thus, the newly developed method meets the need of large quantities of TMAg for practical application, and at the same time the more purified preparations can be used for analytical purposes.

Dye-ligand affinity systems.

PubMed

Denizli, A; Pişkin, E

2001-10-30

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography.

PubMed

Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui

2012-11-01

Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

Purification, Characterization, and Sensitivity to Pesticides of Carboxylesterase From Dendrolimus superans (Lepidoptera: Lasiocampidae)

PubMed Central

Zou, Chuan-shan; Cao, Chuan-wang; Zhang, Guo-cai; Wang, Zhi-ying

2014-01-01

Abstract Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 μmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (β-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to β-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or β-NA as substrate. PMID:25525114

Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

PubMed Central

Rodrigo, Lourdes; Gil, Fernando; Hernandez, Antonio F; Lopez, Olga; Pla, Antonio

2003-01-01

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability. PMID:12946270

Selection of imprinted nanoparticles by affinity chromatography.

PubMed

Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

2009-04-15

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

Enhanced production and immunological characterization of recombinant West Nile virus envelope domain III protein.

PubMed

Tripathi, Nagesh K; Karothia, Divyanshi; Shrivastava, Ambuj; Banger, Swati; Kumar, Jyoti S

2018-05-13

West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation. Wet biomass obtained after batch and fed-batch cultivation processes was 11.2 g and 84 g/L of culture respectively. Protein yield after affinity purification was 5.76 mg and 5.81 mg/g wet cell weight after batch and fed-batch processes respectively. The purified WNV EDIII elicited specific antibodies in rabbits, confirming its immunogenicity. Moreover, the antibodies were able to neutralize WNV in vitro. These results established that the refolded and purified WNV EDIII could be a potential vaccine candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

PubMed

Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

2001-10-30

The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular methods for estimating the parameters that characterize the mass-transfer and adsorption mechanisms in affinity systems are described.

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

PubMed

Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

2011-08-01

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. Copyright © 2011 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2017-01-01

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

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

Expression and purification of mouse peptide ESP4 in Escherichia coli.

PubMed

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

2014-04-01

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

Modeling on-column reduction of trisulfide bonds in monoclonal antibodies during protein A chromatography.

PubMed

Ghose, Sanchayita; Rajshekaran, Rupshika; Labanca, Marisa; Conley, Lynn

2017-01-06

Trisulfides can be a common post-translational modification in many recombinant monoclonal antibodies. These are a source of product heterogeneity that add to the complexity of product characterization and hence, need to be reduced for consistent product quality. Trisulfide bonds can be converted to the regular disulfide bonds by incorporating a novel cysteine wash step during Protein A affinity chromatography. An empirical model is developed for this on-column reduction reaction to compare the reaction rates as a function of typical operating parameters such as temperature, cysteine concentration, reaction time and starting level of trisulfides. The model presented here is anticipated to assist in the development of optimal wash conditions for the Protein A step to effectively reduce trisulfides to desired levels. Copyright © 2016 Elsevier B.V. All rights reserved.

Selective retention of basic compounds by metal aquo-ion affinity chromatography.

PubMed

Asakawa, Yoshiki; Yamamoto, Eiichi; Asakawa, Naoki

2014-10-01

A novel metal aquo-ion affinity chromatography has been developed for the analysis of basic compounds using heat-treated silica gel containing hydrated metal cations (metal aquo-ions) as the packing material. The packing materials of the metal aquo-ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo-ions to present cation-exchange ability for basic analytes and the cation-exchange ability for basic analytes depends on pKa of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo-ion affinity chromatography, the on-line solid-phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo-ion affinity chromatography for basic analytes with sufficient capacity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.

PubMed

Alba, Annia; Marcet, Ricardo; Otero, Oscar; Hernández, Hilda M; Figueredo, Mabel; Sarracent, Jorge

2016-02-01

Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.

A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline.

PubMed

Gao, Shujuan; von Schumann, Gerald; Stöckigt, Joachim

2002-10-01

A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

Simple method to assess stability of immobilized peptide ligands against proteases.

PubMed

Giudicessi, Silvana L; Salum, María L; Saavedra, Soledad L; Martínez-Ceron, María C; Cascone, Osvaldo; Erra-Balsells, Rosa; Camperi, Silvia A

2017-09-01

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin-bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide-ChemMatrix resin. Peptidyl-resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C-terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

High yield of recombinant human Apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatography.

PubMed

Narasimhan Janakiraman, Vignesh; Noubhani, Abdelmajid; Venkataraman, Krishnan; Vijayalakshmi, Mookambeswaran; Santarelli, Xavier

2016-01-01

A vast majority of the cardioprotective properties exhibited by High-Density Lipoprotein (HDL) is mediated by its major protein component Apolipoprotein A-I (ApoA1). In order to develop a simplified bioprocess for producing recombinant human Apolipoprotein A-I (rhApoA1) in its near-native form, rhApoA1was expressed without the use of an affinity tag in view of its potential therapeutic applications. Expressed in Pichia pastoris at expression levels of 58.2 mg ApoA1 per litre of culture in a reproducible manner, the target protein was purified by mixed-mode chromatography using Capto™ MMC ligand with a purity and recovery of 84% and 68%, respectively. ApoA1 purification was scaled up to Mixed-mode Expanded Bed Adsorption chromatography to establish an 'on-line' process for the efficient capture of rhApoA1 directly from the P. pastoris expression broth. A polishing step using anion exchange chromatography enabled the recovery of ApoA1 up to 96% purity. Purified ApoA1 was identified and verified by RPLC-ESI-Q-TOF mass spectrometry. This two-step process would reduce processing times and therefore costs in comparison to the twelve-step procedure currently used for recovering rhApoA1 from P. pastoris. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

PubMed Central

Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

2011-01-01

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

Affinity monolith chromatography: A review of general principles and applications.

PubMed

Li, Zhao; Rodriguez, Elliott; Azaria, Shiden; Pekarek, Allegra; Hage, David S

2017-11-01

Affinity monolith chromatography, or AMC, is a liquid chromatographic method in which the support is a monolith and the stationary phase is a biological-binding agent or related mimic. AMC has become popular for the isolation of biochemicals, for the measurement of various analytes, and for studying biological interactions. This review will examine the principles and applications of AMC. The materials that have been used to prepare AMC columns will be discussed, which have included various organic polymers, silica, agarose, and cryogels. Immobilization schemes that have been used in AMC will also be considered. Various binding agents and applications that have been reported for AMC will then be described. These applications will include the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, dye-ligand affinity chromatography, and immobilized metal-ion affinity chromatography. The use of AMC with chiral stationary phases and as a tool to characterize biological interactions will also be examined. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical theory of chromatography: new outlooks for affinity chromatography.

PubMed Central

Denizot, F C; Delaage, M A

1975-01-01

We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods. PMID:1061072

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

PubMed

Tao, Pingyang; Poddar, Saumen; Sun, Zuchen; Hage, David S; Chen, Jianzhong

2018-02-02

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets. Copyright © 2018 Elsevier Inc. All rights reserved.

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

PubMed Central

Man-Kupisinska, Aleksandra; Michalski, Mateusz; Maciejewska, Anna; Swierzko, Anna S.; Cedzynski, Maciej; Lugowski, Czeslaw; Lukasiewicz, Jolanta

2016-01-01

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3. PMID:27232184

The development of a purification procedure for saxitoxin-induced protein.

PubMed

Smith, D S; Kitts, D D; Fenske, B; Owen, T G; Shyng, S

1995-02-01

A simple economical procedure for purifying saxitoxin-induced protein (SIP) from crude extracts of the small shore crab, Hemigrapsus oregenesis, was developed. (NH4)2SO4 precipitation, chymotrypsin digestion, heat treatment, gel filtration and ion-exchange-chromatography procedures were evaluated in purifying SIP. An enzyme immunoassay was used to determine the SIP yield and relative purity at each step of three procedures, thus permitting an assessment of the conditions required for maximum recovery. Response surface analysis was used in an attempt to determine the optimum temperature and exposure time for the heat treatment. A 20 min incubation at 65 degrees C was confirmed by electrophoretic analysis to be the best combination of time and temperature for achieving both an acceptable yield and purity of SIP. SIP in desalted concentrate was shown to be resistant to chymotrypsin proteolysis; however, this enzyme had deleterious effects on SIP purification at later stages of the procedure. The omission of the chymotrypsin digestion, and the inclusion of gel-filtration chromatography in the final clean-up step, resulted in the purification of SIP comparable with that achieved with affinity chromatography.

Cholera Toxin Inhibitors Studied with High-Performance Liquid Affinity Chromatography: A Robust Method to Evaluate Receptor–Ligand Interactions

PubMed Central

Bergström, Maria; Liu, Shuang; Kiick, Kristi L.; Ohlson, Sten

2009-01-01

Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The KD values of galactose and meta-nitrophenyl α-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC50 values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but KD values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct KD values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis. PMID:19152642

Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

PubMed Central

KASAI, Kenichi

2014-01-01

Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

Affinity monolith chromatography: A review of principles and recent analytical applications

PubMed Central

Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

2012-01-01

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

PubMed Central

Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

2016-01-01

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation.

PubMed

Hammerschmidt, Nikolaus; Tscheliessnig, Anne; Sommer, Ralf; Helk, Bernhard; Jungbauer, Alois

2014-06-01

Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra-/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography-based antibody standard purification; (ii) the continuous precipitation-based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation-based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small-scale production in parallel fashion, which is of importance for antibody production in non-privileged countries and for personalized medicine. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

PubMed

Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Svensson, Susanne; Ohlson, Sten; Isaksson, Roland

2013-07-01

In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have a major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

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

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

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

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

PubMed

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

2017-08-01

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

The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

ERIC Educational Resources Information Center

Landman, A. D.; Landman, N. N.

1976-01-01

Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

PubMed Central

Hage, David S.

2017-01-01

BACKGROUND The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically-related binding agent, are two methods that can be used to study these interactions. CONTENT This review looks at various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. SUMMARY The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. PMID:28396561

One-step purification of assembly-competent tubulin from diverse eukaryotic sources

PubMed Central

Widlund, Per O.; Podolski, Marija; Reber, Simone; Alper, Joshua; Storch, Marko; Hyman, Anthony A.; Howard, Jonathon; Drechsel, David N.

2012-01-01

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. PMID:22993214

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

Zhang, Qibin; Tang, Ning; Brock, Jonathan W.

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resultedmore » in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.« less

Chromatographic removal combined with heat, acid and chaotropic inactivation of four model viruses.

PubMed

Valdés, R; Ibarra, Neysi; Ruibal, I; Beldarraín, A; Noa, E; Herrera, N; Alemán, R; Padilla, S; Garcia, J; Pérez, M; Morales, R; Chong, E; Reyes, B; Quiñones, Y; Agraz, A; Herrera, L

2002-07-03

The virus removal of protein A affinity chromatography, inactivation capacity, acid pH and a combination of high temperature with a chaotropic agent was determined in this work. The model viruses studied were sendaivirus, human immunodeficency virus (HIV-IIIb), human poliovirus type-II, human herpesvirus I and canine parvovirus. The protein A affinity chromatography showed a maximum reduction factor of 8 logs in the case of viruses larger than 120 nm size, while for small viruses (18-30 nm) the maximum reduction factor was about 5 logs. Non viral inactivation was observed during the monoclonal antibody elution step. Low pH treatment showed a maximum inactivation factor of 7.1 logs for enveloped viruses. However, a weak inactivation factor (3.4 logs) was obtained for DNA nonenveloped viruses. The combination of high temperature with 3 M KSCN showed a high inactivation factor for all of the viruses studied. The total clearance factor was 23.1, 15.1, 13.6, 20.0 and 16.0 logs for sendaivirus, HIV-IIIb, human poliovirus type-II, human herpesvirus I and canine parvovirus, respectively.

Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells.

PubMed

Rossi, Edmund A; Chang, Chien-Hsing; Losman, Michele J; Sharkey, Robert M; Karacay, Habibe; McBride, William; Cardillo, Thomas M; Hansen, Hans J; Qu, Zhengxing; Horak, Ivan D; Goldenberg, David M

2005-10-01

To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors. hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC. The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor-bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with (111)In. Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumor-to-nontumor ratios as early as 3 hours postinjection. The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.

Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

PubMed

Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

2017-01-01

In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

Pathogenesis-Related Proteins of Tomato 1

PubMed Central

Vera, Pablo; Conejero, Vicente

1988-01-01

An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv “Rutgers”) leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH4)2SO4 fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pI around 9.0. Images Fig. 4 Fig. 8 PMID:16666127

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin.

PubMed

Sanz, J M; Lopez, R; Garcia, J L

1988-05-23

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

Comprehensive proteomic analysis of the human spliceosome

NASA Astrophysics Data System (ADS)

Zhou, Zhaolan; Licklider, Lawrence J.; Gygi, Steven P.; Reed, Robin

2002-09-01

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome `core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify ~145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

PubMed

Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

2014-01-01

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers.

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

PubMed Central

Lalwani, N D; Alvares, K; Reddy, M K; Reddy, M N; Parikh, I; Reddy, J K

1987-01-01

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response. Images PMID:3474650

Affinity chromatography: A versatile technique for antibody purification.

PubMed

Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

2017-03-01

Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Biochemical quality of the pharmaceutically licensed plasma OctaplasLG after implementation of a novel prion protein (PrPSc) removal technology and reduction of the solvent/detergent (S/D) process time.

PubMed

Heger, A; Svae, T-E; Neisser-Svae, A; Jordan, S; Behizad, M; Römisch, J

2009-10-01

A new chromatographic step for the selective binding of pathological prion proteins (PrP(Sc)) to an affinity ligand, developed and optimized for PrP(Sc) capture and attached to synthetic resin particles (PRDT, USA; ProMetic BioSciences Ltd, Isle of Man, UK) was implemented into the manufacturing process of the solvent/detergent (S/D) treated biopharmaceutical quality plasma Octaplas. Pilot batches of Octaplas with the implemented chromatographic step [labelled as OctaplasLG (ligand gel)] were manufactured by Octapharma PPGmbH, Vienna, Austria. The biochemical quality was compared directly after manufacturing as well as after 18 months storage. All samples were tested on global coagulation parameters, fibrinogen levels, activities of coagulation factors and protease inhibitors, ADAMTS13 levels, as well as markers of activated coagulation and fibrinolysis. In addition, von Willebrand factor multimeric analysis was performed. The incorporation of this novel chromatography into the large-scale routine manufacturing process was shown to be technically feasible and the performance of the column was assessed to be excellent. The biochemical studies showed that Octaplas and OctaplasLG produced without and with the new column, respectively, demonstrate an identical biochemical quality. OctaplasLG remained stable over a period of 18 months stored frozen. A parallel reduction of the S/D virus inactivation step from 4-4.5 to 1-1.5 h led to significantly higher activities of plasmin inhibitor. The studies confirmed that the affinity ligand chromatography under the developed conditions can be introduced into the Octaplas manufacturing process, as a mean to reduce potentially present PrP(Sc), without hampering the proven quality of this product.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Bacillus Collagen Like Protein of Anthracis: Immunological and Functional Analyses

DTIC Science & Technology

2007-09-21

heated at 65°C for 30 minutes, diluted, and plated on trypticase soy agar (TSA) to obtain viable counts. Since heat treatment kills the vegetative...purification of that protein by nickel-affinity chromatography are also described in detail elsewhere (Brahmbhatt T.N, lK. Janes, E.S. Stibitz, S.C...Trap Nickel affinity column chromatography with the Fast Phase Liquid Chromatography (FPLC) AKTA system (GE Healthcare, Piscataway, NJ). Rabbit anti

Evaluation of Quantitative Performance of Sequential Immobilized Metal Affinity Chromatographic Enrichment for Phosphopeptides

PubMed Central

Sun, Zeyu; Hamilton, Karyn L.; Reardon, Kenneth F.

2014-01-01

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium-dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. While our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semi-quantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. PMID:24096195

Applications of alginate in bioseparation of proteins.

PubMed

Jain, Sulakshana; Mondal, Kalyani; Gupta, Munishwar N

2006-01-01

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, a and ss amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

Purification of bacteriophage lambda repressor

PubMed Central

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

2013-01-01

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

Design and scaleup of downstream processing of monoclonal antibodies for cancer therapy: from research to clinical proof of principle.

PubMed

Horenstein, Alberto L; Crivellin, Federico; Funaro, Ada; Said, Marcela; Malavasi, Fabio

2003-04-01

Murine monoclonal antibodies (mAb) from cell culture supernatants have been purified in order to acquire clinical grade for in vivo cancer treatment. The starting material was purified by high performance liquid chromatography (HPLC) systems ranging from the analytical scale process to a scaleup to 1 g per batch. Three columns (Protein A affinity chromatography with single-step elution, hydroxyapatite (HA) chromatography followed by linear gradient elution and endotoxin removing-gel chromatography), exploiting different properties of the mAb were applied. The final batches of antibody were subjected to a large panel of tests for the purpose of evaluating the efficacy of the downstream processing. The resulting data have allowed us to determine the maximum number of times the column can be used and to precisely and thoroughly characterize antibody integrity, specificity, and potency according to in-house reference standards. The optimized bioprocessing is rapid, efficient, and reproducible. Not less importantly, all the techniques applied are characterized by costs which are affordable to medium-sized laboratories. They represent the basis for implementing immunotherapeutic protocols transferable to clinical medicine.

Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis.

PubMed

Mahn, Andrea; Ismail, Maritza

2011-11-15

Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE. Copyright © 2011 Elsevier B.V. All rights reserved.

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

PubMed

Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

2016-01-01

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

Solubilization and purification of melatonin receptors from lizard brain

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

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Bromelain: from production to commercialisation.

PubMed

Ramli, Aizi Nor Mazila; Aznan, Tuan Norsyalieza Tuan; Illias, Rosli Md

2017-03-01

Bromelain is a mixture of proteolytic enzymes found in pineapple (Ananas comosus) plants. It can be found in several parts of the pineapple plant, including the stem, fruit, leaves and peel. High demand for bromelain has resulted in gradual increases in bromelain production. These increases have led to the need for a bromelain production strategy that yields more purified bromelain at a lower cost and with fewer production steps. Previously, bromelain was purified by conventional centrifugation, ultrafiltration and lyophilisation. Recently, the development of more modern purification techniques such as gel filtration, ion exchange chromatography, affinity chromatography, aqueous two-phase extraction and reverse micelle chromatography has resulted in increased industrial bromelain production worldwide. In addition, recombinant DNA technology has emerged as an alternative strategy for producing large amounts of ultrapure bromelain. An up-to-date compilation of data regarding the commercialisation of bromelain in the clinical, pharmaceutical and industrial fields is provided in this review. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

PubMed

Moureaux, T; Leydecker, M T; Meyer, C

1989-02-15

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

PubMed

Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin

2017-04-01

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

Protein purification by aminosquarylium cyanine dye-affinity chromatography.

PubMed

Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

2013-12-01

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.

Characterization of Extracellular Proteins in Tomato Fruit using Lectin Affinity Chromatography and LC-MALDI-MS/MS analysis

USDA-ARS?s Scientific Manuscript database

The large-scale isolation and analysis of glycoproteins by lectin affinity chromatography coupled with mass spectrometry has become a powerful tool to monitor changes in the “glycoproteome” of mammalian cells. Thus far, however, this approach has not been used extensively for the analysis of plant g...

Production of a pokeweed antiviral protein (PAP)-containing immunotoxin, B43-PAP, directed against the CD19 human B lineage lymphoid differentiation antigen in highly purified form for human clinical trials.

PubMed

Myers, D E; Irvin, J D; Smith, R S; Kuebelbeck, V M; Uckun, F M

1991-02-15

We describe a standardized method for the preparation and purification of a potent immunotoxin against B-lineage leukemia/lymphoma cells, constructed with the ribosome inhibitory single chain plant toxin pokeweed antiviral protein (PAP) and a murine IgG1 monoclonal antibody (MoAb) specific for the human B lineage differentiation antigen CD19 for human clinical trials. PAP was prepared from spring leaves of Phytolacca americana plants by ammonium sulfate precipitation and purified to homogeneity by successive steps of ion exchange chromatography. B43 MoAb was produced in vitro by hollow fiber technology and purified to homogeneity by affinity chromatography. PAP toxin and B43 MoAb were modified via their free amino groups prior to their intermolecular conjugation. 2-iminothiolane was used to introduce reactive sulfhydryl groups into PAP and N-succinimidyl 3-(2-pyridyldithio) propionate was used to introduce 2-pyridyl disulfide bonds into B43 MoAb. Modified PAP was reacted with modified B43 MoAb resulting in a sulfhydryl-disulfide exchange reaction and yielding disulfide linked PAP-B43 MoAb conjugates, which we refer to as B43-PAP immunotoxin. B43-PAP immunotoxin was subjected to preparative gel filtration chromatography and cation exchange chromatography to obtain a highly purified, sterile, and pyrogen-free immunotoxin preparation with less than 5% free antibody contamination and less than 0.5% free PAP contamination. The final product displayed a high affinity for and a very potent anti-leukemic activity against B lineage leukemia cells. With slight modifications, the procedures detailed in this report should be generally applicable to preparation of other PAP-MoAb conjugates for treatment of cancer or AIDS.

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli.

PubMed

Delli-Bovi, Teegan A; Spalding, Maroya D; Prigge, Sean T

2010-10-11

Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

Affinity Chromatography in Nonionic Detergent Solutions

NASA Astrophysics Data System (ADS)

Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

1980-10-01

Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

PubMed

Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

1994-04-07

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid.

PubMed

Piletska, Elena V; Villoslada, Fernando Navarro; Chianella, Iva; Bossi, Alessandra; Karim, Kal; Whitcombe, Michael J; Piletsky, Sergey A; Doucette, Gregory J; Ramsdell, John S

2008-03-03

A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

PubMed

Yamada, H; Fukumura, T; Ito, Y; Imoto, T

1985-04-01

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

PubMed

Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

2010-12-10

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-02-01

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

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

PubMed

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

2013-11-14

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2004-08-01

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

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

PubMed Central

Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

2013-01-01

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O-GlcNAc)-modified counterparts. Solid-phase enzymatic dephosphorylation proved to be a viable tool to condition O-GlcNAcylated peptide in mixtures with phosphopeptides for selective affinity purification. Acetylation, as an integral step of the sample-preparation method, precluded reduction in recovery of the thiolation substrate caused by intrapeptide lysine-dehydroalanine cross-link formation. The solid-phase analytical platform provides robustness and simplicity of operation using equipment readily available in most biological laboratories and is expected to accommodate additional chemistries to expand the scope of solid-phase serial derivatization for protein structural characterization. PMID:23997662

Comparing Multi-Step IMAC and Multi-Step TiO2 Methods for Phosphopeptide Enrichment

PubMed Central

Yue, Xiaoshan; Schunter, Alissa; Hummon, Amanda B.

2016-01-01

Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multi-step enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multi-phosphopeptides, as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment, or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multi-step enrichment. PMID:26237447

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

PubMed Central

Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

2014-01-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation.

PubMed

Savary, B J

2001-08-01

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

PubMed Central

Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PubMed

Walkup, Ward G; Kennedy, Mary B

2014-06-01

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

PubMed Central

de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra

2016-01-01

Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

Affinity purification and mass spectrometry: an attractive choice to investigate protein-protein interactions in plant immunity

USDA-ARS?s Scientific Manuscript database

Affinity purification of protein complexes from biological tissues, followed by liquid chromatography- tandem mass spectrometry (AP-MS/MS), has ballooned in recent years due to sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, improvements in affinity purifica...

Affinity Proteomics for Fast, Sensitive, Quantitative Analysis of Proteins in Plasma.

PubMed

O'Grady, John P; Meyer, Kevin W; Poe, Derrick N

2017-01-01

The improving efficacy of many biological therapeutics and identification of low-level biomarkers are driving the analytical proteomics community to deal with extremely high levels of sample complexity relative to their analytes. Many protein quantitation and biomarker validation procedures utilize an immunoaffinity enrichment step to purify the sample and maximize the sensitivity of the corresponding liquid chromatography tandem mass spectrometry measurements. In order to generate surrogate peptides with better mass spectrometric properties, protein enrichment is followed by a proteolytic cleavage step. This is often a time-consuming multistep process. Presented here is a workflow which enables rapid protein enrichment and proteolytic cleavage to be performed in a single, easy-to-use reactor. Using this strategy Klotho, a low-abundance biomarker found in plasma, can be accurately quantitated using a protocol that takes under 5 h from start to finish.

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography.

PubMed

Saavedra, Soledad L; Martínez Ceron, María C; Giudicessi, Silvana L; Forno, Guillermina; Bosco, María Belén; Marani, Mariela M; Erra-Balsells, Rosa; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

2018-04-25

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling*

PubMed Central

Kennedy, Jacob J.; Yan, Ping; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Moore, Heather D.; Lin, Chenwei; Pogosova-Agadjanyan, Era L.; Stirewalt, Derek L.; Reding, Kerryn W.; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

2016-01-01

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

An overview of the kinetic parameters of class B beta-lactamases.

PubMed Central

Felici, A; Amicosante, G; Oratore, A; Strom, R; Ledent, P; Joris, B; Fanuel, L; Frère, J M

1993-01-01

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase'. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column. Images Figure 1 PMID:8471035

Approaches to High-Performance Preparative Chromatography of Proteins

NASA Astrophysics Data System (ADS)

Sun, Yan; Liu, Fu-Feng; Shi, Qing-Hong

Preparative liquid chromatography is widely used for the purification of chemical and biological substances. Different from high-performance liquid chromatography for the analysis of many different components at minimized sample loading, high-performance preparative chromatography is of much larger scale and should be of high resolution and high capacity at high operation speed and low to moderate pressure drop. There are various approaches to this end. For biochemical engineers, the traditional way is to model and optimize a purification process to make it exert its maximum capability. For high-performance separations, however, we need to improve chromatographic technology itself. We herein discuss four approaches in this review, mainly based on the recent studies in our group. The first is the development of high-performance matrices, because packing material is the central component of chromatography. Progress in the fabrication of superporous materials in both beaded and monolithic forms are reviewed. The second topic is the discovery and design of affinity ligands for proteins. In most chromatographic methods, proteins are separated based on their interactions with the ligands attached to the surface of porous media. A target-specific ligand can offer selective purification of desired proteins. Third, electrochromatography is discussed. An electric field applied to a chromatographic column can induce additional separation mechanisms besides chromatography, and result in electrokinetic transport of protein molecules and/or the fluid inside pores, thus leading to high-performance separations. Finally, expanded-bed adsorption is described for process integration to reduce separation steps and process time.

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

PubMed

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

2017-04-01

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

Targeting Human Serum Fucome by an Integrated Liquid-phase Multi Column Platform Operating in “Cascade” to Facilitate Comparative Mass Spectrometric Analysis of Disease-Free and Breast Cancer Sera

PubMed Central

Selvaraju, Subhashini; Rassi, Ziad El

2013-01-01

A fully integrated platform was developed for capturing/fractionating human fucome from disease-free and breast cancer sera. It comprised multicolumn operated by HPLC pumps and switching valves for the simultaneous depletion of high abundance proteins via affinity-based subtraction and the capturing of fucosylated glycoproteins via lectin affinity chromatography followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) were utilized. The platform allowed the “cascading” of the serum sample from column-to-column in the liquid phase with no sample manipulation between the various steps. This guaranteed no sample loss and no propagation of experimental biases between the various columns. Finally, the fucome was fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to trypsinolysis for LC-MS/MS analysis. This permitted the identification of the differentially expressed proteins (DEP) in breast cancer serum yielding a broad panel of 35 DEP from the combined LTA and AAL captured proteins and a narrower panel of 8 DEP that were commonly differentially expressed in both LTA and AAL fractions, which are considered as more representative of cancer altered fucome. PMID:23533108

Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

PubMed

Nilsson, O Rickard; Lannergård, Jonas; Morgan, B Paul; Lindahl, Gunnar; Gustafsson, Mattias C U

2013-01-01

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

PubMed

Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

2017-06-23

The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

New process for purifying high purity α1-antitrypsin from Cohn Fraction IV by chromatography: A promising method for the better utilization of plasma.

PubMed

Huangfu, Chaoji; Zhang, Jinchao; Ma, Yuyuan; Jia, Junting; Lv, Maomin; Zhao, Xiong; Zhang, Jingang

2017-03-01

α1-antitrypsin (AAT) is a 52kDa serine protease inhibitor that is abundant in plasma. It is synthesized mainly by hepatic cells, and widely used to treat patients with emphysema due to congenital deficiency of AAT. A new isolation method for the purification of AAT from Cohn Fraction IV (Cohn F IV) is described. Cohn F IV is usually discarded as a byproduct from Cohn process. Using Cohn F IV as starting material does not interfere with the production of other plasma proteins and the cost of purification could be reduced greatly. Parameters of each step during purification were optimized, 15% polyethyleneglycol (PEG) concentration and pH 5.2 for PEG precipitation, elution with 0.05M sodium acetate and pH 4.7 for ion-exchange chromatography, and two steps blue sepharose affinity chromatography were chosen for AAT purification. The final protein with purity of 98.17%, specific activity of 3893.29 IU/mg, and yield of 28.35%, was achieved. Western blotting was applied for qualitative identification of final product, which specifically reacted with goat anti-human AAT antibody. LC-ESI-MS/MS was also employed to confirm the final protein. High performance liquid chromatography was used to analyze the composition of purified protein suggesting that pure protein was achieved. The molecular weight of AAT is 51062.77Da which was identified by LC-MS-MS. The manufacturing process described here may make better use of human plasma with Cohn F IV as starting material. The simple process described in this study is simple and inexpensive, it has a potential value for large scale production. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

Ryan, Daniel P; Tremethick, David J

2016-04-01

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

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

PubMed Central

Elliott, J; Blanchard, S G; Wu, W; Miller, J; Strader, C D; Hartig, P; Moore, H P; Racs, J; Raftery, M A

1980-01-01

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 7. PLATE 1 PMID:7387629

Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity.

PubMed

Panteleev, Pavel V; Ovchinnikova, Tatiana V

2017-01-01

Here, we report an efficient procedure for recombinant production and purification of tachyplesin I (THI) with a final yield of 17 mg/L of the culture medium. The peptide was expressed in Escherichia coli as a part of the thioredoxin fusion protein. With the use of soluble expression followed by immobilized metal-ion affinity chromatography, the recombinant protein cleavage and reversed-phase high-performance liquid chromatography, a yield of THI did not exceed 6.5 mg/L of the culture medium. Further optimization studies were carried out to improve the protein expression level and simplify purification procedure of the target peptide. To achieve better yield of the peptide, we used high-cell-density bacterial expression. The formed inclusion bodies were highly enriched with the fusion protein, which allowed us to perform direct chemical cleavage of the inclusion bodies solubilized in 6 M guanidine-HCl with subsequent selective precipitation of proteins with trifluoroacetic acid. This enabled us to avoid an extra step of purification by immobilized metal-ion affinity chromatography. The developed procedure has made it possible to obtain biologically active THI and was used for screening a number of its mutant analogs. As a result, several selective and nonhemolytic analogs were developed. Significant reduction in hemolytic activity without losing antimicrobial activity was achieved by substitution of tyrosine or isoleucine residue in the β-turn region of the molecule with hydrophilic serine. The present study affords further insight into molecular mechanism of antimicrobial action of tachyplesin and gains a better understanding of structure-activity relationships in its analogs. This is aimed at searching for novel antibiotics on the basis of antimicrobial peptides with reduced cytotoxicity. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

PubMed

Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

2015-03-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

PubMed

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

2006-04-13

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

Single-step affinity purification for fungal proteomics.

PubMed

Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

2010-05-01

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

The integrated simulation and assessment of the impacts of process change in biotherapeutic antibody production.

PubMed

Chhatre, Sunil; Jones, Carl; Francis, Richard; O'Donovan, Kieran; Titchener-Hooker, Nigel; Newcombe, Anthony; Keshavarz-Moore, Eli

2006-01-01

Growing commercial pressures in the pharmaceutical industry are establishing a need for robust computer simulations of whole bioprocesses to allow rapid prediction of the effects of changes made to manufacturing operations. This paper presents an integrated process simulation that models the cGMP manufacture of the FDA-approved biotherapeutic CroFab, an IgG fragment used to treat rattlesnake envenomation (Protherics U.K. Limited, Blaenwaun, Ffostrasol, Llandysul, Wales, U.K.). Initially, the product is isolated from ovine serum by precipitation and centrifugation, before enzymatic digestion of the IgG to produce FAB and FC fragments. These are purified by ion exchange and affinity chromatography to remove the FC and non-specific FAB fragments from the final venom-specific FAB product. The model was constructed in a discrete event simulation environment and used to determine the potential impact of a series of changes to the process, such as increasing the step efficiencies or volumes of chromatographic matrices, upon product yields and process times. The study indicated that the overall FAB yield was particularly sensitive to changes in the digestive and affinity chromatographic step efficiencies, which have a predicted 30% greater impact on process FAB yield than do the precipitation or centrifugation stages. The study showed that increasing the volume of affinity matrix has a negligible impact upon total process time. Although results such as these would require experimental verification within the physical constraints of the process and the facility, the model predictions are still useful in allowing rapid "what-if" scenario analysis of the likely impacts of process changes within such an integrated production process.

Separation of an associated 90K heat shock protein from the glucocorticoid receptor complex

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

Miller-Diener, A.; Kirsch, T.; Grove, B.

1986-05-01

A 90K heat shock protein(HSP), observed to copurify with the glucocorticoid receptor(GR), can be separated from the complex by 2 methods, allowing investigation of the role of HSP on kinase activity that was previously reported to be inherent to purified activated GR. Na/sub 2/MoO/sub 4/ stabilized unactivated rat hepatic GR complexes have been purified to >10,000-fold using a purification scheme that involves batchwise treatment of cytosol with phosphocellulose/DNAcellulose, elution from an affinity resin, gel filtration and ion exchange chromatography. Samples were subjected to 10-20% gradient SDS-PAGE. Proteins were transferred to nitrocellulose and blotted against monoclonal antibodies to GR(3A6), HSP ormore » nonspecific IgM/G. Immunoblots indicated that HSP was separated from unactivated GR complexes at the affinity step prior to elution of GR with active steroid. GR eluted from the resin with /sup 3/H Triamcinolone acetonide or /sup 3/H Dexamethasone mesylate had an apparent M/sub r/ = 94-96,000 for the steroid binding subunit and is recognized by 3A6. Purification of GR minus the affinity step resulted in copurification of HSP throughout the procedure. However, after Sephadex G75 filtration and subsequent incubation at 25/sup 0/C, 30 min., HSP was separated from activated (DNA binding) GR on DEAE cellulose-52. HSP did not enhance or inhibit /sup 32/P incorporation of the 94K steroid binding subunit nor did it affect phosphorylation of histones by GR.« less

Plasma fibronectin: three steps to purification and stability.

PubMed

Poulouin, L; Gallet, O; Rouahi, M; Imhoff, J M

1999-10-01

Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing condition. No proteolytic activities were detected by zymography at acidic or neutral pH. Furthermore, the fibronectin preparation was stable over time up to 456 h at 37 degrees C in the presence of calcium, zinc, or mercury. This preparation of very stable fibronectin, called highly purified fibronectin (hpFN), gave a yield of 7.00 +/- 0.77 mg of fibronectin per gram of cryoprecipitated plasma and 0.16 mg of fibronectin per milliliter of fresh citrated, giving a yield of 32 to 53% (from presumed fibronectin concentration). This preparation may be useful for cellular tests and interaction analysis. Copyright 1999 Academic Press.

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

PubMed

Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

1995-07-01

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography.

PubMed

Ghose, Sanchayita; Nagrath, Deepak; Hubbard, Brian; Brooks, Clayton; Cramer, Steven M

2004-01-01

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.

A simplified methylcoenzyme M methylreductase assay with artificial electron donors and different preparations of component C from Methanobacterium thermoautotrophicum delta H.

PubMed Central

Hartzell, P L; Escalante-Semerena, J C; Bobik, T A; Wolfe, R S

1988-01-01

Different preparations of the methylreductase were tested in a simplified methylcoenzyme M methylreductase assay with artificial electron donors under a nitrogen atmosphere. ATP and Mg2+ stimulated the reaction. Tris(2,2'-bipyridine)ruthenium (II), chromous chloride, chromous acetate, titanium III citrate, 2,8-diaminoacridine, formamidinesulfinic acid, cob(I)alamin (B12s), and dithiothreitol were tested as electron donors; the most effective donor was titanium III citrate. Methylreductase (component C) was prepared by 80% ammonium sulfate precipitation, 70% ammonium sulfate precipitation, phenyl-Sepharose chromatography, Mono Q column chromatography, DEAE-cellulose column chromatography, or tetrahydromethanopterin affinity column chromatography. Methylreductase preparations which were able to catalyze methanogenesis in the simplified reaction mixture contained contaminating proteins. Homogeneous component C obtained from a tetrahydromethanopterin affinity column was not active in the simplified assay but was active in a methylreductase assay that contained additional protein components. Images PMID:3372480

Purification of human alpha uterine protein.

PubMed

Sutcliffe, R G; Bolton, A E; Sharp, F; Nicholson, L V; MacKinnon, R

1980-03-01

Human alpha uterine protein (AUP) has been prepared from extracts of decudua by antibody affinity chromatography, DEAE Sepharose chromatography and by filtration through Sephadex G-150. This procedure yielded a protein fraction containing AUP, which was labelled with 125I by chloramine T. When analysed by SDS gel electrophoresis this radioiodinated protein fraction was found to contain predominantly a single species of protein which was precipitated by antibodies against AUP in antibody-antigen crossed electrophoresis. Rabbit anti-AUP precipitated 55-65% of the tracer in a double-antibody system. Sephadex G150 gel filtration of AUP obtained before and after affinity chromatography provided a molecular weight estimate of 50000. Since SDS gel electrophoresis revealed a polypeptide molecular weight of 23000-25000, it is suggested that AUP is a dimer.

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

Method of identity analyte-binding peptides

DOEpatents

Kauvar, Lawrence M.

1990-01-01

A method for affinity chromatography or adsorption of a designated analyte utilizes a paralog as the affinity partner. The immobilized paralog can be used in purification or analysis of the analyte; the paralog can also be used as a substitute for antibody in an immunoassay. The paralog is identified by screening candidate peptide sequences of 4-20 amino acids for specific affinity to the analyte.

Cellular Targets of Dietary Polyphenol Resveratrol

DTIC Science & Technology

2005-03-01

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

New exopolysaccharides produced by Aureobasidium pullulans grown on glucosamine.

PubMed

Cescutti, Paola; Pupulin, Raffaella; Delben, Franco; Abbate, Maria; Dentini, Mariella; Sparapano, Lorenzo; Rizzo, Roberto; Crescenzi, Vittorio

2002-07-16

The polysaccharides produced by Aureobasidium pullulans, grown using glucosamine as the carbon source, were investigated by means of methylation analysis, affinity chromatography and NMR spectroscopy. The results indicated that, besides a small amount of pullulan, this micro-organism was capable of producing-in low yields-mixtures of at least two different complex polysaccharides containing mainly mannose and galactose. (1)H NMR spectra of two fractions obtained by lectin affinity chromatography indicated that one polymer was constituted exclusively of mannose residues while the other contained both galactofuranosyl and mannopyranosyl residues.

Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

PubMed

Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

2009-07-01

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

PubMed

Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

2015-11-01

Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

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

Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.

A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was appliedmore » to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.« less

Some parameters relevant to affinity chromatography on immobilized nucleotides

PubMed Central

Lowe, C. R.; Harvey, M. J.; Craven, D. B.; Dean, P. D. G.

1973-01-01

1. The suitability of cellulose and Sepharose as supports for affinity chromatography of two groups of cofactor-linked enzymes, dehydrogenases and kinases, was examined. Sepharose was found to be superior. 2. The selective capacities of the columns were measured by frontal analysis and are discussed in relation to the nucleotide contents. 3. The effect of various concentrations of enzyme and of non-specific protein on the performance of the affinity columns, and the effects of equilibration time, flow rate, sample volume and dilution of the nucleotide were examined. 4. The effect of interposing polymethylene and polyglycine extension arms between the matrix backbone and the nucleotide was investigated for several cofactor-dependent enzymes. Maximum binding was observed with an extension arm 0.8–1nm long. PMID:4354739

Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes.

PubMed

Matoba, Nobuyuki; Griffin, Tagan A; Mittman, Michele; Doran, Jeffrey D; Alfsen, Annette; Montefiori, David C; Hanson, Carl V; Bomsel, Morgane; Mor, Tsafrir S

2008-05-01

CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

PubMed

Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

2016-09-01

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Method of identity analyte-binding peptides

DOEpatents

Kauvar, L.M.

1990-10-16

A method for affinity chromatography or adsorption of a designated analyte utilizes a paralog as the affinity partner. The immobilized paralog can be used in purification or analysis of the analyte; the paralog can also be used as a substitute for antibody in an immunoassay. The paralog is identified by screening candidate peptide sequences of 4--20 amino acids for specific affinity to the analyte. 5 figs.

Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

PubMed

Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

2015-01-01

A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

PubMed Central

Gabe, Claire M.; Brookes, Steven J.; Kirkham, Jennifer

2017-01-01

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond. PMID:28670287

Affinity approaches in RNAi-based therapeutics purification.

PubMed

Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

2016-05-15

The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

PubMed

Horejsí, V; Tichá, M; Kocourek, J

1977-09-29

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.

PubMed

Boesch, Austin W; Kappel, James H; Mahan, Alison E; Chu, Thach H; Crowley, Andrew R; Osei-Owusu, Nana Y; Alter, Galit; Ackerman, Margaret E

2018-05-01

As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques. © 2018 Wiley Periodicals, Inc.

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

PubMed Central

Chung, Wai Keen; Freed, Alexander S.; Holstein, Melissa A.; McCallum, Scott A.; Cramer, Steven M.

2010-01-01

NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems. PMID:20837551

Protein complex purification from Thermoplasma acidophilum using a phage display library.

PubMed

Hubert, Agnes; Mitani, Yasuo; Tamura, Tomohiro; Boicu, Marius; Nagy, István

2014-03-01

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. Copyright © 2013 Elsevier B.V. All rights reserved.

Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5.

PubMed

Kim, M J; Park, W S; Lee, H S; Kim, T J; Shin, J H; Yoo, S H; Cheong, T K; Ryu, S; Kim, J C; Kim, J W; Moon, T W; Robyt, J F; Park, K H

2000-01-01

The cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 (CDase I-5) was expressed in Escherichia coli and the purified enzyme was used for characterization of the enzyme action. The hydrolysis products were monitored by both HPLC and high-performance ion chromatography analysis that enable the kinetic analysis of the cyclomaltodextrin (CD)-degrading reaction. Analysis of the kinetics of cyclomaltodextrin hydrolysis by CDase I-5 indicated that ring-opening of the cyclomaltodextrin was the major limiting step and that CDase I-5 preferentially degraded the linear maltodextrin chain by removing the maltose unit. The substrate binding affinity of the enzyme was almost same for those of cyclomaltodextrins while the rate of ring-opening was the fastest for cyclomaltoheptaose. Acarbose and methyl 6-amino-6-deoxy-alpha-d-glucopyranoside were relatively strong competitive inhibitors with K(i) values of 1.24 x 10(-3) and 8.44 x 10(-1) mM, respectively. Both inhibitors are likely to inhibit the ring-opening step of the CD degradation reaction. Copyright 2000 Academic Press.

An improved affine projection algorithm for active noise cancellation

NASA Astrophysics Data System (ADS)

Zhang, Congyan; Wang, Mingjiang; Han, Yufei; Sun, Yunzhuo

2017-08-01

Affine projection algorithm is a signal reuse algorithm, and it has a good convergence rate compared to other traditional adaptive filtering algorithm. There are two factors that affect the performance of the algorithm, which are step factor and the projection length. In the paper, we propose a new variable step size affine projection algorithm (VSS-APA). It dynamically changes the step size according to certain rules, so that it can get smaller steady-state error and faster convergence speed. Simulation results can prove that its performance is superior to the traditional affine projection algorithm and in the active noise control (ANC) applications, the new algorithm can get very good results.

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-12-01

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2', 5'-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, KM and Vmax values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC50 values in the range of 0.07-30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.

A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model

USDA-ARS?s Scientific Manuscript database

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with differ...

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

PubMed

Serpa, Gisele; Augusto, Elisabeth Fátima Pires; Tamashiro, Wirla Maria Silva Cunha; Ribeiro, Mariana Borçoe; Miranda, Everson Alves; Bueno, Sônia Maria Alves

2005-02-25

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Functional characterization of Penicillium occitanis Pol6 and Penicillium funiculosum GH11 xylanases.

PubMed

Driss, Dorra; Berrin, Jean Guy; Juge, Nathalie; Bhiri, Fatma; Ghorbel, Raoudha; Chaabouni, Semia Ellouz

2013-08-01

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of heteroxylans constituting the lignocellulosic plant cell wall. Xylanases from the GH11 family are considered as true xylanases because of their high substrate specificity. In order to study in depth a crucial difference in the thumb region between two closely related xylanases from Penicillium in terms of kinetic parameters and inhibition sensitivity, the GH11 xylanases from Penicillium occitanis Pol6 (PoXyn3) and from Penicillium funiculosum (PfXynC) were heterologously expressed in Pichia pastoris. The PoXyn3 and PfXynC cDNAs encoding mature xylanases were cloned into pGAPZαA vectors and integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase constitutive promoter. PfXynC was expressed as a His-tagged recombinant protein and purified from the supernatant homogeneity by a one-step purification protocol using immobilized metal affinity chromatography. The recombinant PoXyn3 was purified using a single anion-exchange chromatography. The purified recombinant enzymes were optimally active at 45°C and pH 4.0 for PoXyn3 and 40°C and pH 3.0 for PfXynC. The measured kinetic parameters (k(cat) and Vmax) showed that PfXynC was five times more active than PoXyn3 irrespective of the substrate whereas the apparent affinity (K(m)) was similar. The recombinant enzymes showed distinct sensitivity to the Triticum aestivum xylanase inhibitor TAXI-I. Copyright © 2013 Elsevier Inc. All rights reserved.

Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling.

PubMed

Grom, Matic; Kozorog, Mirijam; Caserman, Simon; Pohar, Andrej; Likozar, Blaž

2018-04-15

Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Litopenaeus vannamei BiP as a novel cellular attachment protein for white spot syndrome virus by using a biotinylation based affinity chromatography method.

PubMed

Yuan, Zengzhi; Chen, Meng; Wang, Jingting; Li, Zhuoyu; Geng, Xuyun; Sun, Jinsheng

2018-05-05

White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target. Copyright © 2018. Published by Elsevier Ltd.

Identification of AOSC-binding proteins in neurons

NASA Astrophysics Data System (ADS)

Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

2008-11-01

Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

PubMed

Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

1998-07-03

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

Tracking control of air-breathing hypersonic vehicles with non-affine dynamics via improved neural back-stepping design.

PubMed

Bu, Xiangwei; He, Guangjun; Wang, Ke

2018-04-01

This study considers the design of a new back-stepping control approach for air-breathing hypersonic vehicle (AHV) non-affine models via neural approximation. The AHV's non-affine dynamics is decomposed into velocity subsystem and altitude subsystem to be controlled separately, and robust adaptive tracking control laws are developed using improved back-stepping designs. Neural networks are applied to estimate the unknown non-affine dynamics, which guarantees the addressed controllers with satisfactory robustness against uncertainties. In comparison with the existing control methodologies, the special contributions are that the non-affine issue is handled by constructing two low-pass filters based on model transformations, and virtual controllers are treated as intermediate variables such that they aren't needed for back-stepping designs any more. Lyapunov techniques are employed to show the uniformly ultimately boundedness of all closed-loop signals. Finally, simulation results are presented to verify the tracking performance and superiorities of the investigated control strategy. Copyright © 2018 ISA. Published by Elsevier Ltd. All rights reserved.

Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

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

Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.

2012-12-07

Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-cmore » containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.« less

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

PubMed

Müller, Tobias K H; Cao, Ping; Ewert, Stephanie; Wohlgemuth, Jonas; Liu, Haiyang; Willett, Thomas C; Theodosiou, Eirini; Thomas, Owen R T; Franzreb, Matthias

2013-04-12

An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling zone and mobile phase were identified as the main parameters affecting TCZR performance. In contrast to conventional systems, which rely on cooling the whole column to effect elution and permit only batch-wise operation, TCZR chromatography generates sharp concentrated elution peaks without tailing effects and appears ideally suited for continuous operation. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of salts on protein-surface interactions: applications for column chromatography.

PubMed

Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

2007-07-01

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

The antigenicity in guinea pigs and monkeys of three mycobacterial polysaccharides purified by affinity chromatography with concanavalin A.

PubMed

Daniel, T M

1975-06-01

The antigenicity of 3 polysaccharides purified from culture filtrates of Mycobacterim tuberculosis by affinity chromatography using a concanavalin A-agarose absorbent was studied. All 3 purified polysaccharides were found to be potent elicitors of delayed skin test reactions in sensitized guinea pigs and in a tuberculos monkey. This antigenicity could not be attributed to contaminating protein. Small dermal reactions were also observed in control guinea pigs. All 3 polysaccharides reacted with precipitating antibody in guinea pig sera, the antigenic specificity observed with the guinea pig sera differing from that demonstrated with reference goat antiserum. The 3 polysaccharides were also demonstrated to contain hemagglutination antigenic sites.

Solid-Phase Extraction Strategies to Surmount Body Fluid Sample Complexity in High-Throughput Mass Spectrometry-Based Proteomics

PubMed Central

Bladergroen, Marco R.; van der Burgt, Yuri E. M.

2015-01-01

For large-scale and standardized applications in mass spectrometry- (MS-) based proteomics automation of each step is essential. Here we present high-throughput sample preparation solutions for balancing the speed of current MS-acquisitions and the time needed for analytical workup of body fluids. The discussed workflows reduce body fluid sample complexity and apply for both bottom-up proteomics experiments and top-down protein characterization approaches. Various sample preparation methods that involve solid-phase extraction (SPE) including affinity enrichment strategies have been automated. Obtained peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations. PMID:25692071

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

NASA Astrophysics Data System (ADS)

Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

2016-07-01

In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

Using cell membrane chromatography and HPLC-TOF/MS method for in vivo study of active components from roots of Aconitum carmichaeli

PubMed Central

Cao, Yan; Chen, Xiao-Fei; Lü, Di-Ya; Dong, Xin; Zhang, Guo-Qing; Chai, Yi-Feng

2012-01-01

An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high Performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis ofthe analysis of the first dimension, retention components of the urine sample were collected into 30 fractions (one fraction per minute). Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine (Songorine, 14-benzoylneoline, Deoxyaconitine, etc.) exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the System suggest that the CMC can be applied to in vivo study. PMID:29403691

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

PubMed Central

Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

2016-01-01

In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

Twin hydroxymethyluracil-A base pair steps define the binding site for the DNA-binding protein TF1.

PubMed

Grove, A; Figueiredo, M L; Galeone, A; Mayol, L; Geiduschek, E P

1997-05-16

The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1-encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (Kd was approximately 3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based largely, if not entirely, on sequence-dependent DNA flexibility.

Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques.

PubMed

Yamamoto, Takenori; Tamaki, Haruna; Katsuda, Chie; Nakatani, Kiwami; Terauchi, Satsuki; Terada, Hiroshi; Shinohara, Yasuo

2013-08-02

Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecularly Imprinted Nanomicrospheres as Matrix Solid-Phase Dispersant Combined with Gas Chromatography for Determination of Four Phosphorothioate Pesticides in Carrot and Yacon

PubMed Central

Zhou, Mengchun; Hu, Nana; Shu, Shaohua; Wang, Mo

2015-01-01

An efficient, rapid, and selective method for sample pretreatment, namely, molecularly imprinted matrix solid-phase dispersion (MI-MSPD) coupled with gas chromatography (GC), was developed for the rapid isolation of four phosphorothioate organophosphorus pesticides (tolclofos-methyl, phoxim, chlorpyrifos, and parathion-methyl) from carrot and yacon samples. New molecularly imprinted polymer nanomicrospheres were synthesized by using typical structural analogue tolclofos-methyl as a dummy template via surface grafting polymerization on nanosilica. Then, these four pesticides in carrot and yacon were extracted and adsorbed using the imprinted nanomicrospheres and further determined by gas chromatography. Under the optimized conditions, a good linearity of four pesticides was obtained in a range of 0.05–17.0 ng·g−1 with R varying from 0.9971 to 0.9996, and the detection limit of the method was 0.012~0.026 ng·g−1 in carrot and yacon samples. The recovery rates at two spiked levels were in the range of 85.4–105.6% with RSD ≤9.6%. The presented MI-MSPD method combined the advantages of MSPD for allowing the extraction, dispersion, and homogenization in two steps and the advantages of MIPs for high affinity and selectivity towards four phosphorothioate pesticides, which could be applied to the determination of pesticide residues in complicated vegetal samples. PMID:25954569

Molecularly imprinted nanomicrospheres as matrix solid-phase dispersant combined with gas chromatography for determination of four phosphorothioate pesticides in carrot and yacon.

PubMed

Zhou, Mengchun; Hu, Nana; Shu, Shaohua; Wang, Mo

2015-01-01

An efficient, rapid, and selective method for sample pretreatment, namely, molecularly imprinted matrix solid-phase dispersion (MI-MSPD) coupled with gas chromatography (GC), was developed for the rapid isolation of four phosphorothioate organophosphorus pesticides (tolclofos-methyl, phoxim, chlorpyrifos, and parathion-methyl) from carrot and yacon samples. New molecularly imprinted polymer nanomicrospheres were synthesized by using typical structural analogue tolclofos-methyl as a dummy template via surface grafting polymerization on nanosilica. Then, these four pesticides in carrot and yacon were extracted and adsorbed using the imprinted nanomicrospheres and further determined by gas chromatography. Under the optimized conditions, a good linearity of four pesticides was obtained in a range of 0.05-17.0 ng·g(-1) with R varying from 0.9971 to 0.9996, and the detection limit of the method was 0.012~0.026 ng·g(-1) in carrot and yacon samples. The recovery rates at two spiked levels were in the range of 85.4-105.6% with RSD ≤9.6%. The presented MI-MSPD method combined the advantages of MSPD for allowing the extraction, dispersion, and homogenization in two steps and the advantages of MIPs for high affinity and selectivity towards four phosphorothioate pesticides, which could be applied to the determination of pesticide residues in complicated vegetal samples.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

PubMed

Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

2012-11-01

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Fragment screening for drug leads by weak affinity chromatography (WAC-MS).

PubMed

Ohlson, Sten; Duong-Thi, Minh-Dao

2018-02-23

Fragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of K d and irrelevant non-specific interactions are abundant in this area of transient interactions. Fortunately, there are methods that can study weak interactions quite efficiently of which NMR, surface plasmon resonance (SPR) and X-ray crystallography are the most prominent. Now, a new technology based on zonal affinity chromatography, weak affinity chromatography (WAC), has been introduced which has remedied many of the problems with other technologies. By combining WAC with mass spectrometry (WAC-MS), it is a powerful tool to identify binders quantitatively in terms of affinity and kinetics either from fragment libraries or from complex mixtures of biological extracts. As WAC-MS can be multiplexed by analysing mixtures of fragments (20-100 fragments) in one sample, this approach yields high throughput, where a whole library of e.g. >2000 fragments can be analysed quantitatively within a day. WAC-MS is easy to perform, where the robustness and quality of HPLC is fully utilized. This review will highlight the rationale behind the application of WAC-MS for fragment screening in drug discovery. Copyright © 2018 Elsevier Inc. All rights reserved.

An inorganic boronate affinity in-needle monolithic device for specific capture of cis-diol containing compounds.

PubMed

Jin, Shanxia; Zhang, Wei; Yang, Qin; Dai, Lili; Zhou, Ping

2018-02-01

In this work, inorganic boronate affinity monolith was prepared by in situ synthesis in 0.33mm i.d. stainless steel needle through sol-gel process using tetraethoxysilane and tetrabutyl orthotitanate as the co-precursors. The morphology, structure and composition of the monolith were characterized. In contrast to conventional boronate affinity materials, inorganic boric acid was used as affinity ligand. Different compounds were used for the evaluation of the boronate affinity of this inorganic monolithic material. The monolith exhibited good selectivity towards cis-diol containing compounds. Recovery of greater than 90% was achieved for in-needle extraction of catechol under neutral conditions. Owing to the hydrophilic property of the monolith, the procedure of affinity chromatography could be performed in aqueous solution. This monolithic in-needle device will be useful for boronate affinity extraction of small-volume samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome.

PubMed

Pieper, Rembert; Su, Qin; Gatlin, Christine L; Huang, Shih-Ting; Anderson, N Leigh; Steiner, Sandra

2003-04-01

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

PubMed

Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

2017-09-01

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

PubMed

Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

2006-04-01

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

PubMed

Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

1993-12-01

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

PubMed

Merten, O W; Reiter, S; Scheirer, W; Katinger, H

1983-01-01

The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.

PubMed

Turková, J; Bláha, K; Malaníková, M; Vancurová, D; Svec, F; Kálal, J

1978-05-11

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.

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

PubMed

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

2001-07-01

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

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

PubMed Central

Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

2012-01-01

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study

PubMed Central

Jamalapuram, Seshulatha; Vuppala, Pradeep K.; Mesangeau, Christophe; McCurdy, Christopher R.; Avery, Bonnie A.

2014-01-01

A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLCTM BEH HILIC column (1.7 µm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats. PMID:22406103

Developing procedures for the large-scale purification of human serum butyrylcholinesterase.

PubMed

Saxena, Ashima; Luo, Chunyuan; Doctor, Bhupendra P

2008-10-01

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.

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

PubMed Central

Kaur, Jasvir; Reinhardt, Dieter P.

2012-01-01

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

High-throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry.

PubMed

Razavi, Morteza; Frick, Lauren E; LaMarr, William A; Pope, Matthew E; Miller, Christine A; Anderson, N Leigh; Pearson, Terry W

2012-12-07

We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.

Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

PubMed

Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

2001-02-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

PubMed Central

Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

2001-01-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

Interferon Induced Transfer of Viral Resistance

DTIC Science & Technology

1982-02-01

released from the cell membrane. We have also shown that CM’s activity is removed by a gelatin /sepharose affinity column which selectively binds...interferon preparation adsorbing to the WISH cells, interferon was subjected to gelatin /sepharose affinity chromatography to remove endogenous...caused an increase in the amount of H-.amnino acids incorporated into a gelatin binding protein, presumably fibronectin. This suggests that in addition to

Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.)

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Richardson, D. P.

1991-01-01

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp.

PubMed

Albrecht, Melanie; Alessandri, Stefano; Conti, Amedeo; Reuter, Andreas; Lauer, Iris; Vieths, Stefan; Reese, Gerald

2008-11-01

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.

Lanthanide-IMAC enrichment of carbohydrates and polyols.

PubMed

Schemeth, Dieter; Rainer, Matthias; Messner, Christoph B; Rode, Bernd M; Bonn, Günther K

2014-03-01

In this study a new type of immobilized metal ion affinity chromatography resin for the enrichment of carbohydrates and polyols was synthesized by radical polymerization reaction of vinyl phosphonic acid and 1,4-butandiole dimethacrylate using azo-bis-isobutyronitrile as radical initiator. Interaction between the chelated trivalent lanthanide ions and negatively charged hydroxyl groups of carbohydrates and polyols was observed by applying high pH values. The new method was evaluated by single standard solutions, mixtures of standards, honey and a more complex extract of Cynara scolymus. The washing step was accomplished by acetonitrile in excess volumes. Elution of enriched carbohydrates was successfully performed with deionized water. The subsequent analysis was carried out with matrix-free laser desorption/ionization-time of flight mass spectrometry involving a TiO2 -coated steel target, especially suitable for the measurement of low-molecular-weight substances. Quantitative analysis of the sugar alcohol xylitol as well as the determination of the maximal loading capacity was performed by gas chromatography in conjunction with mass spectrometric detection after chemical derivatization. In a parallel approach quantum mechanical geometry optimizations were performed in order to compare the coordination behavior of various trivalent lanthanide ions. Copyright © 2013 John Wiley & Sons, Ltd.

Gamma-Aminobutyric Acid Production Using Immobilized Glutamate Decarboxylase Followed by Downstream Processing with Cation Exchange Chromatography

PubMed Central

Lee, Seungwoon; Ahn, Jungoh; Kim, Yeon-Gu; Jung, Joon-Ki; Lee, Hongweon; Lee, Eun Gyo

2013-01-01

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step. PMID:23322022

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

PubMed Central

2009-01-01

Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine. PMID:19930574

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

PubMed

Lombardi, Raffaele; Circelli, Patrizia; Villani, Maria Elena; Buriani, Giampaolo; Nardi, Luca; Coppola, Valentina; Bianco, Linda; Benvenuto, Eugenio; Donini, Marcello; Marusic, Carla

2009-11-20

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.

Preparative two-step purification of recombinant H1.0 linker histone and its domains.

PubMed

Ivic, Nives; Bilokapic, Silvija; Halic, Mario

2017-01-01

H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1.0 overexpressed in Escherichia coli, as well as its domains: N-terminal/globular domain and C-terminal intrinsically disordered domain. This purification protocol relies on a simple affinity chromatography step followed by cation exchange due to the highly basic properties of histone proteins. Therefore, this protocol can also be applied to other linker histones. Highly pure proteins in amounts sufficient for most biochemical experiments can be obtained. The functional quality of purified H1.0 histone and its domains has been confirmed by pull-down, gel-mobility shift assays and the nuclear import assay.

Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography.

PubMed

Lima, Juliana Maria; Salmazo Vieira, Plínio; Cavalcante de Oliveira, Arthur Henrique; Cardoso, Carmen Lúcia

2016-08-07

Nucleoside diphosphate kinase from Leishmania spp. (LmNDKb) has recently been described as a potential drug target to treat leishmaniasis disease. Therefore, screening of LmNDKb ligands requires methodologies that mimic the conditions under which LmNDKb acts in biological systems. Here, we compare two label-free methodologies that could help screen LmNDKb ligands and measure NDKb activity: an offline LC-UV assay for soluble LmNDKb and an online two-dimensional LC-UV system based on LmNDKb immobilised on a silica capillary. The target enzyme was immobilised on the silica capillary via Schiff base formation (to give LmNDKb-ICER-Schiff) or affinity attachment (to give LmNDKb-ICER-His). Several aspects of the ICERs resulting from these procedures were compared, namely kinetic parameters, stability, and procedure steps. Both the LmNDKb immobilisation routes minimised the conformational changes and preserved the substrate binding sites. However, considering the number of steps involved in the immobilisation procedure, the cost of reagents, and the stability of the immobilised enzyme, immobilisation via Schiff base formation proved to be the optimal procedure.

Molecularly Imprinted Polymers for Ochratoxin A Extraction and Analysis

PubMed Central

Yu, Jorn C. C.; Lai, Edward P. C.

2010-01-01

Molecularly imprinted polymers (MIPs) are considered as polymeric materials that mimic the functionality of antibodies. MIPs have been utilized for a wide variety of applications in chromatography, solid phase extraction, immunoassays, and sensor recognition. In this article, recent advances of MIPs for the extraction and analysis of ochratoxins are discussed. Selection of functional monomers to bind ochratoxin A (OTA) with high affinities, optimization of extraction procedures, and limitations of MIPs are compared from different reports. The most relevant examples in the literature are described to clearly show how useful these materials are. Strategies on MIP preparation and schemes of analytical methods are also reviewed in order to suggest the next step that would make better use of MIPs in the field of ochratoxin research. The review ends by outlining the remaining issues and impediments. PMID:22069649

Protein profile of Lupinus texensis phloem sap exudates: searching for Fe- and Zn-containing proteins.

PubMed

Lattanzio, Giuseppe; Andaluz, Sofía; Matros, Andrea; Calvete, Juan José; Kehr, Julia; Abadía, Anunciación; Abadía, Javier; López-Millán, Ana-Flor

2013-08-01

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI-MS and ESI-MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19-21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe-containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe-binding proteins in phloem sap: a metallothionein-like protein type 2B identified in the Fe-affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem-specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn-binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

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

PubMed

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

2003-07-01

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

Comparative Normal/Failing Rat Myocardium Cell Membrane Chromatographic Analysis System for Screening Specific Components That Counteract Doxorubicin-Induced Heart Failure from Acontium carmichaeli

PubMed Central

2015-01-01

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K+ channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models. PMID:24731167

Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein

PubMed Central

McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

2014-01-01

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. PMID:24811361

Method development of enantiomer separations by affinity capillary electrophoresis, cyclodextrin electrokinetic chromatography and capillary electrophoresis-mass spectrometry.

PubMed

Tanaka, Yoshihide

2002-07-01

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 mumol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

PubMed

Martinović, Tamara; Andjelković, Uroš; Klobučar, Marko; Černigoj, Urh; Vidič, Jana; Lučić, Marina; Pavelić, Krešimir; Josić, Djuro

2017-11-01

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

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

Turley, E.A.; Moore, D.; Hayden, L.J.

1987-06-02

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

Smart Hydrogel Particles: Biomarker Harvesting: One-step affinity purification, size exclusion, and protection against degradation

PubMed Central

Luchini, Alessandra; Geho, David H.; Bishop, Barney; Tran, Duy; Xia, Cassandra; Dufour, Robert; Jones, Clint; Espina, Virginia; Patanarut, Alexis; Zhu, Weidong; Ross, Mark; Tessitore, Alessandra; Petricoin, Emanuel; Liotta, Lance A.

2010-01-01

Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis. PMID:18076201

Challenges and opportunities in the purification of recombinant tagged proteins.

PubMed

Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

2014-01-01

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. Copyright © 2013 Elsevier Inc. All rights reserved.

Hydroxylapatite chromatography.

PubMed

Broadhurst, A V

2001-05-01

Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low-pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step-gradient elution.

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

PubMed

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

2010-02-01

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

Phospholipid bilayer affinities and solvation characteristics by electrokinetic chromatography with a nanodisc pseudostationary phase.

PubMed

Penny, William M; Steele, Harmen B; Ross, J B Alexander; Palmer, Christopher P

2017-03-01

Phospholipid bilayer nanodiscs composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and synthetic maleic acid-styrene copolymer belts have been introduced as a pseudostationary phase (PSP) in electrokinetic chromatography and demonstrated good performance. The nanodiscs provide a suitable migration range and high theoretical plate counts. Using this nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was determined and characterized. Good correlation is observed between retention factors and octanol water partition coefficients for particular categories of solutes, but the general correlation is weak primarily because the nanodiscs show stronger affinity than octanol for hydrogen bond donors. This suggests that a more appropriate application of this technology is to measure and characterize interactions between solutes and lipid bilayers directly. Linear solvation energy relationship analysis of the nanodisc-solute interactions in this study demonstrates that the nanodiscs provide a solvation environment with low cohesivity and weak hydrogen bond donating ability, and provide relatively strong hydrogen bond acceptor strength. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

From proteomics to systems biology: MAPA, MASS WESTERN, PROMEX, and COVAIN as a user-oriented platform.

PubMed

Weckwerth, Wolfram; Wienkoop, Stefanie; Hoehenwarter, Wolfgang; Egelhofer, Volker; Sun, Xiaoliang

2014-01-01

Genome sequencing and systems biology are revolutionizing life sciences. Proteomics emerged as a fundamental technique of this novel research area as it is the basis for gene function analysis and modeling of dynamic protein networks. Here a complete proteomics platform suited for functional genomics and systems biology is presented. The strategy includes MAPA (mass accuracy precursor alignment; http://www.univie.ac.at/mosys/software.html ) as a rapid exploratory analysis step; MASS WESTERN for targeted proteomics; COVAIN ( http://www.univie.ac.at/mosys/software.html ) for multivariate statistical analysis, data integration, and data mining; and PROMEX ( http://www.univie.ac.at/mosys/databases.html ) as a database module for proteogenomics and proteotypic peptides for targeted analysis. Moreover, the presented platform can also be utilized to integrate metabolomics and transcriptomics data for the analysis of metabolite-protein-transcript correlations and time course analysis using COVAIN. Examples for the integration of MAPA and MASS WESTERN data, proteogenomic and metabolic modeling approaches for functional genomics, phosphoproteomics by integration of MOAC (metal-oxide affinity chromatography) with MAPA, and the integration of metabolomics, transcriptomics, proteomics, and physiological data using this platform are presented. All software and step-by-step tutorials for data processing and data mining can be downloaded from http://www.univie.ac.at/mosys/software.html.

Generation of Murine Monoclonal Antibodies by Hybridoma Technology.

PubMed

Holzlöhner, Pamela; Hanack, Katja

2017-01-02

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Isolation and characterization of recombinant murine Wnt3a.

PubMed

Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A; Ryan, Robert O

2015-02-01

Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation and characterization of recombinant murine Wnt3a

PubMed Central

Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A.; Ryan, Robert O.

2014-01-01

Wnt proteins are a family of morphogens that possess potent biological activity. Structure – function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30 mM methyl-β-cyclodextrin (MβCD). Wnt3a recovered in MβCD-containing buffer was soluble and biologically active. Insofar as MβCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. PMID:25448592

Purification and characterization of rat liver nuclear thyroid hormone receptors.

PubMed Central

Ichikawa, K; DeGroot, L J

1987-01-01

Nuclear thyroid hormone receptor was purified to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding capacity per mg of protein with 2.5-5.2% recovery by sequentially using hydroxylapatite column chromatography, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column chromatography, DEAE-Sephadex column chromatography, and heparin-Sepharose column chromatography. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, we reproducibly obtained 6.4-14.7 micrograms of receptor protein with 4.2-4.9% purity from 4-5 kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate, which was observed to diminish binding of receptor to heparin-Sepharose or DNA-cellulose. This effect was specific for pyridoxal 5'-phosphate, since related compounds were not effective. Purified receptor bound T3 with high affinity (6.0 X 10(9) liter/mol), and the order of affinity of iodothyronine analogues to purified receptor was identical to that observed with crude receptor preparations [3,5,3'-triiodothyroacetic acid greater than L-T3 greater than D-3,5,3'-triiodothyronine (D-T3) greater than L-thyroxine greater than D-thyroxine]. Purified receptor had a sedimentation coefficient of 3.4 S, Stokes radius of 34 A, and calculated molecular mass of 49,000. Among several bands identified by silver staining after electrophoresis in NaDodSO4/polyacrylamide gels, one 49,000-Da protein showed photoaffinity labeling with [125I]thyroxine that was displaceable with excess unlabeled T3. The tryptic fragment and endogenous proteinase-digested fragment of the affinity-labeled receptor showed saturable binding in 27,000-Da and 36,000-Da peptides, respectively. These molecular masses are in agreement with estimates from gel filtration and gradient sedimentation, indicating that affinity labeling occurred at the hormone binding domain of nuclear thyroid hormone receptor. This procedure reproducibly provides classical native rat liver T3 nuclear receptor in useful quantity and purity and of the highest specific activity so far reported. Images PMID:3472213

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

PubMed

Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

2016-01-01

Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

Temperature effect on affinity chromatography of two lectins from the seeds of Ricinus communis

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

Hsu, H.W.; Davis, D.S.; Wei, C.H.

1976-06-01

Specific adsorption capacity of Sepharose 4B in affinity chromatography for two purified galactose-binding lectins, designated as III/sub L/ and III/sub H/, from the seed of Ricinus communis (castor bean) was measured from 7 to 24/sup 0/C. The adsorption coefficients for these two protein fractions as a function of temperature were also obtained. It was found that there is a characteristic transition of adsorption coefficient at 18/sup 0/C for both lectins. Adsorption coefficients between Sepharose 4B and these two lectins were also expressed in terms of ..delta..G, ..delta..H, and ..delta..S. It is suggested that the difference in the temperature dependence ofmore » the binding energy of these two lectins may be used for their separation at selected temperature.« less

Prion removal effect of a specific affinity ligand introduced into the manufacturing process of the pharmaceutical quality solvent/detergent (S/D)-treated plasma OctaplasLG.

PubMed

Neisser-Svae, A; Bailey, A; Gregori, L; Heger, A; Jordan, S; Behizad, M; Reichl, H; Römisch, J; Svae, T-E

2009-10-01

A new chromatographic step for the selective binding of abnormal prion protein (PrP(Sc)) was developed, and optimization for PrP(Sc) capture was achieved by binding to an affinity ligand attached to synthetic resin particles. This step was implemented into the manufacturing process of the solvent/detergent (S/D)-treated biopharmaceutical quality plasma Octaplas to further improve the safety margin in terms of risk for variant Creutzfeldt-Jakob disease (vCJD) transmission. Intermediates and Octaplas final container material, spiked with hamster brain-derived PrP(Sc)-containing fractions, were used for experiments to establish the feasibility of introducing this novel chromatography step. The binding capacity per millilitre of ligand gel was determined under the selected manufacturing conditions. In addition, the specificity of the ligand gel to bind PrP(Sc) from human sources was investigated. A validated Western blot test was used for the identification and quantification of PrP(Sc). A reduction factor of > or = 3.0 log(10) could be demonstrated by Western blotting, utilizing the relevant Octaplas matrix from manufacturing. In this particular cell-free plasma solution, the PrP(Sc) binding capacity of the selected gel was very high (> or = 6 log(10) ID(50)/ml, equivalent to roughly 10 log(10) ID(50)/column at manufacturing scale). The gel binds specifically PrP(Sc) from both animal (hamster and mouse) and human (sporadic and variant CJD) sources. This new single-use, disposable PrP(Sc)-harvesting gel ensures a very high capacity in terms of removing the pathogenic agent causing vCJD from the new generation OctaplasLG, in the event that prions can be found in plasma from donors incubating the disease and thereby contaminating the raw material plasma used for manufacturing.

Isolation, purification, and partial characterization of a membrane-bound Cl-/HCO3--activated ATPase complex from rat brain with sensitivity to GABAAergic ligands.

PubMed

Menzikov, Sergey A

2017-02-07

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

PubMed

Golovko, A O; Koroleva, O N; Drutsa, V L

2017-12-01

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His) 6 -tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

Heavy metal ion inhibition studies of human, sheep and fish α-carbonic anhydrases.

PubMed

Demirdağ, Ramazan; Yerlikaya, Emrah; Şentürk, Murat; Küfrevioğlu, Ö İrfan; Supuran, Claudiu T

2013-04-01

Carbonic anhydrases (CAs, EC 4.2.1.1) were purified from sheep kidney (sCA IV), from the liver of the teleost fish Dicentrarchus labrax (dCA) and from human erythrocytes (hCA I and hCA II). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The kinetic parameters of these enzymes were determined for their esterase activity with 4-nitrophenyl acetate as substrate. The following metal ions, Pb(2+), Co(2+), Hg(2+), Cd(2+), Zn(2+), Se(2+), Cu(2+), Al(3+) and Mn(3+) showed inhibitory effects on these enzymes. The tested metal ions inhibited these CAs competitively in the low milimolar/submillimolar range. The susceptibility to various cations inhibitors differs significantly between these vertebrate α-CAs and is probably due to their binding to His64 or the histidine cluster.

Employing immuno-affinity for the analysis of various microbial metabolites of the mycotoxin deoxynivalenol.

PubMed

Zhu, Yan; Hassan, Yousef I; Shao, Suqin; Zhou, Ting

2018-06-29

Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly detected in grains infested with Fusarium species. The maximum tolerated levels of DON in the majority of world's countries are restricted to 0.75 mg kg -1 within the human food chain and to less than 1-5 mg kg -1 in animal feed depending on the feed material and/or animal species due to DON's short and long-term adverse effects on human health and animal productivity. The ability to accurately analyze DON and some of its fungal/bacterial metabolites is increasingly gaining a paramount importance in food/feed analysis and research. In this study, we used the immuno-affinity approach to enrich and detect DON and three of its bacterial metabolites, namely 3-epi-DON, 3-keto-DON, and deepoxy-DON (DOM-1). The optimized enrichment step coupled with high performance liquid chromatography can accurately and reproducibly quantify the aforementioned metabolites in feed matrixes (silage extract as an example in this case). It minimizes any background interface and provides a fast and easy-to-operate protocol for the analytical determination of such metabolites. More importantly, the presented data demonstrates the ability of the utilized monoclonal antibody, generated originally to capture DON in Enzyme-Linked Immunosorbent Assays (ELISA), to cross react with three less/non-toxic DON metabolites. This raises the concerns about the genuine need to account for such cross-reactivity when DON contamination is assessed through an immuno-affinity based analyses using the investigated antibody. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation*

PubMed Central

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J.

2014-01-01

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. PMID:24958729

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

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

PubMed

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

2010-02-01

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

Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue

NASA Technical Reports Server (NTRS)

Karuppiah, N.; Vadlamudi, B.; Kaufman, P. B.

1989-01-01

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (beta-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organo-mercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, Km of 12 millimolar, and a Vmax of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, Km of millimolar, and a Vmax of 9 micromole per minute per milligram of protein.

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

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.

Purification and characterization of paraoxon hydrolase from rat liver.

PubMed Central

Rodrigo, L; Gil, F; Hernandez, A F; Marina, A; Vazquez, J; Pla, A

1997-01-01

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45 000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM. PMID:9032442

Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

PubMed

McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

2014-07-01

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. © 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

PubMed

Saito, Maiko; Kurosawa, Yae; Okuyama, Tsuneo

2012-02-01

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Affinity Interaction between Hexamer Peptide Ligand HWRGWV and Immunoglobulin G Studied by Quartz Crystal Microbalance and Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Shen, Fei

Immunoglobulins (Ig), also referred to as antibodies, act as protective agents against pathogens trying to invade an organism. Human immunoglobulin G (hIgG), as the most prominent immunoglobulin presented in serum and other human fluids, has broad applications in fields like immunotherapy and clinical diagnostics. Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG purifaction and detection. However, drawbacks associated with these two protein ligands have motivated searches for alternative affinity ligands. The hexamer peptide ligand HWRGWV identified from a one-bead-one-peptide combinatorial library synthesized on chromatography resins has demonstrated high affinity and specificity to the Fc fragment of hIgG. A chromatography resin with HWRGWV can purify human IgG (hIgG) from complete minimum essential medium (cMEM) with purities and yields as high as 95%, which are comparable to using Protein A as affinity ligand (4). As a short peptide ligand, HWRGWV can be produced at relatively low costs under good manufacturing practices (GMP) conditions, it is highly robust, less immunogenic and allows for milder elution conditions for the bound antibody (3, 5). Although this short peptide ligand has exhibited promising properties for IgG capture and purification, limited information is available on the intrinsic mechanisms of affinity interaction between the peptide ligand and target protein. In this study, the affinity interaction between hIgG and peptide ligand immobilized on solid surfaces was studied by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). Compared with previous methods employed for the peptide characterization, QCM and SPR can provide direct measurements of equilibrium adsorption isotherms and rates of adsorption, allowing a complete kinetic and thermodynamics analyses of the ligand-target interactions. New methods were developed to modify gold and silica surfaces of QCM and SPR sensors for the immobilization of peptide ligands with low nonspecific binding. The silica surface was first modified by the formation of self-assembling monolayer (SAM) of 3-amino-propyl triethoxy silane as an anchor layer. Short chains of poly(ethylene glycol) (PEG) with Fmoc-protected amino groups at one end and carboxyl groups at the other end were then coupled through the carboxyl terminal to the amino groups on the silane. The short PEG chains served as spacer arms to reduce nonspecific binding to the substrate. The gold surface was modified by a two-component SAM using mixtures of HS(CH 2)11(CH2CH2O)6NH2 and HS(CH2)11(CH2CH2O)3OH. The advantage of using a modified silica surface is its relatively higher stability than the SAM on gold during the peptide functionalization step, however the SPR sensors do not work on silica surfaces. In addition, the modification process of the gold surface is relatively simple compared with that of the silica surface. The peptide immobilization process was optimized with silica surfaces and the best conditions were applied for the immobilization on gold surfaces. The results of surface modifications and peptide immobilizations were characterized by various surface analysis techniques including, ellipsometry, contact angle goniometer, chemical force microscopy (CFM), x-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS). QCM and SPR results indicated that this peptide ligand HWRGWV immobilized on modified silica or gold surfaces has high affinity and specificity to hIgG binding even in a complex medium such as cMEM. Both thermodynamic and kinetic parameters of affinity interaction were obtained by the analysis of QCM and SPR data. Compared with QCM, SPR is more suitable for quantitative analysis of the protein binding, which is essential for the investigation of thermodynamics and kinetics parameters. The maximum binding capacity (4.15 mg m-2 ) and the dissociation constant (1.83 muM) derived from SPR data are both close to those obtained with chromatography techniques. The association and dissociation rate constants (0.68 m3 mol-1 s-1 and 1.24 s-1 respectively) were acquired for the first time for the affinity binding of IgG on peptide ligand HWRGWV functionalized surface. Although QCM is not as quantitative as SPR, it provides additional information on the status of the adsorbed layers. For instance, the dissipation measurement of QCM indicated that no significant denaturation of adsorbed hIgG occurred during the adsorption process. In addition, it was shown that the peptide ligand immobilized on modified silica surfaces has similar affinity and binding characteristics for IgG adsorption as on modified gold surfaces. In summary, new surface modification strategies were developed to study the affinity interaction between peptide ligands and target biomolecules. The use of Fc-specific binding peptides on QCM and SPR sensors could result in new devices for IgG concentration determination and also have promise as platforms for the development of immunosensors.

Neuregulin: First Steps Towards a Structure

NASA Technical Reports Server (NTRS)

Ferree, D. S.; Malone, C. C.; Karr, L. J.

2003-01-01

Neuregulins are growth factor domain proteins with diverse bioactivities, such as cell proliferation, receptor binding, and differentiation. Neureguh- 1 binds to two members of the ErbB class I tyrosine kinase receptors, ErbB3 and ErbB4. A number of human cancers overexpress the ErbB receptors, and neuregulin can modulate the growth of certain cancer types. Neuregulin-1 has been shown to promote the migration of invasive gliomas of the central nervous system. Neuregulin has also been implicated in schizophrenia, multiple sclerosis and abortive cardiac abnormalities. The full function of neuregulin-1 is not known. In this study we are inserting a cDNA clone obtained from American Type Culture Collection into E.coli expression vectors to express neuregulin- 1 protein. Metal chelate affinity chromatography is used for recombinant protein purification. Crystallization screening will proceed for X-ray diffraction studies following expression, optimization, and protein purification. In spite of medical and scholarly interest in the neuregulins, there are currently no high-resolution structures available for these proteins. Here we present the first steps toward attaining a high-resolution structure of neuregulin- 1, which will help enable us to better understand its function

Functional and proteomic comparison of different techniques to produce equine anti-tetanus immunoglobulin F(ab')2 fragments.

PubMed

Zhang, Xue-Jun; Li, Hai-Ling; Deng, Da-Yi; Ji, Chong; Yao, Xiao-Dong; Liu, Jia-Xin

2018-05-29

Tetanus is still a major cause of human deaths in several developing countries. In particular, the neonatal form remains a significant public health problem. According to the World Health Organization, administration of tetanus toxoid is recommended for neonatal tetanus patients. Furthermore, tetanus antitoxin or anti-tetanus immunoglobulin (Ig) are used for mild case or intensive care. This paper discusses a novel purification technique for improving equine anti-tetanus Ig production. First, equine plasma dealt with two steps salting out with ammonium sulfate; second, ultrafiltration concentration liquid purified by one successive protein G based affinity chromatography steps; finally, the purified F(ab')2 fragments was characterized using biochemical and proteomic methods and shown to be pure and homogeneous. Compared with the original technique product, specific activity increased by 80% (about 90,000 IU/g) and recovery of F(ab')2 is approximately equal 75%. Furthermore, Proteomic profiling of total technique process is demonstrated by nano-HPLC-MS and bioinformatics analysis. New technique to produce equine anti-tetanus immunoglobulin F(ab')2 fragments from crude plasma in high quality and yield. And it also could be used for industrial amplification. Copyright © 2018 Elsevier B.V. All rights reserved.

The Cutting Edge of Affinity Electrophoresis Technology

PubMed Central

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2015-01-01

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

Modular microfluidics for point-of-care protein purifications

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

Millet, L. J.; Lucheon, J. D.; Standaert, R. F.

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

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

Ooi, G.T.; Herington, A.C.

1986-05-29

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When /sup 125/I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, followingmore » further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands.« less

Detection of glycoproteins in the Acanthamoeba plasma membrane

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

Paatero, G.I.L.; Gahmberg, C.G.

1988-11-01

In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presencemore » of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.« less

Studies on gonadotropin receptor of rat ovary and testis

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

Zhang, Q.

1989-01-01

The subunit structure of the testicular LH/hCG receptor was studied by a chemical cross-linking technique. Leydig cells isolated from rat testis were incubated with {sup 125}I-hCG, following which the bound {sup 125}I-hCG was covalently cross-linked to the receptor on the cell surface with a cleavable or a non-cleavable cross-linking reagent. The hormone-receptor complex was extracted and then either subjected to gel permeation chromatography under nondenaturing conditions, or resolved by SDS-polyacrylamide gel electrophoresis, followed by autoradiographic analysis. The ovarian LH/hCG receptor was studied with luteal cells from pseudopregnant rats. Purification of the receptor was achieved by ligand affinity chromatography following detergentmore » solubilization of the plasma membrane. The purified hCG receptor displayed properties identical to the membrane bound receptor with regard to binding specificity and affinity, and exhibited a molecular weight of approximately 130,000 dalton.« less

Modular microfluidics for point-of-care protein purifications.

PubMed

Millet, L J; Lucheon, J D; Standaert, R F; Retterer, S T; Doktycz, M J

2015-04-21

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

PubMed

Konami, Y; Yamamoto, K; Osawa, T

1991-02-01

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

A Research Module for the Organic Chemistry Laboratory: Multistep Synthesis of a Fluorous Dye Molecule

PubMed Central

2014-01-01

A multi-session research-like module has been developed for use in the undergraduate organic teaching laboratory curriculum. Students are tasked with planning and executing the synthesis of a novel fluorous dye molecule and using it to explore a fluorous affinity chromatography separation technique, which is the first implementation of this technique in a teaching laboratory. Key elements of the project include gradually introducing students to the use of the chemical literature to facilitate their searching, as well as deliberate constraints designed to force them to think critically about reaction design and optimization in organic chemistry. The project also introduces students to some advanced laboratory practices such as Schlenk techniques, degassing of reaction mixtures, affinity chromatography, and microwave-assisted chemistry. This provides students a teaching laboratory experience that closely mirrors authentic synthetic organic chemistry practice in laboratories throughout the world. PMID:24501431

Modular microfluidics for point-of-care protein purifications

DOE PAGES

Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; ...

2015-01-01

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

A Research Module for the Organic Chemistry Laboratory: Multistep Synthesis of a Fluorous Dye Molecule.

PubMed

Slade, Michael C; Raker, Jeffrey R; Kobilka, Brandon; Pohl, Nicola L B

2014-01-14

A multi-session research-like module has been developed for use in the undergraduate organic teaching laboratory curriculum. Students are tasked with planning and executing the synthesis of a novel fluorous dye molecule and using it to explore a fluorous affinity chromatography separation technique, which is the first implementation of this technique in a teaching laboratory. Key elements of the project include gradually introducing students to the use of the chemical literature to facilitate their searching, as well as deliberate constraints designed to force them to think critically about reaction design and optimization in organic chemistry. The project also introduces students to some advanced laboratory practices such as Schlenk techniques, degassing of reaction mixtures, affinity chromatography, and microwave-assisted chemistry. This provides students a teaching laboratory experience that closely mirrors authentic synthetic organic chemistry practice in laboratories throughout the world.

68Ga-THP-PSMA: A PET Imaging Agent for Prostate Cancer Offering Rapid, Room-Temperature, 1-Step Kit-Based Radiolabeling.

PubMed

Young, Jennifer D; Abbate, Vincenzo; Imberti, Cinzia; Meszaros, Levente K; Ma, Michelle T; Terry, Samantha Y A; Hider, Robert C; Mullen, Greg E; Blower, Philip J

2017-08-01

The clinical impact and accessibility of 68 Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, 1-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of 99m Tc radiopharmaceuticals. Currently, chelating agents used in 68 Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a 68 Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of 68 Ga generator eluate to a cold kit. Methods: A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with 68 Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin-layer chromatography and high-performance liquid chromatography. The product was subjected to in vitro studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their nonexpressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were performed in mice bearing xenografts of the same cell lines, comparing 68 Ga-THP-PSMA with 68 Ga-HBED-CC-PSMA. Results: Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by high-performance liquid chromatography that was stable in human serum for more than 6 h and showed specific binding to PSMA-expressing cells (concentration giving 50% inhibition of 361 ± 60 nM). In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 percentage injected dose per cubic centimeter at 40-60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution, and pharmacokinetics were not significantly different from those of 68 Ga-HBED-CC-PSMA except for reduced uptake in the spleen. Conclusion: 68 Ga-THP-PSMA has equivalent imaging properties but greatly simplified radiolabeling compared with other 68 Ga-PSMA conjugates. THP offers the prospect of rapid, simple, 1-step, room-temperature syringe-and-vial radiolabeling of 68 Ga radiopharmaceuticals. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography

PubMed Central

Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

2018-01-01

ABSTRACT Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples. PMID:29381421

Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

DOE PAGES

Springer, David L.; Auberry, Deanna L.; Ahram, Mamoun; ...

2004-01-01

To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsinmore » digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.« less

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography.

PubMed

Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

2018-04-01

Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

Identification and measurement of chlorinated organic pesticides in water by electron-capture gas chromatography

USGS Publications Warehouse

Lamar, William L.; Goerlitz, Donald F.; Law, LeRoy M.

1965-01-01

Pesticides, in minute quantities, may affect the regimen of streams, and because they may concentrate in sediments, aquatic organisms, and edible aquatic foods, their detection and their measurement in the parts-per-trillion range are considered essential. In 1964 the U.S. Geological Survey at Menlo Park, Calif., began research on methods for monitoring pesticides in water. Two systems were selected--electron-capture gas chromatography and microcoulometric-titration gas chromatography. Studies on these systems are now in progress. This report provides current information on the development and application of an electron-capture gas chromatographic procedure. This method is a convenient and extremely sensitive procedure for the detection and measurement of organic pesticides having high electron affinities, notably the chlorinated organic pesticides. The electron-affinity detector is extremely sensitive to these substances but it is not as sensitive to many other compounds. By this method, the chlorinated organic pesticide may be determined on a sample of convenient size in concentrations as low as the parts-per-trillion range. To insure greater accuracy in the identifications, the pesticides reported were separated and identified by their retention times on two different types of gas chromatographic columns.

Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

NASA Astrophysics Data System (ADS)

Ivanov, A. I.; Zhbankov, R. G.; Korolenko, E. A.; Korolik, E. V.; Meleshchenko, L. A.; Sarnatskaya, V. V.; Nikolaev, V. G.; Nikolaichik, V. V.; Yushko, L. A.

1997-01-01

Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin.

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

Estimation of biological variation and reference change value of glycated hemoglobin (HbA(1c)) when two analytical methods are used.

PubMed

Ucar, Fatma; Erden, Gonul; Ginis, Zeynep; Ozturk, Gulfer; Sezer, Sevilay; Gurler, Mukaddes; Guneyk, Ahmet

2013-10-01

Available data on biological variation of HbA1c revealed marked heterogeneity. We therefore investigated and estimated the components of biological variation for HbA1c in a group of healthy individuals by applying a recommended and strictly designed study protocol using two different assay methods. Each month, samples were derived on the same day, for three months. Four EDTA whole blood samples were collected from each individual (20 women, 9 men; 20-45 years of age) and stored at -80°C until analysis. HbA1c values were measured by both high performance liquid chromatography (HPLC) (Shimadzu, Prominence, Japan) and boronate affinity chromatography methods (Trinity Biotech, Premier Hb9210, Ireland). All samples were assayed in duplicate in a single batch for each assay method. Estimations were calculated according to the formulas described by Fraser and Harris. The within subject (CV(I))-between subject (CV(G)) biological variations were 1.17% and 5.58%, respectively for HPLC. The calculated CV(I) and CV(G) were 2.15% and 4.03%, respectively for boronate affinity chromatography. Reference change value (RCV) for HPLC and boronate affinity chromatography was 5.4% and 10.4% respectively and individuality index of HbA(1c) was 0.35 and 0.93 respectively. This study for the first time described the components of biological variation for HbA1c in healthy individuals by two different assay methods. Obtained findings showed that the difference between CV(A) values of the methods might considerably affect RCV. These data regarding biological variation of HbA(1c) could be useful for a better evaluation of HbA(1c) test results in clinical interpretation. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Hupresin Retains Binding Capacity for Butyrylcholinesterase and Acetylcholinesterase after Sanitation with Sodium Hydroxide.

PubMed

Onder, Seda; David, Emilie; Tacal, Ozden; Schopfer, Lawrence M; Lockridge, Oksana

2017-01-01

Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10-15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.

Purification and Characterization of a Mucin Specific Mycelial Lectin from Aspergillus gorakhpurensis: Application for Mitogenic and Antimicrobial Activity

PubMed Central

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Background Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Methods Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Results Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5–9.5, while optimum temperature for lectin activity was 20–30°C. Lectin was stable within a pH range of 7.0–10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. Conclusion This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin. PMID:25286160

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

PubMed

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

PubMed

Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

2001-08-24

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

PubMed Central

Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

2013-01-01

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

Prostate cell membrane chromatography-liquid chromatography-mass spectrometry for screening of active constituents from Uncaria rhynchophylla.

PubMed

He, Jianyu; Han, Shengli; Yang, Fangfang; Zhou, Nan; Wang, Sicen

2013-01-01

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model.

PubMed

Boto, R E F; Anyanwu, U; Sousa, F; Almeida, P; Queiroz, J A

2009-09-01

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright (c) 2009 John Wiley & Sons, Ltd.

Artefactually low glycated haemoglobin in a patient with severe hypertriglyceridaemia

PubMed Central

Garrib, A; Griffiths, W; Eldridge, P; Hatton, R; Worsley, A; Crook, M

2003-01-01

This report describes a case of artefactually low glycated haemoglobin (Hb) in a patient with type II diabetes and severe hypertriglyceridaemia. The effect of hypertriglyceridaemia on glycated Hb determination using the Abbott Vision method was investigated in a series of patients with diabetes. The interference of triglycerides in glycated Hb assays was also investigated by two other methods, the Beckman Synchron CX4 delta immunoturbidimetric method, and the Primus affinity chromatography high performance liquid chromatography assay. PMID:12719463

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream.

PubMed

Weber, Martin R; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E; Zijlstra, Andries; Quigley, James P; Staflin, Karin; Eliceiri, Brian P; Krueger, Joseph S; Marchese, Patrizia; Ruggeri, Zaverio M; Felding, Brunhilde H

2016-04-01

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease. © 2016 Elsevier Ltd. All rights reserved.

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream

PubMed Central

Weber, Martin R.; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E.; Zijlstra, Andries; Quigley, James P.; Staflin, Karin; Eliceiri, Brian P.; Krueger, Joseph S.; Marchese, Patricia; Ruggeri, Zaverio M.; Felding, Brunhilde H.

2016-01-01

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease. PMID:27067975

Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

PubMed

Leedman, P J; Newman, J D; Harrison, L C

1989-07-01

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

PubMed Central

Falck, David; de Vlieger, Jon S. B.; Niessen, Wilfried M. A.; Kool, Jeroen; Honing, Maarten; Irth, Hubertus

2010-01-01

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures. Figure P38 α online screening platform Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4087-8) contains supplementary material, which is available to authorized users. PMID:20730527

Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.

PubMed

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J

2014-08-08

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 5K

USDA-ARS?s Scientific Manuscript database

Cyclodextrin glycosyltransferase (CGTase) from the thermophilic anaerobe Thermoanaerobacter sp. 5K was purified and characterized. The enzyme was purified with ammonium sulfate precipitation followed by a-CD-bound, epoxy-activated Sepharose 6B affinity chromatography. Molecular weight of the purifie...

Molecular cloning, expression and molecular modeling of chemosensory protein from Spodoptera litura and its binding properties with Rhodojaponin III.

PubMed

Zhang, Yanbo; Dong, Xiaolin; Liu, Jinxiang; Hu, Meiying; Zhong, Guohua; Geng, Peng; Yi, Xin

2012-01-01

Insects stimulate specific behaviors by the correct recognition of the chemicals in the external environment. Rhodojaponin III is a botanical grayanoid diterpenid oviposition deterrent isolated from Rhododendron molle. In this study we aimed to determine whether the CSPs involved in the recognition of Rhodojaponin III. A full-length cDNA encoding chemosensory protein was isolated from the antennae of Spodoptera litura Fabricius (CSPSlit, GenBank Accession No. DQ007458). The full-length cDNA of NlFoxA is 1789 bp and has an open reading frame (ORF) of 473 bp, encoding a protein of 126 amino acids, Northern blot analysis revealed that CSPSlit mRNA was mainly expressed in the antennae, legs, wings and female abdomens. A three-dimensional model of CSPSlit was constructed using homology modeling method, and its reliability was evaluated. The active site of CSPSlit was calculated using CDOCKER program indicated that the Tyr24, Ile45, Leu49, Thr64, Leu68, Trp79 and Leu82 were responsible ligand-binding active site on identifying Rhodojaponin III in the CSPSlit. The recombinant CSPSlit protein was expressed in Escherichia coli and purified using single-step Ni-NTA affinity chromatography. Fluorescence emission spectra revealed that the CSPSlit protein had significant affinity to rhodojaponin III. These results mean that CSPSlit is critical for insects identify the Rhodojaponin III.

Molecular Cloning, Expression and Molecular Modeling of Chemosensory Protein from Spodoptera litura and Its Binding Properties with Rhodojaponin III

PubMed Central

Zhang, Yanbo; Dong, Xiaolin; Liu, Jinxiang; Hu, Meiying; Zhong, Guohua; Geng, Peng; Yi, Xin

2012-01-01

Insects stimulate specific behaviors by the correct recognition of the chemicals in the external environment. Rhodojaponin III is a botanical grayanoid diterpenid oviposition deterrent isolated from Rhododendron molle. In this study we aimed to determine whether the CSPs involved in the recognition of Rhodojaponin III. A full-length cDNA encoding chemosensory protein was isolated from the antennae of Spodoptera litura Fabricius (CSPSlit, GenBank Accession No. DQ007458). The full-length cDNA of NlFoxA is 1789 bp and has an open reading frame (ORF) of 473 bp, encoding a protein of 126 amino acids, Northern blot analysis revealed that CSPSlit mRNA was mainly expressed in the antennae, legs, wings and female abdomens. A three-dimensional model of CSPSlit was constructed using homology modeling method, and its reliability was evaluated. The active site of CSPSlit was calculated using CDOCKER program indicated that the Tyr24, Ile45, Leu49, Thr64, Leu68, Trp79 and Leu82 were responsible ligand-binding active site on identifying Rhodojaponin III in the CSPSlit. The recombinant CSPSlit protein was expressed in Escherichia coli and purified using single-step Ni-NTA affinity chromatography. Fluorescence emission spectra revealed that the CSPSlit protein had significant affinity to rhodojaponin III. These results mean that CSPSlit is critical for insects identify the Rhodojaponin III. PMID:23133516

Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein.

PubMed

Balogh, Ria K; Gyurcsik, Béla; Hunyadi-Gulyás, Éva; Christensen, Hans E M; Jancsó, Attila

2016-07-01

Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

PubMed

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Online hyphenation of extraction, Sephadex LH-20 column chromatography, and high-speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step.

PubMed

Zhang, Ying-Qi; Wang, Shan-Shan; Han, Chao; Xu, Jin-Fang; Luo, Jian-Guang; Kong, Ling-Yi

2017-12-01

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH-20 column chromatography combined with high-speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH-20 column directly to cut the nontarget fractions followed by the second-dimensional high-speed countercurrent chromatography, hyphenated by a six-port valve equipped at the post-end of Sephadex LH-20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation of oxidative degradation products of atorvastatin with supercritical fluid chromatography.

PubMed

Klobčar, Slavko; Prosen, Helena

2015-12-01

The isolation of four oxidative degradation products of atorvastatin using preparative high-performance liquid chromatography applying at least two chromatographic steps is known from the literature. In this paper it is shown that the same four impurities could be isolated from similarly prepared mixtures in only one step using supercritical fluid chromatography. The methods for separation were developed and optimized. The preparation of the mixtures was altered in such a way as to enhance the concentration of desired impurities. Appropriate solvents were applied for collection of separated impurities in order to prevent degradation. The structures of the isolated impurities were confirmed and their purity determined. The preparative supercritical fluid chromatography has proven to be superior to preparative HPLC regarding achieved purity of standards applying fewer chromatographic as well as isolation steps. Copyright © 2015 John Wiley & Sons, Ltd.

A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

PubMed

Kim, Heejae; Chen, Wilfred

2016-09-20

Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2018-01-01

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

Immunomodulatory response of mice splenocytes induced by RcaL, a lectin isolated from cobia fish (Rachycentron canadum) serum.

PubMed

Coriolano, Marília Cavalcanti; Silva, Cynarha Daysy Cardoso da; Melo, Cristiane Moutinho Lagos de; Bezerra, Ranilson de Souza; Santos, Athiê Jorge Guerra; Pereira, Valéria Rêgo Alves; Coelho, Luana Cassandra Breitenbach Barroso

2012-11-01

This work reports the isolation of a serum lectin from cobia fish (Rachycentron canadum) named RcaL. Immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production were also performed. RcaL was obtained through precipitation with ammonium sulphate and affinity chromatography on a Concanavalin A-Sepharose 4B column. The ammonium sulphate fraction F3 showed the highest specific hemagglutinating activity and was applied to affinity chromatography. The lectin was eluted with methyl-α-D-mannopyranoside. RcaL showed highest affinity for methyl-α-D-mannopyranoside and D-mannose; eluted fractions of RcaL agglutinated rabbit erythrocytes (titre, 128(-1)) retained 66 % of chromatographed lectin activity, and the obtained purification factor was 1.14. Under reducing conditions, a polypeptide band of 19.2 kDa was revealed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE). PAGE confirmed RcaL as an acidic protein revealed in a single band. Cytotoxic and immunomodulatory assays with RcaL in mice splenocyte cultures showed that the lectin was not cytotoxic and induced higher interferon gamma and nitric oxide production in splenocyte cultures. Purified RcaL induced preferential Th1 response, suggesting that it acts as an immunomodulatory compound.

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

PubMed

Konziase, Benetode

2015-08-01

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

Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

Monocarboxylate and alpha-ketoglutarate carriers from bovine heart mitochondria. Purification by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate.

PubMed

Bolli, R; Nałecz, K A; Azzi, A

1989-10-25

2-Cyano-4-hydroxycinnamate was covalently linked, through a diazo bond, to Sepharose 4B, which had been elongated with a hydrophobic spacer. A Triton X-100 extract from bovine heart mitochondria was pre-purified by hydroxylapatite chromatography and passed through the 2-cyano-4-hydroxycinnamate affinity resin in the presence of 0.7% deoxycholate. At pH 6 and in the presence of 0.2 M sodium chloride, a single polypeptide with an Mr of 34,000 was eluted. Subsequently, at pH 8 and in the presence of 2-cyano-4-hydroxycinnamate, another single protein with an Mr of 31,500 was released. Both proteins were reconstituted into phospholipid vesicles and their transport activities were measured. High, delta pH-dependent, 2-cyanocinnamate-sensitive pyruvate uptake was measured in vesicles containing only the 34-kDa protein. alpha-Ketobutyrate and other alpha-ketomonocarboxylic acids were competitive inhibitors of the pyruvate uptake, whereas di- and tricarboxylates had only small effects. alpha-Ketoglutarate-alpha-ketoglutarate exchange could only be measured in vesicles containing the 31.5-kDa protein. The molecular weight of this protein and its functional properties were similar to those of the alpha-ketoglutarate carrier isolated by a different method (Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369). 2-Cyano-4-hydroxycinnamate inhibited the alpha-ketoglutarate exchange in a noncompetitive manner with an apparent Ki of 0.7 mM. It is concluded that by the described affinity chromatography procedure, two mitochondrial carriers transporting alpha-ketoacids, i.e. the monocarboxylate and the alpha-ketoglutarate carrier, could be purified in a functionally active state.

Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.

PubMed

Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N

2011-05-25

Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

Preparation of hydrophilic polymer-grafted polystyrene beads for hydrophilic interaction chromatography via surface-initiated atom transfer radical polymerization.

PubMed

Dai, Xiaojun; He, Yuan; Wei, Yinmao; Gong, Bolin

2011-11-01

A one-step procedure based on surface-initiated atom transfer radical polymerization (SI-ATRP) to hydrophilize monodisperse poly(chloromethylstyrene-co-divinylbenzene) beads has been presented in this work, using 2-hydroxyl-3-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]propyl 2-methylacrylate (HTMA) as a monomer. The chain length of the grafted poly(HTMA) was controlled via varying the ratio of HTMA to initiator on the surface of the beads. When using the grafted beads as a stationary phase in hydrophilic interaction chromatography (HILIC), good resolution for nucleobases/nucleosides was obtained with acetonitrile aqueous solution as an eluent; while for phenolic acids and glycosides, they could be eluted and separated in the presence of TFA. The retention time of the solutes increased with the amount of the grafted HTMA. The retention mechanisms of solutes were investigated by the effects of mobile phase composition and buffer pH on the retention of solutes. The results illustrated that the retention behaviors of the tested solutes were dominated by hydrogen bonding interaction and electrostatic interaction. From the chemical structure of the ligands, the modified beads could not only be used as a stationary phase in HILIC, but also act as a useful building block to develop new stationary phases for other chromatographic modes such as affinity media. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insight into the biochemical, kinetic and spectroscopic characterization of garlic (Allium sativum) phytocystatin: Implication for cardiovascular disease.

PubMed

Siddiqui, Mohd Faizan; Ahmed, Azaj; Bano, Bilqees

2017-02-01

Phytocystatins are cysteine proteinase inhibitors present in plants. They play crucial role in maintaining protease-anti protease balance and are involved in various endogenous processes. Thus, they are suitable and convenient targets for genetic engineering which makes their isolation and characterisation from different sources the need of the hour. In the present study a phytocystatin has been isolated from garlic (Allium sativum) by a simple two-step process using ammonium sulphate fractionation and gel filtration chromatography on Sephacryl S-100HR with a fold purification of 152.6 and yield 48.9%. A single band on native gel electrophoresis confirms the homogeneity of the purified inhibitor. The molecular weight of the purified inhibitor was found to be 12.5kDa as determined by SDS-PAGE and gel filtration chromatography. The garlic phytocystatin was found to be stable under broad range of pH (6-8) and temperature (30°C-60°C). Kinetic studies suggests that garlic phytocystatins are reversible and non-competitive inhibitors having highest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy revealed significant conformational change upon garlic phytocystatin-papain complex formation. Secondary structure analysis was performed using CD and FTIR. Garlic phytocystatin possesses 33.9% alpha-helical content as assessed by CD spectroscopy. Copyright © 2016 Elsevier B.V. All rights reserved.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

PubMed

Goncharuk, M V; Shul'ga, A A; Ermoliuk, Ia S; Tkach, E N; Goncharuk, S A; Pustovalova, Iu E; Mineev, K S; Bocharov, É V; Maslennikov, I V; Arsen'ev, A S; Kirpichnikov, M P

2011-01-01

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Analytical and semipreparative chiral separation of cis-itraconazole on cellulose stationary phases by high-performance liquid chromatography.

PubMed

Kurka, Ondřej; Kučera, Lukáš; Bednář, Petr

2016-07-01

cis-Itraconazole is a chiral antifungal drug administered as a racemate. The knowledge of properties of individual cis-itraconazole stereoisomers is vital information for medicine and biosciences as different stereoisomers of cis-itraconazole may possess different affinity to certain biological pathways in the human body. For this purpose, either chiral synthesis of enantiomers or chiral separation of racemate can be used. This paper presents a two-step high-performance liquid chromatography approach for the semipreparative isolation of four stereoisomers (two enantiomeric pairs) of itraconazole using polysaccharide stationary phases and volatile organic mobile phases without additives in isocratic mode. The approach used involves the separation of the racemate into three fractions (i.e. two pure stereoisomers and one mixed fraction containing the remaining two stereoisomers) in the first run and consequent separation of the collected mixed fraction in the second one. For this purpose, combination of cellulose tris-(4-methylbenzoate) and cellulose tris-(3,5-dimehylphenylcarbamate) columns with complementary selectivity for cis-itraconazole provided full separation of all four stereoisomers (with purity of each isomer > 97%). The stereoisomers were collected, their optical rotation determined and their identity confirmed based on the results of a previously published study. Pure separated stereoisomers are subjected to further biological studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

PubMed

Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

2015-10-01

Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization.

PubMed

Benavente, Rocio; Pessela, Benevides C; Curiel, Jose Antonio; de las Rivas, Blanca; Muñoz, Rosario; Guisán, Jose Manuel; Mancheño, Jose M; Cardelle-Cobas, Alejandra; Ruiz-Matute, Ana I; Corzo, Nieves

2015-04-30

A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.

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

PubMed

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

2013-01-01

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

Soluble expression and one-step purification of recombinant mouse interferon-λ3 in Escherichia coli.

PubMed

Wang, Y Q; Zhou, M; Zeng, L M; Gao, Q Y; Yuan, X L; Li, Y; Li, M C

2015-02-01

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

Experimental and in silico investigations of organic phosphates and phosphonates sorption on polymer-ceramic monolithic materials and hydroxyapatite.

PubMed

Pietrzyńska, Monika; Zembrzuska, Joanna; Tomczak, Rafał; Mikołajczyk, Jakub; Rusińska-Roszak, Danuta; Voelkel, Adam; Buchwald, Tomasz; Jampílek, Josef; Lukáč, Miloš; Devínsky, Ferdinand

2016-10-10

A method based on experimental and in silico evaluations for investigating interactions of organic phosphates and phosphonates with hydroxyapatite was developed. This quick and easy method is used for determination of differences among organophosphorus compounds of various structures in their mineral binding affinities. Empirical sorption evaluation was carried out using liquid chromatography with tandem mass spectrometry or UV-VIS spectroscopy. Raman spectroscopy was used to confirm sorption of organic phosphates and phosphonates on hydroxyapatite. Polymer-ceramic monolithic material and bulk hydroxyapatite were applied as sorbent materials. Furthermore, a Polymer-ceramic Monolithic In-Needle Extraction device was used to investigate both sorption and desorption steps. Binding energies were computed from the fully optimised structures utilising Density Functional Theory (DFT) at B3LYP/6-31+G(d,p) level. Potential pharmacologic and toxic effects of the tested compounds were estimated by the Prediction of the Activity Spectra of Substances using GeneXplain software. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification of charybdotoxine, a specific inhibitor of the high-conductance Ca/sup 2 +/-activated K/sup +/ channel

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

Smith, C.; Phillips, M.; Miller, C.

1986-11-05

Charybdotoxim is a high-affinity specific inhibitor of the high-conductance Ca/sup 2 +/-activated K/sup +/ channel found in the plasma membranes of many vertebrate cell types. Using Ca/sup 2 +/-activated K/sup +/ channels reconstituted into planar lipid bilayer membranes as an assay, the authors have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under the standard assay conditions, the purified toxin inhibits the Ca/sup 2 +/-activated K/sup +/ channelmore » with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity.« less

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

PubMed

Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

2014-12-03

Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

PubMed Central

Novakovic, Predrag; Huang, Yanyun Y.; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R.; Middleton, Dorothy M.; Loewen, Matthew E.; Kidney, Beverly A.; Simko, Elemir

2015-01-01

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization. PMID:25852227

Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta

PubMed Central

2014-01-01

Background This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDS-PAGE and N-terminal microsequencing. Results Four distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues. Conclusion Our results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family. PMID:24410890

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

PubMed

Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro

2011-01-01

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

NASA Astrophysics Data System (ADS)

Miles, Jeff; Formosa, Tim

1992-02-01

We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

[Separation of osteoclasts by lectin affinity chromatography].

PubMed

Itokazu, M; Tan, A; Tanaka, S

1991-09-01

Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

PubMed

Zhang, Pengpeng; Battchikova, Natalia; Paakkarinen, Virpi; Katoh, Hirokazu; Iwai, Masako; Ikeuchi, Masahiko; Pakrasi, Himadri B; Ogawa, Teruo; Aro, Eva-Mari

2005-09-01

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

One-step purification of nisin A by immunoaffinity chromatography.

PubMed

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-12-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

PubMed

Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

2015-12-07

The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

PubMed

Edupuganti, Soujanya Ratna; Edupuganti, Om Prakash; O'Kennedy, Richard; Defrancq, Eric; Boullanger, Stéphanie

2013-04-01

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

Improved Thermal Modulator for Gas Chromatography

NASA Technical Reports Server (NTRS)

Hasselbrink, Ernest Frederick, Jr.; Hunt, Patrick J.; Sacks, Richard D.

2008-01-01

An improved thermal modulator has been invented for use in a variant of gas chromatography (GC). The variant in question denoted as two-dimensional gas chromatography (2DGC) or GC-GC involves the use of three series-connected chromatographic columns, in the form of capillary tubes coated interiorly with suitable stationary phases (compounds for which different analytes exhibit different degrees of affinity). The two end columns are relatively long and are used as standard GC columns. The thermal modulator includes the middle column, which is relatively short and is not used as a standard GC column: instead, its temperature is modulated to affect timed adsorption and desorption of analyte gases between the two end columns in accordance with a 2DGC protocol.

One-step purification of nisin A by immunoaffinity chromatography.

PubMed Central

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-01-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method. PMID:9406424

Segregation of human peripheral blood lymphocytes according to their affinity for insolubilized histamine. Principal differences between males and females.

PubMed Central

Tartakovsky, B; Segal, S; Shani, A; Hellerstein, S; Weinstein, Y; Bentwich, Z

1979-01-01

An attempt was made to investigate the possible existence of differences in the composition of peripheral blood lymphocytes between males and females. Using affinity chromatography of human peripheral mononuclear cells on insolubilized histamine together with staining by fluoresceinated histamine-rabbit serum albumin (HRSA) we revealed that males possess a significantly higher proportion of mononuclear cells which bind to HRSA. These results are also reflected in sex-related differences in proliferative responses of the HRSA-non-adherent mononuclear cell population to T cell-dependent mitogens antigens and allogeneic mononuclear cells. PMID:160849

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies.

PubMed

Brne, P; Lim, Y-P; Podgornik, A; Barut, M; Pihlar, B; Strancar, A

2009-03-27

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.

Evidence of land plant affinity for the Devonian fossil Protosalvinia (Foerstia)

USGS Publications Warehouse

Romankiw, L.A.; Hatcher, P.G.; Roen, J.B.

1988-01-01

The Devonian plant fossil Protosalvinia (Foerstia) has been examined by solid-state 13C nuclear magnetic resonance spectroscopy (NMR) and pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). Results of these studies reveal that the chemical structure of Protosalvinia is remarkably similar to that of coalified wood. A well-defined phenolic carbon peak in the NMR spectra and the appearance of phenol and alkylated phenols in pyrolysis products are clearly indicative of lignin-like compounds. These data represent significant new information on the chemical nature of Protosalvinia and provide the first substantial organic geochemical evidence for land plant affinity. -Authors

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

PubMed

Pinto, Nuno D S; Uplekar, Shaunak D; Moreira, Antonio R; Rao, Govind; Frey, Douglas D

2017-01-01

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Two-step purification method of vitellogenin from three teleost fish species: rainbow trout (Oncorhynchus mykiss), gudgeon (Gobio gobio) and chub (Leuciscus cephalus).

PubMed

Brion, F; Rogerieux, F; Noury, P; Migeon, B; Flammarion, P; Thybaud, E; Porcher, J M

2000-01-14

A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.

Separation of Albumin, Ceruloplasmin, and Transferrin from Human Plasma.

ERIC Educational Resources Information Center

Barnes, Grady; Frieden, Earl

1982-01-01

Procedures are provided for separating the principal metalloproteins (albumin, ceruloplasmin, and transferrin) from plasma using column chromatographic techniques. The experiment can be completed in two separate three-hour laboratory periods during which column chromatography is illustrated and the effect of pH on charge and affinity of a protein…

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

USDA-ARS?s Scientific Manuscript database

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

Purification and characterization pecan (Carya Illinoinensis) vicilin, a putative food allergen (abstract)

USDA-ARS?s Scientific Manuscript database

The pecan seed storage protein vicilin, a putative food allergen, was recombinantly expressed for and purified by a combination of metal affinity and gel filtration chromatography. The protein was crystallized and studied by crystallography. The obtained crystals belonged to space group P212121 with...

SCREENING PROCESSED MILK FOR VOLATILE ORGANIC COMPOUNDS USING VACUUM DISTILLATION/GAS CHROMATOGRAPHY/MASS SPECTROMETRY

EPA Science Inventory

An adaptation of Office of Solid Waste and Emergency Response' Test Methods for Evaluating Solid Waste Physical/Chemical Methods (SW-846) method 8261 to analyze milk for an expanded list of volatile organic compounds is presented. The milk matriz exhibits a strong affinity for o...

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

EPA Science Inventory

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

A protein with anion exchange properties found in the kidney proximal tubule.

PubMed

Soleimani, M; Bizal, G L; Anderson, C C

1993-09-01

One important mechanism for reabsorption of chloride in the kidney proximal tubule involves anion exchange of chloride for a base. Anion exchange transport systems in general demonstrate sensitivity to inhibition by disulfonic stilbenes, probenecid, furosemide, and the arginyl amino group modifier phenylglyoxal. Using disulfonic stilbene affinity chromatography, we have identified and partially purified a protein with anion exchanger properties in luminal membrane vesicles isolated from rabbit kidney cortex. This protein has a molecular weight of 162 kD. The binding of the 162 kD protein to the stilbene affinity matrix is inhibited by disulfonic stilbenes, probenecid, furosemide, and phenylglyoxal. Reconstitution of the proteins eluted from the affinity matrix into liposomes demonstrates anion exchange activity as assayed by radiolabeled chloride influx. Deletion of the 162 kD protein from the eluted mixture by probenecid diminishes the anion exchanger activity in the reconstituted liposomes. Further purification of the disulfonic stilbene column eluant by Econo-Pac Q ion exchange chromatography resulted in significant enrichment in 162 kD protein abundance and also anion exchange activity in reconstituted liposomes. The results of the above experiments strongly suggest that the 162 kD protein is an anion exchanger. Insight into the functional and molecular characteristics of this protein should provide important information about the mechanism(s) of chloride reabsorption in the kidney proximal tubule.

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities.

PubMed

Lee, M Y; Whyte, W A

1984-05-01

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.

Revealing multi-binding sites for taspine to VEGFR-2 by cell membrane chromatography zonal elution.

PubMed

Du, Hui; Wang, Sicen; Ren, Jing; Lv, Nan; He, Langchong

2012-03-01

A new high-expression vascular endothelial growth factor receptor-2 (VEGFR-2) cell membrane chromatography (CMC) method was developed to investigate the affinity of ligands for VEGFR-2. An HEK293 VEGFR-2/CMC system was applied to specifically recognize ligands acting on VEGFR-2. Sorafenib was used as a mobile phase additive to evaluate the effect of the marker's concentration on the retention of sorafenib and taspine, respectively. The relationship among the retention, the types of binding sites and the affinity of taspine binding to VEGFR-2 has also been concerned. The retention behavior indicated that sorafenib had two major binding regions on VEGFR-2, and that taspine might act as a multi-target VEGFR-2 inhibitor with similar biological activity to sorafenib. The equilibrium dissociation constants (K(D)) obtained from the model are (5.25 ± 0.31) × 10⁻⁷ and (9.88 ± 0.54) × 10⁻⁵ mol L⁻¹ for sorafenib at the high- and low-affinity sites, respectively, and the corresponding values for taspine are (3.88 ± 0.31) × 10⁻⁶ and (7.04 ± 0.49)×10⁻⁵ mol L⁻¹. The two types of binding sites contributed about a 1:2 ratio on the retention of taspine. Copyright © 2012 Elsevier B.V. All rights reserved.

Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

PubMed

Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

2016-04-05

Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

A Variable Step-Size Proportionate Affine Projection Algorithm for Identification of Sparse Impulse Response

NASA Astrophysics Data System (ADS)

Liu, Ligang; Fukumoto, Masahiro; Saiki, Sachio; Zhang, Shiyong

2009-12-01

Proportionate adaptive algorithms have been proposed recently to accelerate convergence for the identification of sparse impulse response. When the excitation signal is colored, especially the speech, the convergence performance of proportionate NLMS algorithms demonstrate slow convergence speed. The proportionate affine projection algorithm (PAPA) is expected to solve this problem by using more information in the input signals. However, its steady-state performance is limited by the constant step-size parameter. In this article we propose a variable step-size PAPA by canceling the a posteriori estimation error. This can result in high convergence speed using a large step size when the identification error is large, and can then considerably decrease the steady-state misalignment using a small step size after the adaptive filter has converged. Simulation results show that the proposed approach can greatly improve the steady-state misalignment without sacrificing the fast convergence of PAPA.

Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability.

PubMed

Takakura, Yoshimitsu; Sofuku, Kozue; Tsunashima, Masako

2013-03-10

A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinity purification of biotinylated biomolecules. Although several proteins, chemically or genetically modified from avidin or streptavidin, with reversible biotin-binding have been reported, they have been problematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin in biotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the serine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin 2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound to biotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coli by means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin 2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotin was 2.8-4.4×10(-7)M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, such as high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating that tamavidin 2-REV is a powerful tool for the purification of biotinylated biomolecules. Copyright © 2013 Elsevier B.V. All rights reserved.

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

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

James, W.M.; Emerick, M.C.; Agnew, W.S.

1989-07-11

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated bindingmore » and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.« less

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

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

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

2012-04-01

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus,more » led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.« less

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

PubMed

Salehi, Nasrin; Peng, Ching-An

2016-07-08

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

Separation and characterization of polycyclic aromatic hydrocarbons and alkylphenols in coal derived solvents

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

Hurtubise, R.J.; Allen, T.W.; Hussain, A.

1981-03-29

Dry-column chromatography with an aluminum oxide stationary phase and a n-hexane-ether (19:1) mobile phase was used to separate polycyclic aromatic hydrocarbons (PAH) by ring size. Prior to the dry-column chromatography step, the coal derived solvents were added to an acid treated silica gel column and eluted with chloroform. This step removed pyridine-type nitrogen heterocycles. After separation of the individual ring fractions, the fractions were further separated by either thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). If TLC was used, then after separation fluorescence profiles of each PAH ring fraction distributed on 30%-acetylated cellulose chromatoplates were obtained withmore » a spectrodensitometer. Measurement of fluorescence peak heights gave an approximate measure of the amount of the 3-, 4-, 5-, and 6- ring PAH. For HPLC separation, the 3- and 4- ring PAH fractions obtained from the dry-column chromatography step were separated with a ..mu..-Bondapak C/sub 18/ column and methanol:water (65:35) mobile phase. The HPLC separated PAH were characterized by chromatographic correlation factors and corrected fluorescence excitation spectra. Alkylphenols were identified in coal recycle solvent sample following separation by HPLC.« less

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

PubMed Central

Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

2011-01-01

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4. The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach. PMID:21968976

Affinity extraction of emerging contaminants from water based on bovine serum albumin as a binding agent.

PubMed

Papastavros, Efthimia; Remmers, Rachael A; Snow, Daniel D; Cassada, David A; Hage, David S

2018-03-01

Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides, and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on-line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions than a traditional C 18 support for most of the tested emerging contaminants. Potential advantages of using these protein-based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

PubMed Central

Serrano, A.; Cordoba, F.; Gonzalez-Reyes, J. A.; Navas, P.; Villalba, J. M.

1994-01-01

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate. PMID:12232306

Column chromatography as a useful step in purification of diatom pigments.

PubMed

Tokarek, Wiktor; Listwan, Stanisław; Pagacz, Joanna; Leśniak, Piotr; Latowski, Dariusz

2016-01-01

Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies.

Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin.

PubMed Central

Brett, M; Findlay, J B

1983-01-01

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported. PMID:6224479

Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

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

Senogles, S.E.; Caron, M.G.

1986-05-01

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

Immunoregulation.

DTIC Science & Technology

1981-05-01

variety of antigens, KLH, GAT, TGAL and antigens from pathogenic bacteria such as Streptococcus mutans . Furthermore, we now have these systems...histocompatibility complex; PBL, peripheral blood lymphocytes; SAI/II, Streptococcus mutans antigen I/II complex; MHFSAI/II, monkey helper factor specific...from Streptococcus mutans . Helper activity was removed from supernatants of monkey cells by affinity chromatography on Sepharose 4B insolubilized

Purification and Analysis of Colorful Hypothetical Open Reading Frames: An Inexpensive Gateway Laboratory

ERIC Educational Resources Information Center

DeSantis, Kara A.; Reinking, Jeffrey L.

2011-01-01

This laboratory exercise is an inquiry-based investigation developed around the core experiment where students, working alone or in groups, each purify and analyze their own prescreened colored proteins using immobilized metal affinity chromatography (IMAC). Here, we present reagents and protocols that allow 12 different proteins to be purified in…

A Versatile and Inexpensive Enzyme Purification Experiment for Undergraduate Biochemistry Labs.

ERIC Educational Resources Information Center

Farrell, Shawn O.; Choo, Darryl

1989-01-01

Develops an experiment that could be done in two- to three-hour blocks and does not rely on cold room procedures for most of the purification. Describes the materials, methods, and results of the purification of bovine heart lactate dehydrogenase using ammonium sulfate fractionation, dialysis, and separation using affinity chromatography and…

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

PubMed

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

2010-01-01

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

Liquid-phase-based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis – An update covering the period 2011-2014

PubMed Central

Puangpila, Chanida; Mayadunne, Erandi; Rassi, Ziad El

2015-01-01

This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field. PMID:25287967

Characterization of mouse natural killer cell activating factor (NKAF) induced by OK-432: evidence for interferon- and interleukin 2-independent NK cell activation.

PubMed Central

Ichimura, O.; Suzuki, S.; Sugawara, Y.; Osawa, T.

1984-01-01

The bacterial immunopotentiator OK-432 induced natural killer cell activating factor (NKAF) from mouse spleen cells. OK-432-induced NKAF showed a single peak with an apparent mol. wt of 70 Kd by Sephadex G-100 chromatography and OK-432-induced interleukin 2 (IL-2) had the same mol. wt as NKAF. However, OK-432-induced interferon (IFN) showed molecular heterogeneity with two peaks at 90 Kd and 45 Kd. Further purification was achieved by Blue Sepharose affinity chromatography which copurified NKAF and IFN. The affinity-purified NKAF, however, was stable to heat (56 degrees C) and acid (pH 2) treatments. Moreover, anti-IFN failed to abolish NKAF activity and this activity was not absorbed by IL-2 dependent T cells. From isoelectric focusing analysis, a dissociation of NKAF and IFN was observed over the range of pI 6.5 to 8.0. Based on these results, KNAF appears to be a new kind of cytokine distinguishable from IFN and IL-2. PMID:6204667

Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death.

PubMed

Zamulaeva, I A; Lekakh, I V; Kiseleva, V I; Gabai, V L; Saenko, A S; Shevchenko, A S; Poverenny, A M

1997-08-18

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion-exchange chromatography and were then affinity-isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2 +/- 10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7 +/- 7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

NASA Astrophysics Data System (ADS)

Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

2013-03-01

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

[Assays of HbA1c and Amadori products in human biology].

PubMed

Gillery, P

2014-09-01

Different Amadori products, formed during the early steps of the non-enzymatic glycation of proteins, may be assayed in current practice in human biology. The most important marker is HbA1c, resulting from the binding of glucose to the N-terminal extremity of HbA beta chains. HbA1c may be evaluated by various techniques (ion exchange or affinity high performance liquid chromatography, capillary electrophoresis, immunoassay, enzymatic technique) and is considered the best marker of diabetic patient survey. Due to its irreversible and cumulative formation, it provides a retrospective information on the glycemic balance over the four to eight weeks preceding blood collection. It benefits from an international standardization, based on a reference method using liquid chromatography coupled to capillary electrophoresis or mass spectrometry, maintained by an international network of reference laboratories. When HbA1c assay cannot be used (anemia, hemolysis, hemoglobinopathy) or when a shorter period of glycemic equilibrium must be evaluated (child and adolescent, pregnancy, therapeutic changes), other Amadori products may be assayed, like plasma fructosamine (all plasma glycated proteins) or glycated albumin. Nevertheless, these assays are less used in practice, because their semiological value has been less evidenced. Besides, fructosamine assay lacks specificity, and glycated albumin assay has been described recently. An expanding use of HbA1c assay is expected, especially for the diagnosis of diabetes mellitus and the evaluation of other risks, especially cardiovascular ones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Purification and characterization of a novel phloretin-2'-O-glycosyltransferase favoring phloridzin biosynthesis.

PubMed

Zhang, Tingjing; Liang, Jianqiang; Wang, Panxue; Xu, Ying; Wang, Yutang; Wei, Xinyuan; Fan, Mingtao

2016-10-12

Phloretin-2'-O-glycosyltransferase (P2'GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2'GT of Malus x domestica (MdP2'GT) with a specific activity of 46.82 μkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified MdP2'GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2'GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make MdP2'GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2'GT displayed the lowest K m and the highest k cat /K m for phloretin and UDPG compared to all previously reported P2'GTs, making MdP2'GT favor phloridzin synthesis the most.

Purification and characterization of a novel phloretin-2′-O-glycosyltransferase favoring phloridzin biosynthesis

PubMed Central

Zhang, Tingjing; Liang, Jianqiang; Wang, Panxue; Xu, Ying; Wang, Yutang; Wei, Xinyuan; Fan, Mingtao

2016-01-01

Phloretin-2′-O-glycosyltransferase (P2′GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2′GT of Malus x domestica (MdP2′GT) with a specific activity of 46.82 μkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified MdP2′GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2′GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make MdP2′GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2′GT displayed the lowest Km and the highest kcat/Km for phloretin and UDPG compared to all previously reported P2′GTs, making MdP2′GT favor phloridzin synthesis the most. PMID:27731384

The search for and identification of amino acids, nucleobases and nucleosides in samples returned from Mars

NASA Technical Reports Server (NTRS)

Gehrke, Charles W.; Ponnamperuma, Cyril; Kuo, Kenneth C.; Stalling, David L.; Zumwalt, Robert W.

1989-01-01

An investigation of the returned Mars samples for biologically important organic compounds, with emphasis on amino acid, the puring and pyrimidine bases, and nucleosides is proposed. These studies would be conducted on subsurface samples obtained by drilling past the surface oxidizing layer with emphasis on samples containing the larges quantities of organic carbon as determined by the rover gas chromatographic mass spectrometer (GCMS). Extraction of these molecules from the returned samples will be performed using the hydrothermal extraction technique described by Cheng and Ponnamperuma. More rigorous extraction methods will be developed and evaluated. For analysis of the extract for free amino acids or amino acids present in a bound or peptidic form, aliquots will be analyzed by capillary GCMS both before and after hydrolysis with 6N hydrochloric acid. Establishment of the presence of amino acids would then lead to the next logical step which would be the use of chiral stationary gas chromatography phases to determine the enatiomeic composition of the amino acids present, and thus potentially establish their biotic or abiotic origin. Confirmational analyses for amino acids would include ion-exchange and reversed-phase liquid chromatographic analysis. For analyses of the returned Mars samples for nucleobases and nucleosides, affinity and reversed-phase liquid chromatography would be utilized. This technology coupled with scanning UV detection for identification, presents a powerful tool for nucleobase and nucleoside analysis. Mass spectrometric analysis of these compounds would confirm their presence in samples returned form Mars.

Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini.

PubMed

Barata, Ricardo Andrade; Andrade, Milton Hercules Guerra; Rodrigues, Roberta Dias; Castro, Ieso Miranda

2002-01-01

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.

Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.

PubMed

Yaacob, Mohd Adilin; Hasan, Wan Atiqah Najiah Wan; Ali, Mohd Shukuri Mohamad; Rahman, Raja Noor Zaliha Raja Abdul; Salleh, Abu Bakar; Basri, Mahiran; Leow, Thean Chor

2014-01-01

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.

Probes for narcotic receptor mediated phenomena. 43. Synthesis of the ortho-a and para-a, and improved synthesis and optical resolution of the ortho-b and para–b oxide-bridged phenylmorphans: Compounds with moderate to low opioid-receptor affinity

PubMed Central

Li, Feng; Folk, John E.; Cheng, Kejun; Kurimura, Muneaki; Deck, Jason A.; Deschamps, Jeffrey R.; Rothman, Richard B.; Dersch, Christina M.; Jacobson, Arthur E.; Rice, Kenner C.

2011-01-01

N-Phenethyl-substituted ortho-a and para-a oxide-bridged phenylmorphans have been obtained through an improved synthesis and their binding affinity examined at the various opioid receptors. Although the N-phenethyl substituent showed much greater affinity for μ- and κ-opioid receptors than their N-methyl relatives (e.g., Ki = 167 nM and 171 nM at μ- and κ-receptors vs >2800 and 7500 nM for the N-methyl ortho-a oxide-bridged phenylmorphan), the a-isomers were not examined further because of their relatively low affinity. The N-phenethyl substituted ortho-b and para-b oxide-bridged phenylmorphans were also synthesized and their enantiomers were obtained using supercritical fluid chromatography. Of the four enantiomers, only the (+)-ortho-b isomer had moderate affinity for μ- and κ-receptors (Ki = 49 and 42 nM, respectively, and it was found to also have moderate μ- and κ-opioid antagonist activity in the [35S]GTP-γ-S assay (Ke = 31 and 26 nM). PMID:21684752

Adsorption of ethoxylated styrene oxide and polyacrylic acid and mixtures there of on organic pigment.

PubMed

Wijting, W K; Laven, J; van Benthem, R A T M; de With, G

2008-11-01

The adsorption of two polymeric surfactants on an organic pigment was investigated. As surfactants the anionic polyacrylic acid sodium salt (PANa, M(W)=15,000) and a non-ionic block copolymer surfactant based on styrene oxide (SO) and ethylene oxide (EO) (M(W)=1500) were used. The adsorption behavior was analyzed by size exclusion chromatography of the supernatant after centrifugation of the pigment dispersions. It was found that PANa has no affinity to the pigment, whereas SO-EO has a strong affinity to the pigment surface. Competitive adsorption of PANa and SO-EO was not observed. Addition of SO-EO yields stable dispersions.

Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System.

PubMed

Henseleit, Anja; Pohl, Carolin; Kaltenbach, Hans-Michael; Hettwer, Karina; Simon, Kirsten; Uhlig, Steffen; Haustein, Natalie; Bley, Thomas; Boschke, Elke

2015-01-19

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System

PubMed Central

Henseleit, Anja; Pohl, Carolin; Kaltenbach, Hans-Michael; Hettwer, Karina; Simon, Kirsten; Uhlig, Steffen; Haustein, Natalie; Bley, Thomas; Boschke, Elke

2015-01-01

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step. PMID:25607476

Robust estimation-free prescribed performance back-stepping control of air-breathing hypersonic vehicles without affine models

NASA Astrophysics Data System (ADS)

Bu, Xiangwei; Wu, Xiaoyan; Huang, Jiaqi; Wei, Daozhi

2016-11-01

This paper investigates the design of a novel estimation-free prescribed performance non-affine control strategy for the longitudinal dynamics of an air-breathing hypersonic vehicle (AHV) via back-stepping. The proposed control scheme is capable of guaranteeing tracking errors of velocity, altitude, flight-path angle, pitch angle and pitch rate with prescribed performance. By prescribed performance, we mean that the tracking error is limited to a predefined arbitrarily small residual set, with convergence rate no less than a certain constant, exhibiting maximum overshoot less than a given value. Unlike traditional back-stepping designs, there is no need of an affine model in this paper. Moreover, both the tedious analytic and numerical computations of time derivatives of virtual control laws are completely avoided. In contrast to estimation-based strategies, the presented estimation-free controller possesses much lower computational costs, while successfully eliminating the potential problem of parameter drifting. Owing to its independence on an accurate AHV model, the studied methodology exhibits excellent robustness against system uncertainties. Finally, simulation results from a fully nonlinear model clarify and verify the design.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

PubMed

Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

2016-02-01

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Accelerated solvent extraction followed by on-line solid-phase extraction coupled to ion trap LC/MS/MS for analysis of benzalkonium chlorides in sediment samples

USGS Publications Warehouse

Ferrer, I.; Furlong, E.T.

2002-01-01

Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed by ASE, were 85% for C12BAC and 79% for C14-BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 ??g/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification.

PubMed

Shin, Min Jae; Tan, Lihan; Jeong, Min Ho; Kim, Ji-Heung; Choe, Woo-Seok

2011-08-05

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis.

PubMed

King, Cory; Patel, Rekha; Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Gu, Zhenyu; Liu, Hongcheng

2018-05-15

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-11-01

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

Relative binding affinities of monolignols to horseradish peroxidase

DOE PAGES

Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; ...

2016-07-22

Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group andmore » a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.« less

d-Fructose-Decorated Poly(ethylene imine) for Human Breast Cancer Cell Targeting.

PubMed

Englert, Christoph; Pröhl, Michael; Czaplewska, Justyna A; Fritzsche, Carolin; Preußger, Elisabeth; Schubert, Ulrich S; Traeger, Anja; Gottschaldt, Michael

2017-08-01

The high affinity of GLUT5 transporter for d-fructose in breast cancer cells has been discussed intensely. In this contribution, high molar mass linear poly(ethylene imine) (LPEI) is functionalized with d-fructose moieties to combine the selectivity for the GLUT5 transporter with the delivery potential of PEI for genetic material. The four-step synthesis of a thiol-group bearing d-fructose enables the decoration of a cationic polymer backbone with d-fructose via thiol-ene photoaddition. The functionalization of LPEI is confirmed by 2D NMR techniques, elemental analysis, and size exclusion chromatography. Importantly, a d-fructose decoration of 16% renders the polymers water-soluble and eliminates the cytotoxicity of PEI in noncancer L929 cells, accompanied by a reduced unspecific cellular uptake of the genetic material. In contrast, the cytotoxicity as well as the cell specific uptake is increased for triple negative MDA-MB-231 breast cancer cells. Therefore, the introduction of d-fructose shows superior potential for cell targeting, which can be assumed to be GLUT5 dependent. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

PubMed

Rojo, Liliana; García-Carreño, Fernando; de Los Angeles Navarrete del Toro, Maria

2013-02-01

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Hippophae rhamnoides N-glycoproteome analysis: a small step towards sea buckthorn proteome mining.

PubMed

Sougrakpam, Yaiphabi; Deswal, Renu

2016-10-01

Hippophae rhamnoides is a hardy shrub capable of growing under extreme environmental conditions namely, high salt, drought and cold. Its ability to grow under extreme conditions and its wide application in pharmaceutical and nutraceutical industry calls for its in-depth analysis. N-glycoproteome mining by con A affinity chromatography from seedling was attempted. The glycoproteome was resolved on first and second dimension gel electrophoresis. A total of 48 spots were detected and 10 non-redundant proteins were identified by MALDI-TOF/TOF. Arabidopsis thaliana protein disulfide isomerase-like 1-4 (ATPDIL1-4) electron transporter, protein disulphide isomerase, calreticulin 1 (CRT1), glycosyl hydrolase family 38 (GH 38) protein, phantastica, maturase k, Arabidopsis trithorax related protein 6 (ATXR 6), cysteine protease inhibitor were identified out of which ATXR 6, phantastica and putative ATPDIL1-4 electron transporter are novel glycoproteins. Calcium binding protein CRT1 was validated for its calcium binding by stains all staining. GO analysis showed involvement of GH 38 and ATXR 6 in glycan and lysine degradation pathways. This is to our knowledge the first report of glycoproteome analysis for any Elaeagnaceae member.

Cloning and expression of gamma carbonic anhydrase from Serratia sp. ISTD04 for sequestration of carbon dioxide and formation of calcite.

PubMed

Srivastava, Shaili; Bharti, Randhir Kumar; Verma, Praveen Kumar; Thakur, Indu Shekhar

2015-01-01

Bacterial strains isolated from marble mines rock and enriched in the chemostat culture with different concentrations of sodium bicarbonate. The enriched consortium had six bacterial isolates. One of bacterium isolate showed carbonic anhydrase (CA) activity by catalyzing the reversible hydration reaction of carbon dioxide to bicarbonate. The bacterium was identified as Serratia sp. by 16S rRNA sequence analysis. The carbonic anhydrase gene from Serratia sp. was found to be homologous with gamma carbonic anhydrase. The carbonic anhydrase gene was cloned in PET21b(+) and expressed it in recombinant Escherichia coli BL21 (DE3) with His-tag at the C-terminus. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. Expected size of carbonic anhydrase was approximately 29 kDa in SDS-PAGE gel. Recombinant carbonic anhydrase enzyme was used for biomineralization-based conversion of atmospheric CO2 into valuable calcite minerals. The calcification was confirmed by using XRD, FTIR, EDX and SEM analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

An Iron 13S-Lipoxygenase with an α-Linolenic Acid Specific Hydroperoxidase Activity from Fusarium oxysporum

PubMed Central

Brodhun, Florian; Cristobal-Sarramian, Alvaro; Zabel, Sebastian; Newie, Julia; Hamberg, Mats; Feussner, Ivo

2013-01-01

Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. Fusarium oxysporum) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in F. oxysporum, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in E. coli, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C18-polyunsaturated fatty acids to 13S-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives. PMID:23741422

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of pseudo death-effector domain of HIPPI, a molecular partner of Huntingtin-interacting protein HIP-1

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

Banerjee, Manisha; Majumder, Pritha; Bhattacharyya, Nitai P.

2006-12-01

A pseudo death-effector domain (pDED) of HIPPI, a partner of Huntingtin-interacting protein HIP1, has been cloned, overexpressed and crystallized. The crystals of pDED-HIPPI diffracted to 2.2 Å. The formation of a heterodimer between Huntingtin-interacting protein-1 (HIP-1) and its novel partner HIPPI (HIP-1 protein interactor) through their pseudo death-effector domains (pDEDs) is a key step that recruits caspase-8 and initiates apoptosis. This could be one of the pathways by which apoptosis is increased in Huntington’s disease (HD). A construct consisting of the pDED of HIPPI has been cloned and overexpressed as 6NH-tagged protein and purified by Ni–NTA affinity chromatography. Crystals ofmore » the pDED of HIPPI were grown in space group P4{sub 1}, with unit-cell parameters a = b = 77.42, c = 33.31 Å and a calculated Matthews coefficient of 1.88 Å{sup 3} Da{sup −1} (33% solvent content) with two molecules per asymmetric unit.« less

Characterization of adsorption on the stationary phase using high-performance immunoaffinity chromatography.

PubMed

Nielsen, R G; Wilson, G S

1987-12-25

Low-level adsorption on the stationary phase has been studied using immunochemical reagents. An immunoaffinity column has been evaluated using affinity-purified radioisotope-labeled monoclonal antibodies. Recovery experiments including continuous immunosorbent monitoring have been performed. Proper characterization of an immunoaffinity separation can result in the recovery of immunologically active material in high yield.

Glycation of antibodies: Modification, methods and potential effects on biological functions.

PubMed

Wei, Bingchuan; Berning, Kelsey; Quan, Cynthia; Zhang, Yonghua Taylor

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

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.

Analytical Glycobiology at High Sensitivity: Current Approaches and Directions

PubMed Central

Novotny, Milos V.; Alley, William R.; Mann, Benjamin F.

2013-01-01

This review summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures. The main analytical techniques used in the fields of glycomics and glycoproteomics involve different modes of mass spectrometry and their combinations with capillary separation methods such as microcolumn liquid chromatography and capillary electrophoresis. The needs for high-sensitivity measurements have been emphasized in the oligosaccharide profiling used in the field of biomarker discovery through MALDI mass spectrometry. High-sensitivity profiling of both glycans and glycopeptides from biological fluids and tissue extracts has been aided significantly through lectin preconcentration and the uses of affinity chromatography. PMID:22945852

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

Substrate binding ability of chemically inactivated pectinase for the substrate pectic acid.

PubMed

Chiba, Y; Kobayashi, M

1995-07-01

Pectinase (polygalacturonase) was purified from a commercial pectinase preparation from a mold. Substrate binding of pectinase was measured by centrifugal affinity chromatography using an immobilized substrate, pectic acid. Desorption of pectinase from the affinity matrix with the substrate pectin and pectic acid gave Kd values of 5.3 and 8.5 mg/ml, respectively. Chemical modification of pectinase by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and diethyl pyrocarbonate (DEP) caused a loss of most of the enzyme activity, but the substrate binding ability was not impaired. Thus, the pectinase preparation was digested with lysyl endopeptidase and the resulting peptides were treated with pectic acid-affinity gel. Three peptide fragments, which were recovered from the affinity column and sequenced, were identical to sequences in the second pectinase gene from Aspergillus niger. The first peptide contained 17 amino acids, Asp101-Ser117, and the second and third peptides corresponded to 18 amino acids of Asn152-Asp169. These results indicate that the inactivated pectinase retained substrate binding ability and would function as an acidic polysaccharide recognizing protein.

Diagonal chromatography to study plant protein modifications.

PubMed

Walton, Alan; Tsiatsiani, Liana; Jacques, Silke; Stes, Elisabeth; Messens, Joris; Van Breusegem, Frank; Goormachtig, Sofie; Gevaert, Kris

2016-08-01

An interesting asset of diagonal chromatography, which we have introduced for contemporary proteome research, is its high versatility concerning proteomic applications. Indeed, the peptide modification or sorting step that is required between consecutive peptide separations can easily be altered and thereby allows for the enrichment of specific, though different types of peptides. Here, we focus on the application of diagonal chromatography for the study of modifications of plant proteins. In particular, we show how diagonal chromatography allows for studying proteins processed by proteases, protein ubiquitination, and the oxidation of protein-bound methionines. We discuss the actual sorting steps needed for each of these applications and the obtained results. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation and identification of calcium-chelating peptides from Pacific cod skin gelatin and their binding properties with calcium.

PubMed

Wu, Wenfei; Li, Bafang; Hou, Hu; Zhang, Hongwei; Zhao, Xue

2017-12-13

A calcium-chelating peptide is considered to have the ability to improve calcium absorption. In this study, Pacific cod skin gelatin hydrolysates treated with trypsin for 120 min exhibited higher calcium-chelating activity. Sequential chromatography, involving hydroxyapatite affinity chromatography and reversed phase high performance liquid chromatography, was used for the purification of calcium-chelating peptides. Two novel peptides with the typical characteristics of collagen were sequenced as GDKGESGEAGER and GEKGEGGHR based on LC-HRMS/MS, which showed a high affinity to calcium. Calcium-peptide complexation was further characterized by ESI-MS (MS and MS/MS) and FTIR spectroscopy. The results showed that the complexation of the two peptides with calcium was conducted mainly at the ratio of 1 : 1. The amino terminal group and the peptide bond of the peptide backbone as well as the amino group of the lysine side chain and the carboxylate of the glutamate side chain were the possible calcium binding sites for the two peptides. Meanwhile, several amino acid side chain groups, including the hydroxyl (Ser) and carboxylate (Asp) of GDKGESGEAGER and the imine (His) of GEKGEGGHR, were crucial in the complexation. The arginine residue in GEKGEGGHR also participated in the calcium coordination. Additionally, several active fragments with calcium-chelating activity were obtained using MS/MS spectra, including GDKGESGEAGE, GEAGER, GEK, EKG and KGE. This study suggests that gelatin-derived peptides have the potential to be used as a calcium-chelating ingredient to combat calcium deficiency.

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.

PubMed

Santos, João H P M; Costa, Iris M; Molino, João V D; Leite, Mariana S M; Pimenta, Marcela V; Coutinho, João A P; Pessoa, Adalberto; Ventura, Sónia P M; Lopes, André M; Monteiro, Gisele

2017-03-01

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His) 6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni 2+ -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg -1 ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017. © 2016 American Institute of Chemical Engineers.

Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

PubMed

Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

2018-04-25

Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

PubMed

Lalev, A I; Abeyrathne, P D; Nazar, R N

2000-09-08

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. Copyright 2000 Academic Press.

Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements.

PubMed

Gjoka, Xhorxhi; Gantier, Rene; Schofield, Mark

2017-01-20

The goal of this study was to adapt a batch mAb purification chromatography platform for continuous operation. The experiments and rationale used to convert from batch to continuous operation are described. Experimental data was used to design chromatography methods for continuous operation that would exceed the threshold for critical quality attributes and minimize the consumables required as compared to batch mode of operation. Four unit operations comprising of Protein A capture, viral inactivation, flow-through anion exchange (AEX), and mixed-mode cation exchange chromatography (MMCEX) were integrated across two Cadence BioSMB PD multi-column chromatography systems in order to process a 25L volume of harvested cell culture fluid (HCCF) in less than 12h. Transfer from batch to continuous resulted in an increase in productivity of the Protein A step from 13 to 50g/L/h and of the MMCEX step from 10 to 60g/L/h with no impact on the purification process performance in term of contaminant removal (4.5 log reduction of host cell proteins, 50% reduction in soluble product aggregates) and overall chromatography process yield of recovery (75%). The increase in productivity, combined with continuous operation, reduced the resin volume required for Protein A and MMCEX chromatography by more than 95% compared to batch. The volume of AEX membrane required for flow through operation was reduced by 74%. Moreover, the continuous process required 44% less buffer than an equivalent batch process. This significant reduction in consumables enables cost-effective, disposable, single-use manufacturing. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of Bacillus anthracis Protective Antigen in Bacillus megaterium

DTIC Science & Technology

2004-03-01

was easily purified to homogeneity in a single step by ion exchange chromatography. N-terminal amino acid sequencing of the final product confirmed...and this material was purified in a single step by ion-exchange chromatography. N-terminal amino acid sequencing definitively proved that the rPA was...production of a poly-D-glutamic acid capsule, encoded by pXO2, is essential for immune evasion and cellular survival in the host [3,4]. The lethal effects

Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

PubMed

Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

2007-01-01

Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made are freely available online at the URL http://www.jenner.ac.uk/MHCPred.

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

PubMed

Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

2017-06-23

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

UPTAKE OF RADIONUCLIDE METALS BY SPME FIBERS

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

Duff, M; S Crump, S; Robert02 Ray, R

2006-08-28

The Federal Bureau of Investigation (FBI) Laboratory currently does not have on site facilities for handling radioactive evidentiary materials and there are no established FBI methods or procedures for decontaminating high explosive (HE) and fire debris (FD) evidence while maintaining evidentiary value. One experimental method for the isolation of HE and FD residue involves using solid phase microextraction or SPME fibers to remove residue of interest. Due to their high affinity for organics, SPME fibers should have little affinity for most metals. However, no studies have measured the affinity of radionuclides for SPME fibers. The focus of this research wasmore » to examine the affinity of dissolved radionuclide ({sup 239/240}Pu, {sup 238}U, {sup 237}Np, {sup 85}Sr, {sup 133}Ba, {sup 137}Cs, {sup 60}Co and {sup 226}Ra) and stable radionuclide surrogate metals (Sr, Co, Ir, Re, Ni, Ba, Cs, Nb, Zr, Ru, and Nd) for SPME fibers at the exposure conditions that favor the uptake of HE and FD residues. Our results from radiochemical and mass spectrometric analyses indicate these metals have little measurable affinity for these SPME fibers during conditions that are conducive to HE and FD residue uptake with subsequent analysis by liquid or gas phase chromatography with mass spectrometric detection.« less

SOLID PHASE MICROEXTRACTION SAMPLING OF HIGH EXPLOSIVE RESIDUES IN THE PRESENCE OF RADIONUCLIDES AND RADIONUCLIDE SURROGATE METALS

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

Duff, M; S Crump, S; Robert02 Ray, R

2007-04-13

The Federal Bureau of Investigation (FBI) Laboratory currently does not have on site facilities for handling radioactive evidentiary materials and there are no established FBI methods or procedures for decontaminating high explosive (HE) evidence while maintaining evidentiary value. One experimental method for the isolation of HE residue involves using solid phase microextraction or SPME fibers to remove residue of interest. Due to their high affinity for organics, SPME fibers should have little affinity for most metals. However, no studies have measured the affinity of radionuclides for SPME fibers. The focus of this research was to examine the affinity of dissolvedmore » radionuclide ({sup 239/240}Pu, {sup 238}U, {sup 237}Np, {sup 85}Sr, {sup 133}Ba, {sup 137}Cs, {sup 60}Co and {sup 226}Ra) and stable radionuclide surrogate metals (Sr, Co, Ir, Re, Ni, Ba, Cs, Nb, Zr, Ru, and Nd) for SPME fibers at the exposure conditions that favor the uptake of HE residues. Our results from radiochemical and mass spectrometric analyses indicate these metals have little measurable affinity for these SPME fibers during conditions that are conducive to HE residue uptake with subsequent analysis by liquid or gas phase chromatography with mass spectrometric detection.« less

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

A Mixed Approach to Similarity Metric Selection in Affinity Propagation-Based WiFi Fingerprinting Indoor Positioning.

PubMed

Caso, Giuseppe; de Nardis, Luca; di Benedetto, Maria-Gabriella

2015-10-30

The weighted k-nearest neighbors (WkNN) algorithm is by far the most popular choice in the design of fingerprinting indoor positioning systems based on WiFi received signal strength (RSS). WkNN estimates the position of a target device by selecting k reference points (RPs) based on the similarity of their fingerprints with the measured RSS values. The position of the target device is then obtained as a weighted sum of the positions of the k RPs. Two-step WkNN positioning algorithms were recently proposed, in which RPs are divided into clusters using the affinity propagation clustering algorithm, and one representative for each cluster is selected. Only cluster representatives are then considered during the position estimation, leading to a significant computational complexity reduction compared to traditional, flat WkNN. Flat and two-step WkNN share the issue of properly selecting the similarity metric so as to guarantee good positioning accuracy: in two-step WkNN, in particular, the metric impacts three different steps in the position estimation, that is cluster formation, cluster selection and RP selection and weighting. So far, however, the only similarity metric considered in the literature was the one proposed in the original formulation of the affinity propagation algorithm. This paper fills this gap by comparing different metrics and, based on this comparison, proposes a novel mixed approach in which different metrics are adopted in the different steps of the position estimation procedure. The analysis is supported by an extensive experimental campaign carried out in a multi-floor 3D indoor positioning testbed. The impact of similarity metrics and their combinations on the structure and size of the resulting clusters, 3D positioning accuracy and computational complexity are investigated. Results show that the adoption of metrics different from the one proposed in the original affinity propagation algorithm and, in particular, the combination of different metrics can significantly improve the positioning accuracy while preserving the efficiency in computational complexity typical of two-step algorithms.

A Mixed Approach to Similarity Metric Selection in Affinity Propagation-Based WiFi Fingerprinting Indoor Positioning

PubMed Central

Caso, Giuseppe; de Nardis, Luca; di Benedetto, Maria-Gabriella

2015-01-01

The weighted k-nearest neighbors (WkNN) algorithm is by far the most popular choice in the design of fingerprinting indoor positioning systems based on WiFi received signal strength (RSS). WkNN estimates the position of a target device by selecting k reference points (RPs) based on the similarity of their fingerprints with the measured RSS values. The position of the target device is then obtained as a weighted sum of the positions of the k RPs. Two-step WkNN positioning algorithms were recently proposed, in which RPs are divided into clusters using the affinity propagation clustering algorithm, and one representative for each cluster is selected. Only cluster representatives are then considered during the position estimation, leading to a significant computational complexity reduction compared to traditional, flat WkNN. Flat and two-step WkNN share the issue of properly selecting the similarity metric so as to guarantee good positioning accuracy: in two-step WkNN, in particular, the metric impacts three different steps in the position estimation, that is cluster formation, cluster selection and RP selection and weighting. So far, however, the only similarity metric considered in the literature was the one proposed in the original formulation of the affinity propagation algorithm. This paper fills this gap by comparing different metrics and, based on this comparison, proposes a novel mixed approach in which different metrics are adopted in the different steps of the position estimation procedure. The analysis is supported by an extensive experimental campaign carried out in a multi-floor 3D indoor positioning testbed. The impact of similarity metrics and their combinations on the structure and size of the resulting clusters, 3D positioning accuracy and computational complexity are investigated. Results show that the adoption of metrics different from the one proposed in the original affinity propagation algorithm and, in particular, the combination of different metrics can significantly improve the positioning accuracy while preserving the efficiency in computational complexity typical of two-step algorithms. PMID:26528984

The Functions of BRCA2 in Homologous Recombinational Repair

DTIC Science & Technology

2004-07-01

chromatography with hydroxyapatite , Q-Sepharose, heparin affinity and MonoQ column (Fig. 4.). We have been able to obtain about 10 mg of the purified Rad51...and DNA- PKcs (the XR -1, xrs5/6, and V3 cell lines, respectively) are highly sensitive to IR in G1 and early S phases, compared to the wild-type, but

Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

ERIC Educational Resources Information Center

de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

2008-01-01

This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

Using Affinity Chromatography to Investigate Novel Protein-Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course

ERIC Educational Resources Information Center

Belanger, Kenneth D.

2009-01-01

Inquiry-driven lab exercises require students to think carefully about a question, carry out an investigation of that question, and critically analyze the results of their investigation. Here, we describe the implementation and assessment of an inquiry-based laboratory exercise in which students obtain and analyze novel data that contribute to our…

MODEL FOR NON-EQUILIBRIUM BINDING AND AFFINITY CHROMATOGRAPHY WITH CHARACTERIZATION OF 8-HYDROXYQUINOLINE IMMOBILIZED ON CONTROLLED PORE GLASS USING A FLOW INJECTION SYSTEM WITH A PACKED MICRO-COLUMN. (R826694C651)

EPA Science Inventory

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

Purification of aflatoxin B1 antibody for the development of aflatoxin biosensor

NASA Astrophysics Data System (ADS)

Prihantoro, E. A. B.; Saepudin, E.; Ivandini, T. A.

2017-07-01

Aflatoxin B1 (AFB1) is produced from agricultural products especially peanuts overgrown with aspergillus flavus during the post-harvest process. Aflatoxin is classified as a highly toxic and carcinogenic substance to humans by the International Agency for Research on Cancer (IARC), WHO. This research was conducted to develop the AFB1 biosensor using antibody that specifically binds to aflatoxin B1. This antibody was produced by injecting an AFB1 hapten-protein (immunogen) to a rabbit. Antibody was obtained from rabbit's blood serum and purified using Protein A affinity chromatography and precipitation at the isoelectric point. The result showed that purification using protein A contains antibody of 4.0 mg/mL, whereas purification using precipitation at isoelectric pH contains antibody of 0.3 mg/mL. The pure antibody was tested for its specificity against AFB1, tetrahydrofuran (THF), dimethyl formamide (DMF), bovine serum albumin (BSA), and ethanol. The result revealed that THF, BSA, and ethanol were bound to antibody, while DMF showed no interaction. It was concluded that the polyclonal antibody which have been successfully purified from rabbit's blood serum using protein A affinity chromatography and precipitation methods showed an unspecific identification.

High-level expression and purification of heparin-binding epidermal growth factor (HB-EGF) with SUMO fusion.

PubMed

Lu, Wuguang; Cao, Peng; Lei, Huangzong; Zhang, Shuangquan

2010-03-01

Heparin-binding epidermal growth factor (HB-EGF) can stimulate the division of various cell types and has potential clinical applications that stimulate growth and differentiation. HB-EGF has an EGF-like domain typical of all members of the EGF family. The high expression of active HB-EGF in Escherichia coli has not been successful as the protein contains three intra-molecular disulfide bonds, the same as other members of the EGF super family that are difficult to form correctly in the bacterial intracellular environment. This work fused the non-glycosylated HB-EGF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fusion gene SUMO-HBEGF was highly expressed in BL21(DE3) that the soluble SUMO-HBEGF was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to obtain the native HB-EGF, which was further purified by Ni-NTA affinity chromatography. MTT assays indicated the purified HB-EGF, as well as SUMO-HBEGF, had mitogenic activity in a dose-dependent manner.

A macro-enzyme cause of an isolated increase of alkaline phosphatase.

PubMed

Cervinski, Mark A; Lee, Hong Kee; Martin, Isabella W; Gavrilov, Dimitar K

2015-02-02

Macroenzyme complexes of serum enzymes and antibody can increase the circulating enzymatic activity and may lead to unnecessary additional testing and procedures. Laboratory physicians and scientists need to be aware of techniques to identify macroenzyme complexes when suspected. To investigate the possibility of a macro-alkaline phosphatase in the serum of a 74 year old male with persistently increased alkaline phosphatase we coupled a protein A/G agarose affinity chromatography technique with isoenzyme electrophoresis to look for the presence of macro-alkaline phosphatase. The majority of the alkaline phosphatase activity in the patient's serum sample was bound to the column and only a minor fraction (25%) of alkaline phosphatase activity was present in the column flow-through. The alkaline phosphatase activity was also found to co-elute with the immunoglobulins in the patient sample. The alkaline phosphatase activity in a control serum sample concurrently treated in the same manner did not bind to the column and was found in the column flow-through. The use of protein A/G agarose affinity chromatography is a rapid and simple method that can be applied to the investigation of other macro-enzyme complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.

PubMed

Bresolin, I T L; Borsoi-Ribeiro, M; Tamashiro, W M S C; Augusto, E F P; Vijayalakshmi, M A; Bueno, S M A

2010-04-01

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

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

PubMed

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

2003-07-01

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

Crystallization and X-ray analysis of the salmon-egg lectin SEL24K

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

Murata, Kenji; Fisher, Andrew J.; Hedrick, Jerry L., E-mail: jlhedrick@ucdavis.edu

2007-05-01

The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) is released from the egg during the cortical reaction. The lectin functions in blocking polyspermy during the fertilization process. The egg lectin was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The crystal diffracted synchrotron-radiation X-rays to 1.63 Å resolution. The crystal belongsmore » to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 93.0, b = 73.6, c = 113.6 Å, α = 90, β = 92.82, γ = 90°. The crystal is likely to contain eight molecules in the asymmetric unit (V{sub M} = 2.3 Å{sup 3} Da{sup −1}), corresponding to a solvent content of 45.5%. A self-rotation function suggests an arrangement with 222 point symmetry within the asymmetric unit.« less

A highly selective dispersive liquid-liquid microextraction approach based on the unique fluorous affinity for the extraction and detection of per- and polyfluoroalkyl substances coupled with high performance liquid chromatography tandem-mass spectrometry.

PubMed

Wang, Juan; Shi, Yali; Cai, Yaqi

2018-04-06

In the present study, a highly selective fluorous affinity-based dispersive liquid-liquid microextraction (DLLME) technique was developed for the extraction and analysis of per- and polyfluoroalkyl substances (PFASs) followed by high performance liquid chromatography tandem-mass spectrometry. Perfluoro-tert-butanol with multiple C-F bonds was chosen as the extraction solvent, which was injected into the aqueous samples with a dispersive solvent (acetonitrile) in a 120:800 (μL, v/v) mixture for PFASs enrichment. The fluorous affinity-based extraction mechanism was confirmed by the significantly higher extraction recoveries for PFASs containing multiple fluorine atoms than those for compounds with fewer or no fluorine atoms. The extraction recoveries of medium and long-chain PFASs (CF 2  > 5) exceeded 70%, except perfluoroheptanoic acid, while those of short-chain PFASs were lower than 50%, implying that the proposed DLLME may not be suitable for their extraction due to weak fluorous affinity. This highly fluoroselective DLLME technique can greatly decrease the matrix effect that occurs in mass spectrometry detection when applied to the analysis of urine samples. Under the optimum conditions, the relative recoveries of PFASs with CF 2  > 5 ranged from 80.6-121.4% for tap water, river water and urine samples spiked with concentrations of 10, 50 and 100 ng/L. The method limits of quantification for PFASs in water and urine samples were in the range of 0.6-8.7 ng/L. Furthermore, comparable concentrations of PFASs were obtained via DLLME and solid-phase extraction, confirming that the developed DLLME technique is a promising method for the extraction of PFASs in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification.

PubMed

Koziel, David; Michaelis, Uwe; Kruse, Tobias

2018-08-01

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans. Thus, endotoxin removal is important in protein purification for academic research and in GMP manufacturing of biopharmaceuticals. Several methods exist to remove endotoxin, but often require additional downstream-processing steps, decrease protein yield and are costly. These disadvantages can be avoided by using an integrated endotoxin depletion (iED) wash-step that utilizes Triton X-114 (TX114). In this paper, we show that the iED wash-step is broadly applicable in most commonly used chromatographies: it reduces endotoxin by a factor of 10 3 to 10 6 during NiNTA-, MBP-, SAC-, GST-, Protein A and CEX-chromatography but not during AEX or HIC-chromatography. We characterized the iED wash-step using Design of Experiments (DoE) and identified optimal experimental conditions for application scenarios that are relevant to academic research or industrial GMP manufacturing. A single iED wash-step with 0.75% (v/v) TX114 added to the feed and wash buffer can reduce endotoxin levels to below 2 EU/ml or deplete most endotoxin while keeping the manufacturing costs as low as possible. The comprehensive characterization enables academia and industry to widely adopt the iED wash-step for a routine, efficient and cost-effective depletion of endotoxin during protein purification at any scale. Copyright © 2018. Published by Elsevier B.V.

Purification of native M. vogae and H. contortus tubulin by TOG affinity chromatography.

PubMed

Munguía, Beatriz; Teixeira, Ramiro; Veroli, Victoria; Melian, Elisa; Saldaña, Jenny; Minteguiaga, Mahia; Señorale, Mario; Marín, Mónica; Domínguez, Laura

2017-11-01

Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 μg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

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

Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.

2009-06-24

A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from micromolar to low nanomolar.

Bimodal imprint chips for peptide screening: integration of high-throughput sequencing by MS and affinity analyses by surface plasmon resonance imaging.

PubMed

Wang, Weizhi; Li, Menglin; Wei, Zewen; Wang, Zihua; Bu, Xiangli; Lai, Wenjia; Yang, Shu; Gong, He; Zheng, Hui; Wang, Yuqiao; Liu, Ying; Li, Qin; Fang, Qiaojun; Hu, Zhiyuan

2014-04-15

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

Jasmonic acid-amino acid conjugation enzyme assays.

PubMed

Rowe, Martha L; Staswick, Paul E

2013-01-01

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

Improving the convergence rate in affine registration of PET and SPECT brain images using histogram equalization.

PubMed

Salas-Gonzalez, D; Górriz, J M; Ramírez, J; Padilla, P; Illán, I A

2013-01-01

A procedure to improve the convergence rate for affine registration methods of medical brain images when the images differ greatly from the template is presented. The methodology is based on a histogram matching of the source images with respect to the reference brain template before proceeding with the affine registration. The preprocessed source brain images are spatially normalized to a template using a general affine model with 12 parameters. A sum of squared differences between the source images and the template is considered as objective function, and a Gauss-Newton optimization algorithm is used to find the minimum of the cost function. Using histogram equalization as a preprocessing step improves the convergence rate in the affine registration algorithm of brain images as we show in this work using SPECT and PET brain images.

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

PubMed

Sandra, Koen; Steenbeke, Mieke; Vandenheede, Isabel; Vanhoenacker, Gerd; Sandra, Pat

2017-11-10

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.

PubMed

Taguchi, R; Ikezawa, H

1987-10-01

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Han, Tianqing; Ming, Hongyan; Deng, Lili

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

A Dual Protease Approach for Expression and Affinity Purification of Recombinant Proteins

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2016-01-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification. PMID:27105777

A dual protease approach for expression and affinity purification of recombinant proteins.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation.

PubMed

Mohamed Abubakkar, M; Saraboji, K; Ponnuswamy, M N

2013-02-01

Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively.

Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants.

PubMed

Strachan, G; Grant, S D; Learmonth, D; Longstaff, M; Porter, A J; Harris, W J

1998-09-15

Single-chain antibody fragments (scAb), specific for the herbicide atrazine, have been expressed in the bacterium Escherichia coli and in transgenic tobacco plants. The scAb could be purified as a monomer (monovalent) via a hexa-histidine tail or as a dimer (divalent) by antibody affinity chromatography. In competition ELISA, the bacterial scAb showed the same specificity for atrazine and related triazine herbicides as the parental mAb cell line, but both plant and bacterial monomeric scAbs showed increased sensitivity to free atrazine. Surface plasmon resonance (BIAcore 2000) analysis confirmed that purified scAb, derived from plant or bacteria, retained similar association rates as the mAb. However, the monomeric plant and bacterial scAbs showed a lower affinity for immobilised antigen, than the equivalent dimeric scAbs or mAb. This decrease in affinity was due to a 10 fold slower dissociation rate and is likely due to loss of the avidity contribution of dimeric molecules.

Decorin is a Zn(2+) Metalloprotein

NASA Technical Reports Server (NTRS)

Yang, Vivian W.-C.; LaBrenz, Steven R.; Rosenberg, Lawrence C.; McQuillan, David; Hoeoek, Magnus

1998-01-01

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. We here demonstrate that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells, has a high affinity for Zn(2+). Binding of Zn(2+) to decorin is demonstrated by Zn(2+) chelating chromatography and equilibrium dialyses. The Zn(2+) binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in the spacing reminiscent of a Zn finger. A recombinant 41 amino acid long peptide representing the N-terminal domain of decorin has full Zn(2+) binding activity and binds two Zn(2+) ions with an average K(D) of 3 x 10(exp -7) M. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn(2+) binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, did not bind Zn(2+) with high affinity.

Introduction of structural affinity handles as a tool in selective nucleic acid separations

NASA Technical Reports Server (NTRS)

Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

2011-01-01

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

Separation and purification of enzymes by continuous pH-parametric pumping

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

Huang, S.Y.; Lin, C.K.; Juang, L.Y.

1985-10-01

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. e-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield,more » e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes. 19 references.« less

Variable pressure ionization detector for gas chromatography

DOEpatents

Buchanan, Michelle V.; Wise, Marcus B.

1988-01-01

Method and apparatus for differentiating organic compounds based on their electron affinity. An electron capture detector cell (ECD) is operated at pressures ranging from atmospheric to less than 1 torr. Through variation of the pressure within the ECD cell, the organic compounds are induced to either capture or emit electrons. Differentiation of isomeric compounds can be obtianed when, at a given pressure, one isomer is in the emission mode and the other is in the capture mode. Output of the ECD is recorded by chromatogram. The invention also includes a method for obtaining the zero-crossing pressure of a compound, defined as the pressure at which the competing emission and capture reactions are balanced and which may be correlated to the electron affinity of a compound.

Targeting of Prostate Cancer with Hyaluronan-Binding Proteins

DTIC Science & Technology

2005-06-01

16). Briefly, bovine nasal cartilage (Pel-Freez, Rogers, AR) was shredded with a Sure-Form blade (Stanley). extracted overnight with 4 M guanidine... nasal cartilage by affinity chromatography on hyaluronan- E Sepharose. As shown in Fig. 1, Metastatin consisted of two molecular C 40 factions as...biotinylated 490 pg/ml.) was isolated from the medium of rat fibrosarcoma cells grown tachvplesin (Fig. IB). However, this interaction was

Defining process design space for a hydrophobic interaction chromatography (HIC) purification step: application of quality by design (QbD) principles.

PubMed

Jiang, Canping; Flansburg, Lisa; Ghose, Sanchayita; Jorjorian, Paul; Shukla, Abhinav A

2010-12-15

The concept of design space has been taking root under the quality by design paradigm as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. This paper outlines the development of a design space for a hydrophobic interaction chromatography (HIC) process step. The design space included the impact of raw material lot-to-lot variability and variations in the feed stream from cell culture. A failure modes and effects analysis was employed as the basis for the process characterization exercise. During mapping of the process design space, the multi-dimensional combination of operational variables were studied to quantify the impact on process performance in terms of yield and product quality. Variability in resin hydrophobicity was found to have a significant influence on step yield and high-molecular weight aggregate clearance through the HIC step. A robust operating window was identified for this process step that enabled a higher step yield while ensuring acceptable product quality. © 2010 Wiley Periodicals, Inc.

High-level expression in Escherichia coli, purification and kinetic characterization of Plasmodium falciparum M1-aminopeptidase.

PubMed

González-Bacerio, Jorge; Osuna, Joel; Ponce, Amaia; Fando, Rafael; Figarella, Katherine; Méndez, Yanira; Charli, Jean-Louis; Chávez, María de Los Á

2014-12-01

Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1mM IPTG at 37°C for 18h. This allowed obtaining 100mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6×His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn(2+) excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure of the beta-galactosidase gene from Thermus sp. strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein.

PubMed

Vian, A; Carrascosa, A V; García, J L; Cortés, E

1998-06-01

The nucleotide sequence of both the bgaA gene, coding for a thermostable beta-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the beta-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric beta-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70 degrees C and a Km value of 1.6 mM with o-nitrophenyl-beta-D-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C.

One-step separation and purification of three lignans and one flavonol from Sinopodophyllum emodi by medium-pressure liquid chromatography and high-speed counter-current chromatography.

PubMed

Wang, Ping; Liu, Yongling; Chen, Tao; Xu, Wenhua; You, Jinmao; Liu, Yongjun; Li, Yulin

2013-01-01

Lignans and flavonols are the primary constituents of Sinopodophyllum emodi and have been used as cathartic, anthelmintic, chemotherapeutic and anti-hypertensive compounds. Although these compounds have been isolated, there have been no reports on the separation of 4'-demethyl podophyllotoxin, podophyllotoxin, deoxypodophyllotoxin and kaempferol in one step by medium-pressure liquid chromatography (MPLC) and high-speed counter-current chromatography (HSCCC). Development of an efficient method for the preparative separation and purification of three lignans and one flavonol from S. emodi. The precipitate of crude extracts was first separated by MPLC into four parts, numbered GJ-1, GJ-2, GJ-3 and GJ-4. GJ-1 was separated and purified by HSCCC using a solvent system composed of n-hexane:ethyl acetate:methanol:water (1.75:1.5:1:0.75, v/v/v/v). The purities of the target compounds were assessed using high-performance liquid chromatography (HPLC) and chemical structures were identified by (1) H-NMR and (13) C-NMR. The HSCCC and MPLC methods were successfully used for the preparative separation and purification of 4'-demethyl podophyllotoxin (8.5 mg, 92.4%), podophyllotoxin (40.1 mg, 92.1%), deoxypodophyllotoxin (4.6 mg, 98.1%), and kaempferol (1.6 mg, 96.7%) from a 100 mg sample. Three lignans (4'-demethyl podophyllotoxin, podophyllotoxin, deoxypodophyllotoxin) and one flavonol (kaempferol) were successfully isolated by HSCCC and MPLC in one step. Copyright © 2013 John Wiley & Sons, Ltd.

Quantum properties of affine-metric gravity with the cosmological term

NASA Astrophysics Data System (ADS)

Baurov, A. Yu; Pronin, P. I.; Stepanyantz, K. V.

2018-04-01

The paper contains analysis of the one-loop effective action for affine-metric gravity of the Hilbert–Einstein type with the cosmological term. We discuss different approaches to the calculation of the effective action, which depends on two independent variables, namely, the metric tensor and the affine connection. In the one-loop approximation we explain how the effective action can be obtained, if, at the first step of the calculation, the metric tensor is integrated out. It is demonstrated that the result is the same as in the case when one starts by integrating out the connection.

A family of variable step-size affine projection adaptive filter algorithms using statistics of channel impulse response

NASA Astrophysics Data System (ADS)

Shams Esfand Abadi, Mohammad; AbbasZadeh Arani, Seyed Ali Asghar

2011-12-01

This paper extends the recently introduced variable step-size (VSS) approach to the family of adaptive filter algorithms. This method uses prior knowledge of the channel impulse response statistic. Accordingly, optimal step-size vector is obtained by minimizing the mean-square deviation (MSD). The presented algorithms are the VSS affine projection algorithm (VSS-APA), the VSS selective partial update NLMS (VSS-SPU-NLMS), the VSS-SPU-APA, and the VSS selective regressor APA (VSS-SR-APA). In VSS-SPU adaptive algorithms the filter coefficients are partially updated which reduce the computational complexity. In VSS-SR-APA, the optimal selection of input regressors is performed during the adaptation. The presented algorithms have good convergence speed, low steady state mean square error (MSE), and low computational complexity features. We demonstrate the good performance of the proposed algorithms through several simulations in system identification scenario.

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system

PubMed Central

Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

2013-01-01

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5′ and 3′ exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3′ splice site (3′SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3′SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing. PMID:23685439

Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.

PubMed

Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul

2016-03-18

Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

Applying high-throughput methods to develop a purification process for a highly glycosylated protein.

PubMed

Sanaie, Nooshafarin; Cecchini, Douglas; Pieracci, John

2012-10-01

Micro-scale chromatography formats are becoming more routinely used in purification process development because of their ability to rapidly screen large number of process conditions at a time with minimal material. Given the usual constraints that exist on development timelines and resources, these systems can provide a means to maximize process knowledge and process robustness compared to traditional packed column formats. In this work, a high-throughput, 96-well filter plate format was used in the development of the cation exchange and hydrophobic interaction chromatography steps of a purification process designed to alter the glycoform distribution of a small protein. The significant input parameters affecting process performance were rapidly identified for both steps and preliminary operating conditions were identified. These ranges were verified in a packed chromatography column in order to assess the ability of the 96-well plate to predict packed column performance. In both steps, the 96-well plate format consistently led to underestimated glycoform-enrichment levels and to overestimated product recovery rates compared to the column-based approach. These studies demonstrate that the plate format can be used as a screening tool to narrow the operating ranges prior to further optimization on packed chromatography columns. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Liquid chromatography with mass spectrometry method based two-step precursor ion scanning for the structural elucidation of flavonoids.

PubMed

Li, Yong; Pang, Tao; Shi, Junli; Lu, Xiuping; Deng, Jianhua; Lin, Qian

2014-11-01

Plant flavonoids are very important secondary metabolites for insect and virus control of their host plant and are potent nutrients for humans. To be able to understand the bioavailability and functions of plant flavonoids, it is necessary to reveal their exact chemical structures. Liquid chromatography with tandem mass spectrometry is a powerful approach for structural elucidation of metabolites. In this report, a two-step precursor ion scanning based liquid chromatography with tandem mass spectrometry method was developed for the structural elucidation of plant flavonoids. The established method consists of the two-step precursor ions scanning for possible flavonoids extraction, MS(2) fragment spectra acquisition and comparison with an online database, liquid chromatography retention rules correction, and commercial standards verification. The developed method was used for the structure elucidation of flavonoids in flowers and leaves of tobacco (Nicotiana tabacum), and 17 flavonoids were identified in the tobacco variety Yunyan 97. Nine of the 17 identified flavonoids were considered to be found in tobacco flowers or/and leaves for the first time based on the available references. This method was proved to be very effective and can be used for the identification of flavonoids in other plants. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrochemical affinity biosensors for detection of mycotoxins: A review.

PubMed

Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

2013-11-15

This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

Stressless Schwarzschild

NASA Astrophysics Data System (ADS)

Deser, S.

2014-01-01

This self-contained pedagogical simple explicit 6-step derivation of the Schwarzschild solution, in "" formulation and conformal spatial gauge, (almost) avoids all affinity, curvature and index gymnastics.

Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

PubMed Central

Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

2011-01-01

Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process. PMID:21589929

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

PubMed Central

Guerreiro, Juliano R.; Lameu, Claudiana; Oliveira, Eduardo F.; Klitzke, Clécio F.; Melo, Robson L.; Linares, Edlaine; Augusto, Ohara; Fox, Jay W.; Lebrun, Ivo; Serrano, Solange M. T.; Camargo, Antonio C. M.

2009-01-01

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases. PMID:19491403

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

PubMed

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

2015-04-20

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

Mechanism of Selective Nickel Transfer from HypB to HypA, Escherichia coli [NiFe]-Hydrogenase Accessory Proteins.

PubMed

Lacasse, Michael J; Douglas, Colin D; Zamble, Deborah B

2016-12-13

[NiFe]-hydrogenase enzymes catalyze the reversible reduction of protons to molecular hydrogen and serve as a vital component of the metabolism of many pathogens. The synthesis of the bimetallic catalytic center requires a suite of accessory proteins, and the penultimate step, nickel insertion, is facilitated by the metallochaperones HypA and HypB. In Escherichia coli, nickel moves from a site in the GTPase domain of HypB to HypA in a process accelerated by GDP. To determine how the transfer of nickel is controlled, the impacts of HypA and nucleotides on the properties of HypB were examined. Integral to this work was His2Gln HypA, a mutant with attenuated nickel affinity that does not support hydrogenase production in E. coli. This mutation inhibits the translocation of nickel from HypB. H2Q-HypA does not modulate the apparent metal affinity of HypB, but the stoichiometry and stability of the HypB-nickel complex are modulated by the nucleotide. Furthermore, the HypA-HypB interaction was detected by gel filtration chromatography if HypB was loaded with GDP, but not a GTP analogue, and the protein complex dissociated upon binding of nickel to His2 of HypA. In contrast, a nucleotide does not modulate the binding of zinc to HypB, and loading zinc into the GTPase domain of HypB inhibits formation of the complex with HypA. These results demonstrate that GTP hydrolysis controls both metal binding and protein-protein interactions, conferring selective and directional nickel transfer during [NiFe]-hydrogenase biosynthesis.

Requirements for construction of a functional hybrid complex of photosystem I and [NiFe]-hydrogenase.

PubMed

Schwarze, Alexander; Kopczak, Marta J; Rögner, Matthias; Lenz, Oliver

2010-04-01

The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H(2) sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O(2)-tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH(PsaE)) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H(2) production capacity of the purified MBH(PsaE) fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH(PsaE) with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH(PsaE)-PS I complex formation and its light-driven H(2) production capacity by means of spectroelectrochemistry.

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

PubMed

Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

Development and automation of a novel NET-PET tracer: [11C]Me@APPI.

PubMed

Mark, Christina; Bornatowicz, Birgit; Mitterhauser, Markus; Hendl, Matthias; Nics, Lukas; Haeusler, Daniela; Lanzenberger, Rupert; Berger, Michael L; Spreitzer, Helmut; Wadsak, Wolfgang

2013-02-01

The norepinephrine transporter (NET) is an important target for research in neurology and psychology and is involved in the pathophysiology of many neurodegenerative diseases such as Alzheimer's disease and attention deficient hyperactivity disorder. For visualization of NET abundance and deregulation, a novel PET tracer--[(11)C]Me@APPI--has been developed. For precursor synthesis, a 4-step synthesis starting from N-phenyl-o-phenylenediamine was set up. Radiosynthesis was established and optimized using standard methods and subsequently automated in a GE TRACERlabFx C Pro synthesizer. Preclinical testing was performed comprising affinity and selectivity testing on human membranes as well as stability and blood-brain-barrier-penetration using in-vitro models. Precursor molecule (APPI:0) and reference compound (Me@APPI) were synthesized with 26.5% and 21.4% overall yield, respectively. So far, 1.25±0.72 GBq [(11)C]Me@APPI with 54.35±7.80 GBq/μmol specific activity were produced (n=11). Affinity of reference compounds was determined as 8.08±1.75 nM for Me@APPI and 19.31±2.91 nM for APPI:0, respectively (n≥9). IAM-chromatography experiments (n=3) revealed a P(m) value of 1.51±0.34 for Me@APPI. Stability testing using human liver microsomes revealed that 99.5% of the tracer was found to be still intact after 60 minutes (n=4). Present data indicate that [(11)C]Me@APPI has promising properties to become a clinically useful NET-PET-tracer. Further in-vitro and in-vivo evaluations are currently under way. Copyright © 2013 Elsevier Inc. All rights reserved.

Physiological and Molecular Biological Characterization of Intracellular Carbonic Anhydrase from the Marine Diatom Phaeodactylum tricornutum1

PubMed Central

Satoh, Dan; Hiraoka, Yasutaka; Colman, Brian; Matsuda, Yusuke

2001-01-01

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO2-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to β-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO2-binding site appeared to be unique among the β-CAs so far reported. PMID:11500545

A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

PubMed

Torres, Oscar B; Duval, Alexander J; Sulima, Agnieszka; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

2018-06-01

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

PubMed

Sueck, Franziska; Poór, Miklós; Faisal, Zelma; Gertzen, Christoph G W; Cramer, Benedikt; Lemli, Beáta; Kunsági-Máté, Sándor; Gohlke, Holger; Humpf, Hans-Ulrich

2018-06-22

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus . 2′ R -Ochratoxin A (2′ R -OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′ R -OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′ R -OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′ R -OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′ R -OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a log K of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′ R -OTA, a lower, but still high, log K of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′ R -OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′ R -OTA found in human blood samples.

Selection and maturation of antibodies by phage display through fusion to pIX.

PubMed

Tornetta, Mark; Reddy, Ramachandra; Wheeler, John C

2012-09-01

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional expression and purification of recombinant Tx1, a sodium channel blocker neurotoxin from the venom of the Brazilian "armed" spider, Phoneutria nigriventer.

PubMed

Diniz, Marcelo R V; Theakston, R David G; Crampton, Julian M; Nascimento Cordeiro, Marta do; Pimenta, Adriano M C; De Lima, Maria Elena; Diniz, Carlos R

2006-11-01

Tx1 from the venom of the Brazilian spider, Phoneutria nigriventer, is a lethal neurotoxic polypeptide of M(r) 8600 Da with 14 cysteine residues. It is a novel sodium channel blocker which reversibly inhibits sodium currents in CHO cells expressing recombinant sodium (Nav1.2) channels. We cloned and expressed the Tx1 toxin as a thioredoxin fusion product in the cytoplasm of Escherichia coli. After semipurification by immobilized Ni-ion affinity chromatography, the recombinant Tx1 was purified by reverse phase chromatography and characterized. It displayed similar biochemical and pharmacological properties to the native toxin, and it should be useful for further investigation of structure-function relationship of Na channels.

SPR Biosensors in Direct Molecular Fishing: Implications for Protein Interactomics.

PubMed

Florinskaya, Anna; Ershov, Pavel; Mezentsev, Yuri; Kaluzhskiy, Leonid; Yablokov, Evgeniy; Medvedev, Alexei; Ivanov, Alexis

2018-05-18

We have developed an original experimental approach based on the use of surface plasmon resonance (SPR) biosensors, applicable for investigation of potential partners involved in protein⁻protein interactions (PPI) as well as protein⁻peptide or protein⁻small molecule interactions. It is based on combining a SPR biosensor, size exclusion chromatography (SEC), mass spectrometric identification of proteins (LC-MS/MS) and direct molecular fishing employing principles of affinity chromatography for isolation of potential partner proteins from the total lysate of biological samples using immobilized target proteins (or small non-peptide compounds) as ligands. Applicability of this approach has been demonstrated within the frame of the Human Proteome Project (HPP) and PPI regulation by a small non-peptide biologically active compound, isatin.

Selective Precipitation and Purification of Monovalent Proteins Using Oligovalent Ligands and Ammonium Sulfate

PubMed Central

Mirica, Katherine A.; Lockett, Matthew R.; Snyder, Phillip W.; Shapiro, Nathan D.; Mack, Eric T.; Nam, Sarah; Whitesides, George M.

2012-01-01

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: i) the removal of high-molecular weight impurities through the addition of ammonium sulfate to the crude cell lysate; ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins—for which appropriate oligovalent ligands can be synthesized—and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation. PMID:22188202

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate.

PubMed

Mirica, Katherine A; Lockett, Matthew R; Snyder, Phillip W; Shapiro, Nathan D; Mack, Eric T; Nam, Sarah; Whitesides, George M

2012-02-15

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.

Target oriented drugs against leishmania. Annual summary report no. 2, 1 May 1980-30 April 1981

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

Zehavi, U.; El-On, J.

1981-01-31

Excreted Factor (EF) is a carbohydrate-rich material released by different strains of Leishmania during growth. It has antigenic properties similar to those of the intact parasite and plays a role in the infective process. Isolation and purification of EF is necessary for study of its biological function, its use for diagnostic purposes, its use in immunization experiments, the study of its biosynthesis, and the preparation of inhibitors of particular biosynthetic steps. Purification of EF by affinity chromatography was markedly improved by introducing Ricinus lectin (specific for galactose) column. This enabled us to obtain more reliable amino acid and sugar analysismore » and will be instrumental in more advanced physical, chemical, and immunological studies. We have developed a radioimmunoassay for leishmaniasis utilizing purified EF. The assay can distinguish between Leishmania strains and once further developed, should prove most valuable for the diagnosis of the disease. EF plays a role in the infective process of Leishmania. We have now shown that surface carbohydrate, related to EF, plays a role in the initial attachment of Leishmania promastigots to macrophages - a stage that is a prelude to their engulfment by the macrophages followed by multiplication in their cells.« less

Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

PubMed Central

Shoyama, Yukihiro

2011-01-01

To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

PubMed Central

2014-01-01

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

PubMed

Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

2016-01-01

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers.

Hydrolysis of tannic acid catalyzed by immobilized-stabilized derivatives of Tannase from Lactobacillus plantarum.

PubMed

Curiel, Jose Antonio; Betancor, Lorena; de las Rivas, Blanca; Muñoz, Rosario; Guisan, Jose M; Fernández-Lorente, Gloria

2010-05-26

A recombinant tannase from Lactobacillus plantarum , overexpressed in Escherichia coli , was purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports. It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support. The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were 500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme at pH 5.0 and 4 degrees C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the optimal reaction conditions.

Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli.

PubMed

Mello, C B; Nigam, Y; Garcia, E S; Azambuja, P; Newton, R P; Ratcliffe, N A

1999-04-01

We demonstrated that in Rhodnius prolixus haemocyte monolayers, both Trypanosoma cruzi and Trypanosoma rangeli are capable of inducing haemocyte/parasite clump formation. We also purified, by one-step affinity chromatography, a haemolymph galactoside-binding lectin from R. prolixus which we believe could play an important role in the development of T. rangeli in the haemocoel of the insect vector. This lectin markedly enhanced the activation of clump formation by T. rangeli in R. prolixus haemocyte monolayers, with an increase in clump size and haemocyte aggregation. The haemolymph lectin also significantly affected the motilitity and survival of T. rangeli culture short forms, but not the long forms, when they were incubated in vitro. This molecule is also one of the few described in insects with agglutination activity independent of calcium ions. The partial N-terminal amino acid sequence of this lectin demonstrated similarity to a bacterial xylulose kinase and in preliminary experiments the purified haemolymph lectin phosphorylated a tyrosine kinase substrate in a dose-dependent manner. The possible role of this haemolymph lectin in the life cycle of T. rangeli is discussed. Copyright 1999 Academic Press.

Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1.

PubMed

Zou, Yu; Hu, Wenzhong; Jiang, Aili; Tian, Mixia

2015-08-18

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

Comparative chromatography of chloroplast pigment

NASA Technical Reports Server (NTRS)

Grandolfo, M.; Sherma, J.; Strain, H. H.

1969-01-01

Methods for isolation of low concentration pigments of the cocklebur species are described. The methods entail two step chromatography so that the different sorption properties of the various pigments in varying column parameters can be utilized. Columnar and thin layer methods are compared. Many conditions influence separability of the chloroplasts.

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract.

PubMed

Delahaye, E P; Foglietti, M J; Andrieux, C; Chardon-Loriaux, I; Szylit, O; Raibaud, P

1991-01-01

1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

PubMed

Szamel, M; Goppelt, M; Resch, K

1985-12-19

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

An Efficient Protocol for Preparation of Gallic Acid from Terminalia bellirica (Gaertn.) Roxb by Combination of Macroporous Resin and Preparative High-Performance Liquid Chromatography.

PubMed

Zou, Denglang; Chen, Tao; Chen, Chen; Li, Hongmei; Liu, Yongling; Li, Yulin

2016-08-01

In this article, macroporous resin column chromatography and preparative high-performance liquid chromatography were applied for preparation of gallic acid from Terminalia bellirica (Gaertn.) Roxb. In the first step, six kinds of resins were investigated by adsorption and desorption tests and AB-8 macroporous resin was selected for the enrichment of gallic acid. As a result, 20 g of gallic acid at a purity of 71% could be separated from 100 g of crude extract in which the content of gallic acid was 16.7% and the recovery of gallic acid reached 85.0%. In the second step, preparative high-performance liquid chromatography was selected to purify gallic acid. As a result, 640 mg of gallic acid at a purity of 99.1% was obtained from 1 g of sample in 35 min. The results demonstrated that macroporous resin coupled with preparative high-performance liquid chromatography was suitable for preparation of gallic acid from T. bellirica (Gaertn.) Roxb. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

PubMed Central

2015-01-01

Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC–MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC–ESI–MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts. PMID:25134008

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

PubMed

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

2006-10-01

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

Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

NASA Astrophysics Data System (ADS)

Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

2016-10-01

Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

Optimization of Immobilized Gallium (III) Ion Affinity Chromatography for Selective Binding and Recovery of Phosphopeptides from Protein Digests

PubMed Central

Aryal, Uma K.; Olson, Douglas J.H.; Ross, Andrew R.S.

2008-01-01

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides. PMID:19183793

On the molecular interaction between lactoferrin and the dye Red HE-3B. A novel approach for docking a charged and highly flexible molecule to protein surfaces

NASA Astrophysics Data System (ADS)

Grasselli, Mariano; Cascone, Osvaldo; Anspach, F. Birger; Delfino, Jose M.

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

On the molecular interaction between lactoferrin and the dye Red HE-3b. A novel approach for docking a charged and highly flexible molecule to protein surfaces.

PubMed

Grasselli, Mariano; Cascone, Osvaldo; Birger Anspach, F; Delfino, Jose M

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

Optimizing purification process of MIM-I-BAR domain by introducing atomic force microscope and dynamics simulations.

PubMed

Zhang, Yue; Lou, Zhichao; Lin, Xubo; Wang, Qiwei; Cao, Meng; Gu, Ning

2017-09-01

MIM (missing in metastasis) is a member of I-BAR (inverse BAR) domain protein family, which functions as a putative metastasis suppressor. However, methods of gaining high purity MIM-I-BAR protein are barely reported. Here, by optimizing the purification process including changing the conditions of cell lysate and protein elution, we successfully purified MIM protein. The purity of the obtained protein was up to ∼90%. High-resolution atomic force microscope (AFM) provides more visual images, ensuring that we can observe the microenvironment around the target protein, as well as the conformations of the purification products following each purification process. MIM protein with two different sizes were observed on mica surface with AFM. Combining with molecular dynamics simulations, these molecules were revealed as MIM monomer and dimer. Furthermore, our study attaches importance to the usage of imidazole with suitable concentrations during the affinity chromatography process, as well as the removal of excessive imidazole after the affinity chromatography process. All these results indicate that the method described here was successful in purifying MIM protein and maintaining their natural properties, and is supposed to be used to purify other proteins with low solubility. Copyright © 2017. Published by Elsevier B.V.

Determination of 16 polycyclic aromatic hydrocarbons in seawater using molecularly imprinted solid-phase extraction coupled with gas chromatography-mass spectrometry.

PubMed

Song, Xingliang; Li, Jinhua; Xu, Shoufang; Ying, Rongjian; Ma, Jiping; Liao, Chunyang; Liu, Dongyan; Yu, Junbao; Chen, Lingxin

2012-09-15

A method of solid-phase extraction (SPE) using molecularly imprinted polymers (MIPs) as adsorbent coupled with gas chromatography-mass spectrometry (GC-MS) was developed for the determination of 16 types of polycyclic aromatic hydrocarbons (PAHs) in seawater samples. The MIPs were prepared through non-covalent polymerization by using the 16 PAHs mixture as a template based on sol-gel surface imprinting. Compared with the non-imprinted polymers (NIPs), the MIPs exhibited excellent affinity towards 16 PAHs with binding capacity of 111.0-195.0 μg g(-1), and imprinting factor of 1.50-3.12. The significant binding specificity towards PAHs even in the presence of environmental parameters such as dissolved organic matter and various metal ions, suggested that this new imprinting material was capable of removing 93.2% PAHs in natural seawater. High sensitivity was attained, with the low limits of detection for 16 PAHs in natural seawater ranging from 5.2-12.6 ng L(-1). The application of MIPs with high affinity and excellent stereo-selectivity toward PAHs in SPE might offer a more attractive alternative to conventional sorbents for extraction and abatement of PAH-contaminated seawater. Copyright © 2012 Elsevier B.V. All rights reserved.

DEVELOPMENT OF SULFHYDRYL-REACTIVE SILICA FOR PROTEIN IMMOBILIZATION IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Wa, Chunling; Hage, David S.

2008-01-01

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77–81% of this protein to maleimide- or iodacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, non-specific binding, stability and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations. PMID:17297940

Two-step purification of scutellarin from Erigeron breviscapus (vant.) Hand. Mazz. by high-speed counter-current chromatography.

PubMed

Gao, Min; Gu, Ming; Liu, Chun-Zhao

2006-07-11

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.

Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum.

PubMed

Chuang, Wen-Hsiao; Liu, Ping-Chung; Hung, Chia-Yu; Lee, Kuo-Kau

2014-12-01

Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50% protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea water fish and primitive fish), and all these fish species were then clustered into three groups. The cobia α-2-M was closer to that of sea water fish than that of freshwater fish compared basing on its similarity of amino acid sequence and phylogenetic analysis of the partial gene. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

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

2018-01-01

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

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

PubMed

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

2004-06-01

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

Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing.

PubMed

Heider, Susanne; Muzard, Julien; Zaruba, Marianne; Metzner, Christoph

2017-07-01

Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.

Approximated affine projection algorithm for feedback cancellation in hearing aids.

PubMed

Lee, Sangmin; Kim, In-Young; Park, Young-Cheol

2007-09-01

We propose an approximated affine projection (AP) algorithm for feedback cancellation in hearing aids. It is based on the conventional approach using the Gauss-Seidel (GS) iteration, but provides more stable convergence behaviour even with small step sizes. In the proposed algorithm, a residue of the weighted error vector, instead of the current error sample, is used to provide stable convergence. A new learning rate control scheme is also applied to the proposed algorithm to prevent signal cancellation and system instability. The new scheme determines step size in proportion to the prediction factor of the input, so that adaptation is inhibited whenever tone-like signals are present in the input. Simulation results verified the efficiency of the proposed algorithm.

Going Beyond, Going Further: An Inexpensive Experiment for the Introduction of High Performance Liquid Chromatography.

ERIC Educational Resources Information Center

Bidlingmeyer, Brian A.; Warren, F. Vincent, Jr.

1984-01-01

Background information, materials needed, laboratory procedures, and typical results are provided for five high performance liquid chromatography experiments (three isocratic and two step gradient separations). Suggestions for further experimentation are also provided, including quantitative determinations and separation of charged solutes. (JN)

Design of two-column batch-to-batch recirculation to enhance performance in ion-exchange chromatography.

PubMed

Persson, Oliver; Andersson, Niklas; Nilsson, Bernt

2018-01-05

Preparative liquid chromatography is a separation technique widely used in the manufacturing of fine chemicals and pharmaceuticals. A major drawback of traditional single-column batch chromatography step is the trade-off between product purity and process performance. Recirculation of impure product can be utilized to make the trade-off more favorable. The aim of the present study was to investigate the usage of a two-column batch-to-batch recirculation process step to increase the performance compared to single-column batch chromatography at a high purity requirement. The separation of a ternary protein mixture on ion-exchange chromatography columns was used to evaluate the proposed process. The investigation used modelling and simulation of the process step, experimental validation and optimization of the simulated process. In the presented case the yield increases from 45.4% to 93.6% and the productivity increases 3.4 times compared to the performance of a batch run for a nominal case. A rapid concentration build-up product can be seen during the first cycles, before the process reaches a cyclic steady-state with reoccurring concentration profiles. The optimization of the simulation model predicts that the recirculated salt can be used as a flying start of the elution, which would enhance the process performance. The proposed process is more complex than a batch process, but may improve the separation performance, especially while operating at cyclic steady-state. The recirculation of impure fractions reduces the product losses and ensures separation of product to a high degree of purity. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and purification of arctigenin from Fructus Arctii by enzymatic hydrolysis combined with high-speed counter-current chromatography.

PubMed

Liu, Feng; Xi, Xingjun; Wang, Mei; Fan, Li; Geng, Yanling; Wang, Xiao

2014-02-01

Enzymatic hydrolysis pretreatment combined with high-speed counter-current chromatography for the transformation and isolation of arctigenin from Fructus Arctii was successfully developed. In the first step, the extract solution of Fructus Arctii was enzymatic hydrolyzed by β-glucosidase. The optimal hydrolysis conditions were 40°C, pH 5.0, 24 h of hydrolysis time, and 1.25 mg/mL β-glucosidase concentration. Under these conditions, the content of arctigenin was transformed from 2.60 to 12.59 mg/g. In the second step, arctigenin in the hydrolysis products was separated and purified by high-speed counter-current chromatography with a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (10:25:15:20, v/v), and the fraction was analyzed by HPLC, ESI-MS, and (1)H NMR spectroscopy. Finally, 102 mg of arctigenin with a purity of 98.9% was obtained in a one-step separation from 200 mg of hydrolyzed sample. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative study of glycated hemoglobin by ion exchange chromatography and affinity binding nycocard reader in type 2 diabetes mellitus.

PubMed

Gautam, N; Dubey, R K; Jayan, A; Nepaune, Y; Padmavathi, P; Chaudhary, S; Jha, S K; Sinha, A K

2014-12-01

The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus (DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n = 100) who visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa, Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their fasting (164.46 ± 45.33 mg/dl) and random (187.93 ± 78.02 mg/dl) serum glucose level along with clinical history highly suggestive of type 2 DM. The HbA1c values of (7.8 ± 1.9%) and (8.0 ± 2.2%) were found in DM patients as estimated by those two different methods respectively. The highest frequency was observed in HbA1c > 8.0% indicating maximum cases were under very poor glycemic control. However, there were no significant differences observed in HbA1c value showing both methods are comparable in nature and can be used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c determination, the availability of report at the time of the patient visit can be made easy by using Nycocard Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control, clinical decision-making and changes in treatment regimen.

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Feasibility of using continuous chromatography in downstream processing: Comparison of costs and product quality for a hybrid process vs. a conventional batch process.

PubMed

Ötes, Ozan; Flato, Hendrik; Winderl, Johannes; Hubbuch, Jürgen; Capito, Florian

2017-10-10

The protein A capture step is the main cost-driver in downstream processing, with high attrition costs especially when using protein A resin not until end of resin lifetime. Here we describe a feasibility study, transferring a batch downstream process to a hybrid process, aimed at replacing batch protein A capture chromatography with a continuous capture step, while leaving the polishing steps unchanged to minimize required process adaptations compared to a batch process. 35g of antibody were purified using the hybrid approach, resulting in comparable product quality and step yield compared to the batch process. Productivity for the protein A step could be increased up to 420%, reducing buffer amounts by 30-40% and showing robustness for at least 48h continuous run time. Additionally, to enable its potential application in a clinical trial manufacturing environment cost of goods were compared for the protein A step between hybrid process and batch process, showing a 300% cost reduction, depending on processed volumes and batch cycles. Copyright © 2017 Elsevier B.V. All rights reserved.

High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges.

PubMed

Wang, Li; Bahadir, Anzel; Kawai, Masataka

2015-06-01

An increase in ionic strength (IS) lowers Ca(2+) activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca(2+) activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150-300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K 0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k-4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k-2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS.

High Ionic Strength Depresses Muscle Contractility by Decreasing both Force per Cross-bridge and the Number of Strongly Attached Cross-bridges

PubMed Central

Wang, Li; Bahadir, Anzel; Kawai, Masataka

2015-01-01

An increase in ionic strength (IS) lowers Ca2+ activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca2+ activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150 mM – 300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 mM to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k−4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k−2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS. PMID:25836331

Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a StepRank scheme.

PubMed

Bi, Jianjun; Song, Rengang; Yang, Huilan; Li, Bingling; Fan, Jianyong; Liu, Zhongrong; Long, Chaoqin

2011-01-01

Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well.

Purification and characterization of acid trehalase from the yeast suc2 mutant.

PubMed

Mittenbühler, K; Holzer, H

1988-06-15

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.

Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells.

PubMed

Stifter, Sebastian A; Gould, Jodee A; Mangan, Niamh E; Reid, Hugh H; Rossjohn, Jamie; Hertzog, Paul J; de Weerd, Nicole A

2014-02-01

Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ. Copyright © 2013 Elsevier Inc. All rights reserved.

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

PubMed

Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

2015-02-01

Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium. © 2014 Wiley Periodicals, Inc.

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.

What reaches the antenna? How to calibrate odor flux and ligand-receptor affinities.

PubMed

Andersson, Martin N; Schlyter, Fredrik; Hill, Sharon Rose; Dekker, Teun

2012-06-01

Physiological studies on olfaction frequently ignore the airborne quantities of stimuli reaching the sensory organ. We used a gas chromatography-calibrated photoionization detector to estimate quantities released from standard Pasteur pipette stimulus cartridges during repeated puffing of 27 compounds and verified how lack of quantification could obscure olfactory sensory neuron (OSN) affinities. Chemical structure of the stimulus, solvent, dose, storage condition, puff interval, and puff number all influenced airborne quantities. A model including boiling point and lipophilicity, but excluding vapor pressure, predicted airborne quantities from stimuli in paraffin oil on filter paper. We recorded OSN responses of Drosophila melanogaster, Ips typographus, and Culex quinquefasciatus, to known quantities of airborne stimuli. These demonstrate that inferred OSN tuning width, ligand affinity, and classification can be confounded and require stimulus quantification. Additionally, proper dose-response analysis shows that Drosophila AB3A OSNs are not promiscuous, but highly specific for ethyl hexanoate, with other earlier proposed ligands 10- to 10 000-fold less potent. Finally, we reanalyzed published Drosophila OSN data (DoOR) and demonstrate substantial shifts in affinities after compensation for quantity and puff number. We conclude that consistent experimental protocols are necessary for correct OSN classification and present some simple rules that make calibration, even retroactively, readily possible.

Cytotoxicity of seven bisphenol analogues compared to bisphenol A and relationships with membrane affinity data.

PubMed

Russo, Giacomo; Capuozzo, Antonella; Barbato, Francesco; Irace, Carlo; Santamaria, Rita; Grumetto, Lucia

2018-06-01

Bisphenol A (BPA) is a chemical used in numerous industrial applications. Due to its well ascertained toxicity as endocrine disruptor, industries have started to replace it with other bisphenols whose alleged greater safety is scarcely supported by literature studies. In this study, the toxicity of seven BPA analogues was evaluated using both in silico and in vitro techniques, as compared to BPA toxicity. Furthermore, their affinity indexes for phospholipids (i.e. phospholipophilicity) were determined by immobilized artificial membrane liquid chromatography (IAM-LC) and possible relationships with in vitro toxic activity were also investigated. The results on four different cell cultures yielded similar ranking of toxicity for the bisphenols considered, with IC 50 values confirming their poor acute toxicity. As compared to BPA, bisphenol AF, bisphenol B, bisphenol M, and bisphenol A diglycidyl ether resulted more toxic, while bisphenol S, bisphenol F and bisphenol E were found as the less toxic congeners. These results are partly consistent with the scale of phospholipid affinity showing that toxicity increases at increasing membrane affinity. Therefore, phospholipophilicity determination can be assumed as a useful preliminary tool to select less toxic congeners to surrogate BPA in industrial applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Preparative separation of bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze using steam distillation extraction and one step high-speed counter-current chromatography.

PubMed

Wei, Yun; Du, Jilin; Lu, Yuanyuan

2012-10-01

In order to utilize and control the invasive weed, bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze were studied. Steam distillation extraction and one step high-speed counter-current chromatography were applied to separate and purify the caryophyllene oxide, 7,11-dimethyl-3-methylene-1,6,10-dodecatriene, and caryophyllene from essential oil of Flaveria bidentis (L.) Kuntze. The two-phase solvent system containing n-hexane/acetonitrile/ethanol (5:4:3, v/v/v) was selected for the one step separation mode according to the partition coefficient values (K) of the target compounds and the separation factor (α). The purity of each isolated fraction after a single high-speed counter-current chromatography run was determined by high performance liquid chromatography. A 3.2 mg of caryophyllene oxide at a purity of 92.6%, 10.4 mg of 7,11-dimethyl-3-methylene-1,6,10-dodecatriene at a purity of 99.1% and 5.7 mg of caryophyllene at a purity of 98.8% were obtained from 200 mg essential oil of Flaveria bidentis (L.) Kuntze. The chemical structures of these components were identified by GC-MS, (1) H-NMR, and (13) C-NMR. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gradient x Isocratic Elution CCC on the Isolation of Verbascoside and Other Phenylethanoids: Influence of the Complexity of the Matrix.

PubMed

Leitão, Gilda Guimarães; Pinto, Shaft Correa; de Oliveira, Danilo Ribeiro; Timoteo, Patrícia; Guimarães, Michelle Guedes; Cordova, Wilmer H Perera; Leitão, Suzana Guimarães

2015-11-01

Verbascoside is a phenylethanoid glycoside widely distributed in nature, especially among the order Lamiales, occurring in numerous plants that are constituents of folk medicine preparations. This natural compound, previously isolated by our group from the ethyl acetate extract of Lantana trifolia using the gradient approach in countercurrent chromatography, was now isolated from the butanol extract of the same plant and from Lippia alba f. intermedia (Verbenaceae) using countercurrent chromatography in either gradient or isocratic elution modes. The ethyl acetate extract of L. alba, rich in phenylethanoids and flavonoids, was fractionated using countercurrent chromatography in the step-gradient elution approach. The four-step solvent system was composed of n-hexane-ethyl acetate-n-butanol-water (4 : 10 : X : 10), where X = 1 (solvent system A), 3 (solvent system B), 5 (solvent system C), and 7 (solvent system D), and allowed for the isolation of verbascoside along with other phenylethanoids and flavonoids from both plants. Verbascoside and 2'-O-β-apiosylverbascoside were further isolated from the n-butanol extract of L. trifolia using the solvent system ethyl acetate-n-butanol-water 10 : 2 : 10 on an isocratic run. The difference in the complexity of the two plant extracts demanded different purification steps, which included a second high-speed countercurrent chromatography purification using the isocratic elution mode. Georg Thieme Verlag KG Stuttgart · New York.

Structure-activity relationships studies on weakly basic N-arylsulfonylindoles with an antagonistic profile in the 5-HT6 receptor

NASA Astrophysics Data System (ADS)

Mella, Jaime; Villegas, Francisco; Morales-Verdejo, César; Lagos, Carlos F.; Recabarren-Gajardo, Gonzalo

2017-07-01

We recently reported a series of 39 weakly basic N-arylsulfonylindoles as novel 5-HT6 antagonists. Eight of the compounds exhibited moderate to high binding affinities, with 2-(4-(2-Methoxyphenyl)piperazin-1-yl)-1-(1-tosyl-1H-indol-3-yl)ethanol 16 showing the highest binding affinity (pKi = 7.87). Given these encouraging results and as a continuation of our research, we performed an extensive step-by-step search for the best 3D-QSAR model that allows us to rationally propose novel molecules with improved 5-HT6 affinity based on our previously reported series. A comparative molecular similarity indices analysis (CoMSIA) model built on a docking-based alignment was developed, wherein steric, electrostatic, hydrophobic and hydrogen bond properties are correlated with biological activity. The model was validated internally and externally (q2 = 0.721; r2pred = 0.938), and identified the sulfonyl and hydroxyl groups and the piperazine ring among the main regions of the molecules that can be modified to create new 5-HT6 antagonists.

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

PubMed

Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

2015-08-20

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

Problem-solving test: Southwestern blotting.

PubMed

Szeberényi, József

2014-01-01

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

NASA Astrophysics Data System (ADS)

Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

1985-12-01

A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

Adult T-cell leukemia-associated antigen (ATLA): detection of a glycoprotein in cell- and virus-free supernatant.

PubMed

Yamamoto, N; Schneider, J; Hinuma, Y; Hunsmann, G

1982-01-01

A glycoprotein of an apparent molecular mass of 46,000, gp 46, was enriched by affinity chromatography from the virus- and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells. gp 46 was specifically precipitated with sera from patients with adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.