Protein Expression and Purification

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Enhanced production of recombinant thermo-stable lipase in Escherichia coli at high induction temperature.

Publication Date: 2013 May 28 PMID: 23727254
Authors: Surnamevelez, G. M. - Surnamehorta, G. C. - Surnamesilva, G. J. - Surnameiemma, G. R. - Surnamegiordano, G. D. - Surnamezangirolami, G. C.
Journal: Protein Expr Purif

Thermostable microbial lipases are potential candidates for industrial applications such as specialty organic syntheses as well as hydrolysis of fats and oils. In this work, basic biochemical engineering tools were applied to enhance the production of BTL2 lipase cloned in E. coli BL321 under control of the strong temperature-inducible lambdaPL promoter. Initially, surface response analysis was used to assess the influence of growth and induction temperatures on enzyme production, in flask experiments. The results showed that temperatures of 30 and 45 degrees C were the most suitable for growth and induction, respectively, and led to an enzyme specific activity of 706,000 U/gDCW. The most promising induction conditions previously identified were validated in fed-batch cultivation, carried out in a 2 L bioreactor. Specific enzyme activity reached 770,000 U/gDCW, corresponding to 13,000 U per liter of culture medium and a lipase protein concentration of 10.8 g/L. This superior performance on enzyme production was a consequence of the improved response of lambdaPL promoter triggered by the high induction temperature applied (45 oC). These results point out to the importance of taking into account protein structure and stability to adequately design the recombinant protein production strategy for thermally induced promoters.

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Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells.

Publication Date: 2013 May 24 PMID: 23711380
Authors: Surnamekhanna, G. - Surnameimasaki, G. - Surnamechikwana, G. M. - Surnameperez-Miller, G. - Surnamehunter, G. O. - Surnamemosley, G. - Surnametakagi, G. - Surnamehurley, G. D.
Journal: Protein Expr Purif

We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.

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Production and characterization of highly purified recombinant thymosin beta 4 in Escherichia coli.

Publication Date: 2013 May 24 PMID: 23711379
Authors: Surnameli, G. - Surnamema, G. Y. - Surnametang, G. C. - Surnamenie, G. Y. - Surnamehuang, G.
Journal: Protein Expr Purif

Thymosin beta4 (Tbeta4) is a small peptide composed of 43 amino acids. It has many important biological functions, such as promoting cardiac repair and wound healing, and therefore has great potential in clinical applications. In this report, we describe a novel and efficient way to produce highly purified and active Tbeta4. It was expressed in a soluble form using a DsbA and hexahistindine tag in Escherichia coli (E. coli). Using high cell density cultivation, the final biomass concentration was about 50gL-1 dry cell weight with the expression level of the fusion protein being 40%. To obtain highly purified protein, a purification process involving a five-step column procedure was implemented. The purity of Tbeta4 was above 98% and all the host cell related impurities, such as endotoxin, host cell protein and residual DNA levels, were within the permissible range listed in the Chinese Pharmacopoeia. The E-rosette test demonstrated that the bioactivity of purified Tbeta4 was consistent with other published work. This is the first report producing highly purified Tbeta4 from genetically engineered sources.

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Cloning, expression and purification of the SRCR domains of glycoprotein 340.

Publication Date: 2013 May 22 PMID: 23707657
Authors: Surnamepurushotham, G. - Surnamedeivanayagam, G.
Journal: Protein Expr Purif

Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340''s domains and oral streptococcal AgI/II domains, we undertook to express human gp340''s first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

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Purification and enzymatic characterization of alcohol dehydrogenase from Arabidopsis thaliana.

Publication Date: 2013 May 24 PMID: 23707506
Authors: Surnamecheng, G. - Surnamehu, G. - Surnamean, G. - Surnamehuang, G. - Surnamexu, G.
Journal: Protein Expr Purif

Alcohol dehydrogenases (ADH) catalyze the interconversion between alcohols and aldehydes with the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. In this study, for the first time we report an over-expression and purification strategy for the Arabidosis thaliana ADH (AtADH), and characterize its enzymatic properties. AtADH was expressed in an Escherichia coli system, the polyhistidine-tag was removed after the recombinant AtADH protein was purified by metal chelating affinity chromatography. Activity assays demonstrated that AtADH has distinct enzymatic properties when compared with many well-known ADHs. It held peak activity at pH 10.5 and showed broad substrate selectivity for primary and secondary alcohols. The kinetic Km parameters for both ethanol and coenzyme were in the order of mM. This relative low affinity may reflect the need of the plant to maintain a supply of NAD+ in nature. Different from yeast ADH, AtADH showed almost the same activity for short straight chain alcohols and reduced activity for secondary alcohols. This broad spectrum in alcohol selection and the observed higher catalytic activity (high Vmax (EtOH)) may result from the requirement of the single enzyme to accommodate many substrates.

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Purification of recombinant vaccinia virus-expressed monomeric HIV-1 gp120 to apparent homogeneity.

Publication Date: 2013 Jul PMID: 23665667
Authors: Guo, W. - Cleveland, B. - Davenport, T. M. - Lee, K. K. - Hu, S. L.
Journal: Protein Expr Purif

Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS-PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.

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Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat.

Publication Date: 2013 Jul PMID: 23648869
Authors: Zhao, X. - Dong, Y. - Zhao, Z. - Guo, J. - Liu, J. - Huang, P. - Dong, D. - Fan, H. - Guo, Q. - Yang, X. - Xu, J. - Li, J. - Fu, L. - Chen, W.
Journal: Protein Expr Purif

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

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Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs.

Publication Date: 2013 Jul PMID: 23631926
Authors: Serena, M. S. - Geisler, C. - Metz, G. E. - Corva, S. G. - Mortola, E. C. - Larsen, A. - Jarvis, D. L. - Echeverria, M. G.
Journal: Protein Expr Purif

Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky's disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine.

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High-level expression and efficient one-step chromatographic purification of a soluble human leukemia inhibitory factor (LIF) in Escherichia coli.

Publication Date: 2013 Jul PMID: 23628981
Authors: Imaizumi, K. - Nishikawa, S. - Tarui, H. - Akuta, T.
Journal: Protein Expr Purif

Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase(R)) and high-aeration shake flask (Ultra Yield Flask), the yield of soluble proteins was significantly improved in comparison to commonly-used 2xYT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.

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A statistical approach for optimization of RANKL overexpression in Escherichia coli: Purification and characterization of the protein.

Publication Date: 2013 Jul PMID: 23623854
Authors: Papaneophytou, C. P. - Rinotas, V. - Douni, E. - Kontopidis, G.
Journal: Protein Expr Purif

Receptor activator of nuclear factor-kappaB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3mM, a post-induction temperature of 25 degrees C and a post-induction time of 6.5h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-alpha, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1+/-0.5muM.

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Expression, purification and preliminary characterization of glucagon receptor extracellular domain.

Publication Date: 2013 Jun PMID: 23597780
Authors: Wu, L. - Zhai, Y. - Lu, J. - Wang, Q. - Sun, F.
Journal: Protein Expr Purif

Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), one of class B G-protein coupled receptors (GPCRs). Diabetes is a bihormonal disease in which excessive glucagon secretion is a major contributor in the pathogenesis of this disease; elucidation of how glucagon binds to GCGR will facilitate the rational design of the GCGR antagonist for treating diabetic hyperglycemia. Here we report the successful expression and purification of the GCGR extracellular domain (GCGR-ECD) and its fusion protein with the glucagon peptide at its C-terminus (GCGR-ECD-Gc). We utilized the maltose binding protein (MBP) fusion method and disulfide bond isomerase DsbC co-expression approach for the success of the soluble expression of both GCGR-ECD and GCGR-ECD-Gc in Escherichia coli. We also obtained a high yield production of secreted GCGR-ECD with the baculovirus expression system by optimizing its N-terminal secreting signal. We first utilized isothermal titration calorimetry approach to determine the in vitro binding affinities of glucagon to the GCGR-ECD. No significant differences were found between the prokaryotic expressed GCGR-ECD (7.6muM) and the eukaryotic glycosylated one (6.6muM). The observation of the intra ligand-receptor binding within the fusion protein GCGR-ECD-Gc suggests it as a good candidate for further structural study.

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Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide.

Publication Date: 2013 Jun PMID: 23597779
Authors: An, S. J. - Yim, S. S. - Jeong, K. J.
Journal: Protein Expr Purif

Corynebacterium glutamicum is one of the useful hosts for the secretory production of heterologous proteins because of intrinsic attributes such as the presence of few endogenous proteins and proteases in culture medium. Here, we report the development of a new secretory system for the production of heterologous proteins by using the porin B (PorB) signal peptide in C. glutamicum. We examined two different endoxylanases and an antibody fragment (scFv) as model proteins for secretory production. In the flask cultivations, all the examined proteins were successfully produced as active forms into the culture medium with high efficiency. For the high-level production of endoxylanase, fed-batch cultivation was also performed in a lab-scale (5L) bioreactor, and the endoxylanases were efficiently secreted in the culture medium at levels as high as 615mg/L. From the culture supernatant, the secreted endoxylanases could be purified with high purity via one-step affinity column chromatography.

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Construction, expression, and purification of recombinant alphaVbeta5 integrin.

Publication Date: 2013 Jun PMID: 23583935
Authors: Tartaglia, L. J. - Bennett, A. - Woodhouse, A. G. - Aydemir, F. - Muzyczka, N. - Agbandje-McKenna, M.
Journal: Protein Expr Purif

A recombinant integrin expression system has been created for the large-scale production of alphaVbeta5 integrin extracellular domains that take advantage of Fos and Jun dimerization for expression in bacterial, insect, and mammalian cells. This utilizes an all-in-one vector, pQE-TriSystem, with molecular machinery for parallel expression without the need of additional subcloning. Optimal expression in HEK293 cells was determined by a time course analysis. The heterodimer was purified in a one-step nickel column purification scheme, and the sequence and functional state were confirmed by mass spectrometry and inhibition assays, respectively. The yields of alphaVbeta5 integrin obtained are in quantities suitable for multiple applications including structural biology and functional assays.

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Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase.

Publication Date: 2013 Jun PMID: 23583934
Authors: Jaffee, M. B. - Imperiali, B.
Journal: Protein Expr Purif

Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of Escherichia coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein.

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Facile isolation of purple membrane from Halobacterium salinarum via aqueous-two-phase system.

Publication Date: 2013 Jun PMID: 23583309
Authors: Shiu, P. J. - Ju, Y. H. - Chen, H. M. - Lee, C. K.
Journal: Protein Expr Purif

Purple membrane (PM) is a part of cytoplasmic membrane in certain extreme halophilic microorganisms belonging to Domain Archaea. It transduces light energy to generate proton gradient for ATP synthesis in the microorganisms. Bacteriorhodopsin (BR) is the only protein in PM responsible for the generation of proton gradient. Generally, PM was purified from Halobacterium salinarum via a tedious and lengthy sucrose density gradient ultracentrifugation (SGU). In this work, a facile method based on polyethyleneglycol (PEG)-phosphate aqueous-two- phase extraction system (ATPS) was employed to purify PM from cell lysate of H. salinarum. The results showed that PM could be completely recovered from the interface of PEG-phosphate ATPS with BR purity ca 94.1% as measured by UV-visible absorption spectra. In comparison with PM obtained by SGU, the PM isolated by ATPS could achieve the same level of purity and photocurrent activity (ca 177.2nA/mugBR/cm(2)) as analyzed by SDS-PAGE and photocurrent measurement, respectively. The easily scalable and straightforward ATPS procedure demonstrated that PM can be purified and recovered more cost-effectively with a significantly reduced operation time that should lead to broader range applications of PM possible.

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Cloning, purification and metal binding of the HNH motif from colicin E7.

Publication Date: 2013 Jun PMID: 23563167
Authors: Gyurcsik, B. - Czene, A. - Jankovics, H. - Jakab-Simon, N. I. - Slaska-Kiss, K. - Kiss, A. - Kele, Z.
Journal: Protein Expr Purif

The HNH family of endonucleases is characterized by a betabetaalpha metal-finger structural motif. Colicin E7 is a representative member of this family containing the strictly conserved HNH motif at its C-terminus. Structural and biochemical studies suggested that the HNH motif could contain all the residues necessary for metal ion binding and nuclease activity. In this work a 43 amino acid peptide extending from V534 to K576 of colicin E7 and encompassing the HNH motif was cloned and expressed in Escherichia coli as a ubiquitin fusion protein. The N-terminal fusion tag was cleaved off by a specific protease, and the HNH peptide was purified free of ubiquitin. Circular dichroism, fluorescence and mass spectrometry showed that the zinc-ion binding affinity of the purified HNH peptide was much weaker than that of the intact nuclease domain suggesting that the N-terminal part of the nuclease domain is essential for stabilizing the structure of the HNH motif. The coordination sphere of the metal ion was found to be not fully equipped by the ligand - leaving a free coordination site for the substrate. Neither DNA binding nor DNAse activity of the purified HNH peptide was detected. Comparison of the glutathion-S-transferase-fused N-terminal deletion mutants of the colicin E7 nuclease domain suggested that the presence of the DNA-binding site is still not sufficient for the catalytic activity.

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Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases.

Publication Date: 2013 Jun PMID: 23562736
Authors: Blazic, M. - Kovacevic, G. - Prodanovic, O. - Ostafe, R. - Gavrovic-Jankulovic, M. - Fischer, R. - Prodanovic, R.
Journal: Protein Expr Purif

Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.

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Transient expression, purification and characterisation of human full-length PPARgamma2 in HEK293 cells.

Publication Date: 2013 Jun PMID: 23562662
Authors: Liu, J. - Ormo, M. - Nystrom, A. C. - Claesson, J. - Giordanetto, F.
Journal: Protein Expr Purif

Effective anti-diabetic drugs known as thiazolidinediones (e.g. rosiglitazone, pioglitazone) exert their therapeutic effects through their agonistic activity at the peroxisome proliferator-activated receptor gamma (PPARgamma). As a multidomain transcription factor, PPARgamma forms heterodimers with different retinoid X receptors (RXRs) to modulate target gene expression at the transcriptional level in response to natural or synthetic ligands. Difficulties in producing either of the two major human PPARgamma isoforms (PPARgamma1 and PPARgamma2) as pure full-length proteins in adequate quantity has hindered detailed mechanistic studies of PPARgamma and its ancillary protein partners. Here we report an efficient transient expression system to produce recombinant human full-length PPARgamma2 protein. The DNA encoding the human full-length PPARgamma2 was cloned into a mammalian episomal vector and transiently expressed in human embryonic kidney 293 (HEK293-6E) cells with an expression level of 10mg/L culture. Identity of the purified recombinant PPARgamma2 protein was confirmed by mass spectrometry analysis. The purified PPARgamma2 protein was active in ligand binding and could be phosphorylated in vitro by Cdk5/p25 at Ser 273. Further studies showed that selected PPARgamma modulators inhibited Cdk5-mediated PPARgamma2 Ser 273 phosphorylation in vitro. Our results demonstrate the feasibility of producing large quantities of pure and functional human full-length PPARgamma2 suitable for drug discovery applications.

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Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.

Publication Date: 2013 Jun PMID: 23562661
Authors: Gromek, K. A. - Meddaugh, H. R. - Wrobel, R. L. - Suchy, F. P. - Bingman, C. A. - Primm, J. G. - Fox, B. G.
Journal: Protein Expr Purif

Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields approximately 3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.

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Developing a reproducible method for the high-resolution separation of peritoneal dialysate proteins on 2-D gels.

Publication Date: 2013 Jun PMID: 23558012
Authors: Zhang, L. - Wen, Q. - Mao, H. P. - Luo, N. - Rong, R. - Fan, J. J. - Yu, X. Q.
Journal: Protein Expr Purif

PURPOSE: Despite recent progress in the proteomic analysis of peritoneal dialysate effluent (PDE), there remains unresolved problems in the development of an optimal sample preparation method. EXPERIMENTAL DESIGN: We examined five protocols for concentrating PDE proteins and the effects of immobilized pH gradient (IPG) strips with different pH ranges and sample loading techniques. In addition, we examined three kits for depleting high abundance proteins by SDS-PAGE and two-dimensional gel electrophoresis (2-DE). RESULTS: PDE proteins precipitated with 75% acetonitrile (ACN) showed the greatest number of protein spots by 2-DE, with over 800 distinct spots. Higher-resolution images were obtained using IPG strips with a pH range of 4-7. The ProteoPrep immunoaffinity albumin and IgG depletion kit removed high abundance proteins with higher efficiency and more compatibility with isoelectric focusing (IEF). Removing high abundance proteins also increased the resolution and improved the intensity of low abundance proteins. CONCLUSION AND CLINICAL RELEVANCE: High-resolution 2-DE images of PDE proteins were obtained by concentrating samples with 75% ACN, using pH 4-7 IPG strips, and depleting high abundance proteins. This optimized method will enable future studies to discover predictive biomarkers of disease in patients on dialysis.

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Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

Publication Date: 2013 Jun PMID: 23545225
Authors: Zheng, Q. - Xiong, Y. L. - Su, Z. J. - Zhang, Q. H. - Dai, X. Y. - Li, L. Y. - Xiao, X. - Huang, Y. D.
Journal: Protein Expr Purif

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37 degrees C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.

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High level expression, purification and characterization of recombinant CCR5 as a vaccine candidate against HIV.

Publication Date: 2013 Jun PMID: 23542826
Authors: Wu, K. - Xue, X. - Li, M. - Qin, X. - Zhang, C. - Li, W. - Hao, Q. - Wang, Z. - Liu, Q. - Zhang, W. - Zhang, Y.
Journal: Protein Expr Purif

Cysteine-cysteine chemokine receptor type 5 (CCR5) is an important co-receptor for human immunodeficiency virus (HIV) infection and CCR5 neutralizing agents have proven efficient in patients suffering from HIV infection. Here, we expressed and purified various CCR5 vaccines named rCCR5, PADRE-rCCR5, GST-C1 and GST-C2 composed of different epitopes of CCR5. Results showed that vaccines containing multiple epitopes (rCCR5 and PADRE-rCCR5) induced stronger immune responses than single-epitope ones (GST-C1 and GST-C2). In addition, the elicited antibodies can specifically bind CCR5(+) U937 but not CCR5(-) Wish cells. These results demonstrate that the CCR5 vaccines are useful for further research, especially for the in vitro preclinical evaluation of their potential as biological CCR5 neutralizing agents.

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"Salt tolerant" anion exchange chromatography for direct capture of an acidic protein from CHO cell culture.

Publication Date: 2013 Jun PMID: 23537793
Authors: Champagne, J. - Balluet, G. - Gantier, R. - Toueille, M.
Journal: Protein Expr Purif

The present study describes the use of the new HyperCel STAR AX "salt tolerant" anion exchange sorbent for the capture from Chinese Hamster Ovary (CHO) cell culture supernatant (CCS) of an acidic model protein (alpha-amylase). HyperCel STAR AX sorbent and other conventional anion exchangers were evaluated to purify biologically-active enzyme. Salt tolerance of the sorbent allowed reaching 5-fold higher dynamic binding capacity than conventional anion exchange during capture of the enzyme from neat (undiluted) CCS. After optimization of operating conditions, HyperCel STAR AX turned out to be the only sorbent allowing efficient protein capture directly from both neat and diluted CCS with consistent and satisfying purity, yield and productivity. Therefore implementation of the salt tolerant sorbent in industrial purification processes should allow avoiding time and cost consuming steps such as dilution or UF/DF that exclusively aim at establishing suitable conditions for ion exchange step without bringing any added value to the purification process performance. Altogether this study highlights the flexibility and cost-reduction potential brought in process design by the HyperCel STAR AX salt tolerant sorbent.

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Chromatographically-purified capsid proteins of red-spotted grouper nervous necrosis virus expressed in Saccharomyces cerevisiae form virus-like particles.

Publication Date: 2013 Jun PMID: 23537792
Authors: Choi, Y. R. - Kim, H. J. - Lee, J. Y. - Kang, H. A. - Kim, H. J.
Journal: Protein Expr Purif

Nervous necrosis viruses (NNVs) cause mass mortality of marine fish, leading to large economic losses for aquaculturists. A promising vaccine candidate for preventing NNV infection is the NNV virus-like particle (VLP), which is a structure resulting from assembly of recombinant NNV capsid protein. NNV capsid proteins have been expressed in insect cells and the Escherichia coli expression system, and purified by non-scalable protocols such as ultracentrifugation on sucrose and cesium chloride density gradients. In this study, we expressed red-spotted grouper NNV (RGNNV) capsid proteins in Saccharomyces cerevisiae (S. cerevisiae) and developed a chromatography-based method with potential for large-scale vaccine production. The RGNNV capsid protein was successfully purified by a single-step of heparin chromatography. Transmission electron microscopy (TEM) confirmed the high quality of the purified RGNNV capsid protein: it was in the form of VLPs with mean diameters of 25nm, in homogeneous suspension without any aggregation. Moreover, the RGNNV capsid protein elicited anti-RGNNV capsid protein antibodies in mice. We suggest that RGNNV capsid protein expressed in S. cerevisiae and purified by heparin chromatography, is of sufficient quality for use as a vaccine.

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Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli.

Publication Date: 2013 Jun PMID: 23537791
Authors: Jang, S. H. - Jeong, K. J.
Journal: Protein Expr Purif

The C5aR antagonist (C5aRA)(1), which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli. To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37 degrees C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496mg/L of NusA-fused C5aRA could be produced.

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Native signal peptide of human ERp57 disulfide isomerase mediates secretion of active native recombinant ERp57 protein in yeast Saccharomyces cerevisiae.

Publication Date: 2013 Jun PMID: 23528814
Authors: Ciplys, E. - Zitkus, E. - Slibinskas, R.
Journal: Protein Expr Purif

Human ERp57 protein is disulfide isomerase, facilitating proper folding of glycoprotein precursors in the concert with ER lectin chaperones calreticulin and calnexin. Growing amount of data also associates ERp57 with many different functions in subcellular locations outside the ER. Analysis of protein functions requires substantial amounts of correctly folded, biologically active protein, and in this study we introduce yeast Saccharomyces cerevisiae as a perfect host for production of human ERp57. Our data suggest that native signal peptide of human ERp57 protein is recognized and correctly processed in the yeast cells, which leads to protein secretion. Secreted recombinant ERp57 protein possesses native amino acid sequence and is biologically active. Moreover, secretion allows simple one-step purification of recombinant ERp57 protein with the yields reaching up to 10mg/L.

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Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system.

Publication Date: 2013 Jun PMID: 23523699
Authors: Maki, J. L. - Tres Brazell, J. - Teng, X. - Cuny, G. D. - Degterev, A.
Journal: Protein Expr Purif

Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-alpha) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.

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Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000.

Publication Date: 2013 Jun PMID: 23507306
Authors: Elkhalfi, B. - Araya-Garay, J. M. - Rodriguez-Castro, J. - Rey-Mendez, M. - Soukri, A. - Serrano Delgado, A.
Journal: Protein Expr Purif

The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in Escherichia coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles.

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Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae.

Publication Date: 2013 Jul PMID: 23500722
Authors: Xia, L. - Liu, Z. - Ma, J. - Sun, S. - Yang, J. - Zhang, F.
Journal: Protein Expr Purif

CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori. In this study, an antibacterial peptide gene of cecropinXJ was cloned into the pYES2/CT/alpha Factor expression vector and expressed in the Saccharomyces cerevisiae INVSc1 strain. Following an induction of recombinant protein expression in yeast for 120h, the maximum amount of total secreted protein was 1.437g/L. The percentage of recombinant cecropinXJ was estimated to be 79.45% of the total protein. After purification with Ni-NTA agarose column, recombinant cecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms, including Gram-negative and Gram-positive bacteria. Its minimal inhibitory concentration (MIC) against Escherichia coli ATCC25922 was 0.81muM. In addition, transmission electron microscopy (TEM) analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms. CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200muM. Thus, cecropinXJ acts selectively on bacterial membranes. Purified recombinant antibacterial peptide, cecropinXJ, retained a high stability against E. coli ATCC25922 over a temperature range from 4 degrees C to 100 degrees C and a pH range from pH 2.0 to 12.0. Taken together, this study demonstrates that recombinant cecropinXJ can be produced in large quantities in yeast with genetic engineering methods, and that it has strong and rapid antimicrobial activities against all of microorganisms tested. Our results suggest that cecropinXJ is a potential candidate for therapy.

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Design, production, and characterization of a single-chain variable fragment (ScFv) derived from the prostate specific membrane antigen (PSMA) monoclonal antibody J591.

Publication Date: 2013 Jun PMID: 23500147
Authors: Parker, S. A. - Diaz, I. L. - Anderson, K. A. - Batt, C. A.
Journal: Protein Expr Purif

A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6x his-tag was added to facilitate affinity purification. A down-scale 2L methanol-induced P. pastoris fermentation yielded 330mg of total protein following a 96h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications.

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High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

Publication Date: 2013 Jul PMID: 23201280
Authors: Surnametruan, G. - Surnamevasil, G. - Surnamestonehouse, G. - Surnamevasil, G. L. - Surnamepohl, G.
Journal: Protein Expr Purif

The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced mu-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4A, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75A.

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