Protein Expression and Purification Journal Impact Factor & Information

Publisher: Elsevier

Journal description

The power of modern molecular genetics to provide large quantities of proteins that were previously difficult to obtain has sparked an explosion of interest in both practical and theoretical aspects of protein purification. Protein Expression and Purification is dedicated to providing a forum for information about protein isolation based on conventional fractionation as well as techniques employing various molecular biological procedures to increase protein expression.

Current impact factor: 1.51

Impact Factor Rankings

2015 Impact Factor Available summer 2015
2013 / 2014 Impact Factor 1.508
2012 Impact Factor 1.429
2011 Impact Factor 1.587
2010 Impact Factor 1.644
2009 Impact Factor 1.563
2008 Impact Factor 1.621
2007 Impact Factor 1.94
2006 Impact Factor 1.867
2005 Impact Factor 1.553
2004 Impact Factor 1.336
2003 Impact Factor 1.47
2002 Impact Factor 1.375
2001 Impact Factor 1.497
2000 Impact Factor 1.569
1999 Impact Factor 1.416
1998 Impact Factor 1.382
1997 Impact Factor 1.341
1996 Impact Factor 1.413
1995 Impact Factor 1.497
1994 Impact Factor 1.822

Impact factor over time

Impact factor
Year

Additional details

5-year impact 1.51
Cited half-life 7.20
Immediacy index 0.38
Eigenfactor 0.01
Article influence 0.43
Website Protein Expression and Purification website
Other titles Protein expression and purification (Online), Protein expression and purification, Protein expression & purification
ISSN 1096-0279
OCLC 36951598
Material type Document, Periodical, Internet resource
Document type Internet Resource, Computer File, Journal / Magazine / Newspaper

Publisher details

Elsevier

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    • Deposit due to Funding Body, Institutional and Governmental policy or mandate only allowed where separate agreement between repository and the publisher exists.
    • Permitted deposit due to Funding Body, Institutional and Governmental policy or mandate, may be required to comply with embargo periods of 12 months to 48 months .
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    • Publisher's version/PDF cannot be used
    • Articles in some journals can be made Open Access on payment of additional charge
    • NIH Authors articles will be submitted to PubMed Central after 12 months
    • Publisher last contacted on 18/10/2013
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: An unknown D-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coil. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in Escherichia coil BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8 kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.015
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    ABSTRACT: Trypsin is a typical member of serine protease families, specifically cleaving the carboxyl group of peptides at the basic amino acids arginine and lysine. The DNA fragment of porcine trypsin with its propeptide coding sequence was optimized and synthesized according to codon usage bias of Pichia pastoris. The optimized sequence was integrated into genome of P.pastoris GS115 by using the vector pHBM905A. The yield of the recombinant protein was 0.48 mg/ml with a maximum activity of 19.2 U/ml after 96h induction in a 5L fermenter. An optimum activity for the recombinant trypsin was observed at 35°C and pH 8.5. This is the first time to express the porcine trypsinogen with P.pastoris expression system. This report also found that the propeptide was cleaved from the recombinant protein and the enzymogen was transferred into trypsin at the later phase of the fed-batch cultivation. In particular, the activation process can be initiated by changing pH. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.014
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    ABSTRACT: 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide synthetase (PurC) is a key enzyme in the de novo purine biosynthetic pathway of bacteria and an ideal target pathway for the discovery of antimicrobials. Bacillus anthracis (Ba) and Streptococcus pneumoniae (Sp) are two of the bacteria shown to be severe detriments to public health. To be able to carry out the experimentation that leads to drug discovery, high yields of pure soluble recombinant protein must first be obtained. We studied two recombinant PurC proteins from B. anthracis and S. pneumoniae, using Escherichia coli as the host cells. These two proteins, with very similar amino acid sequences, exhibit very different solution properties, leading to a large difference in yields during protein purification under the same conditions. The yield for SpPurC (>50mG per gram of cells) is ten times greater than that for BaPurC (<5mG per gram of cells). The BaPurC samples in solution consisted of oligomers and dimers, with dimers as its functional form. Comparing the yields of dimers, SpPurC is 25 times greater than that for BaPurC (∼2mG per gram of cell). Our studies suggest that the difference in exposed hydrophobic surface area is responsible for the difference in yields under the same conditions. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.05.016
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    ABSTRACT: Single molecule assays were performed on bovine intestinal alkaline phosphatase and the recombinant enzyme expressed in Pichia pastoris using a capillary electrophoresis-based method. The catalytic rates for the bovine and recombinant enzymes were found to be 11000 ± 7000 min(-1) (N=161) and 12000 ± 7000 min(-1) (N=173) respectively. Mean catalytic rates and variances did not differ significantly between the enzyme from both sources. Furthermore, the distribution of catalytic rates were indistinguishable. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.013
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    ABSTRACT: The (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS-PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75Å resolution. The crystal belongs to space group P41212, with unit-cell parameters a=b=82.0Å, c=139.8Å, α=β=γ=90.0°. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.007
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    ABSTRACT: Interleukin-33 (IL-33) is a member of the IL-1 family and the ligand of orphan ST2 molecules. IL-33 is widely expressed in multiple tissues and cells, and mainly involved in regulating Th2 immune and inflammatory responses. Inhibiting IL-33 signaling pathways relieves the symptoms of allergic inflammation, indicating that IL-33 is a potential target for the treatment of allergic diseases. In this study, the recombinant vectors SP-scFv-Fc/pcDNA3.1 and SP-scFv-Fc/PMH3(EN) were constructed to express a human scFv-Fcs against IL-33. The size of the inserted SP-scFv-Fc was approximately 1,540 bp. The RT-PCR results showed that SP-scFv-Fcs were successfully transfected into CHO K1 cells. Western blot analysis indicated specific binding of the expressed scFv-Fcs fusion protein (approximately 60 kDa) with a goat anti-human IgG1 Fc antibody. The expression level of the scFv-Fcs from SP-scFv-Fc/PMH3(EN) was higher than that from SP-scFv-Fc/ pcDNA3.1. A single high-expressing cell line was selected after three rounds of screening and the fusion protein was expressed in a suspension culture in serum-free medium. The level of expression products reached 20 mg/L and the expressed and purified scFvs was further characterized and analyzed for bioactivity and functionality. The recombinant vectors for eukaryotic expression of scFv-Fcs against IL-33 were successfully constructed and the expressed scFv-Fcs was shown to be a suitable candidate for the development of a new therapy for allergic and autoimmune diseases. Copyright © 2015 Elsevier Inc. All rights reserved.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.008
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    ABSTRACT: The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca(2+) and Ni(2+). In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.010
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    ABSTRACT: The gene encoding a novel glucoamylase (GlucaM) from the Corallococcus sp. strain EGB was cloned and heterologous expressed in Escherichia coli BL21(DE3), and the enzymatic characterization of recombinant GlucaM (rGlucaM) was determined in the study. The glucaM had an open reading frame of 1938 bp encoding GlucaM of 645 amino acids with no signal peptide. GlucaM belongs to glycosyl hydrolase family 15 and shares the highest identity 96% with the GH15 glucoamylase of C. coralloides DSM 2259. The rGlucaM with His-tag was purified by the Ni(2+)-NTA resin, with a specific activity from 3.4 U/mg up to 180 U/mg, and the molecular weight of rGlucaM was approximately 73 kDa on SDS-PAGE. The Km and Vmax of rGlucaM for soluble starch were 1.2 mg/mL and 46 U/mg, respectively. rGlucaM was optimally active at pH 7.0 and 50°C and had highly tolerance to high concentrations of salts, detergents, and various organic solvents. rGlucaM hydrolyzed soluble starch to glucose, and hydrolytic activities were also detected with amylopectin, amylase, glycogen, starch (potato), α-cyclodextrin, starch (corn and potato). The analysis of hydrolysis products shown that rGlucaM with α-(1-4),(1-6)-D-glucan glucohydrolase toward substrates. These characteristics indicated that the GlucaM was a new member of glucoamylase family and a potential candidate for industrial application. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.009
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    ABSTRACT: A deep-sea thermophile, Geobacillus sp. 4j, was identified to grow on starch and produce thermostable amylase. N-terminally truncated form of Geobacillus sp. 4j α-amylase (Gs4j-amyA) was fused at its N-terminal end with the signal peptide of outer membrane protein A (OmpA) of Escherichia coli. The enzyme was over-expressed in E. coli BL21 with a maximum extracellular production of 130U/ml in shake flask. The yield of the transformant increased 22-fold as, compared with that of the wild strain. The recombinant enzyme purified to apparent homogeneity by metal-affinity chromatography, exhibited a molecular mass of 62kDa. It displayed the maximal activity at 60-65°C and pH 5.5. Its half-life (t1/2) at 80°C was 4.25h with a temperature deactivation energy of 166.3kJ/mol. Compared to three commonly used commercial α-amylases, the Gs4j-amyA exhibited similar thermostable performance to BLA but better than BAA and BSA. It also showed a universally efficient raw starch hydrolysis performance superior to commercial α-amylases at an acidic pH approaching nature of starch slurry. As a new acidic-resistant thermostable α-amylase, it has the potential to bypass the industrial gelatinization step in raw starch hydrolysis. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; 114. DOI:10.1016/j.pep.2015.06.002
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    ABSTRACT: An open reading frame of the Thermus thermophilus HJ6 hypothetical laccase, which composed of 729 bases, was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli SoluBL21™ cells. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 6M guanidine HCl. The solubilized protein was refolded by a simple on-column refolding procedure using Ni-chelation affinity chromatography and then the refolded protein was purified by gel filtration chromatography. It showed a single band with a molecular mass of 27kDa in SDS-PAGE. The results from UV-visible absorption and electron paramagnetic resonance (EPR) analysis suggested that the enzyme had the typical copper sites, type-1, 2, and 3 Cu(II) of laccase. The purified enzyme exhibited the laccase activity with the optimal catalytic temperature at 75°C. The optimum pH for the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and syringaldazine was 4.5 and 6.0, respectively. The recombinant protein showed high thermostability, and the half-life of heat inactivation was about 50min at 85°C. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest kcat value for guaiacol, and highest kcat/Km for 2,6-dimethoxy-phenol. The enzyme reaction was strongly inhibited by the metal chelators and the thiol compounds. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.004
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    ABSTRACT: The roles of the extracellular domain of type II TGF-β receptor (TBRII-ECD) in physiological processes ranging from development to cancer to wound healing render it an attractive target for exploration with chemical tools. For such applications, large amounts of active soluble protein are needed, but the yields of TBRII-ECD we obtained with current folding protocols were variable. To expedite the identification of alternative folding conditions, we developed an on-plate screen. This assay indicated that effective folding additives included the non-detergent sulfobetaine-201 (NDSB-201). Although NDSB-201 can facilitate protein folding, the mode by which it does so is poorly understood. We postulated that specific interactions between NDSB-201 and TBRII-ECD might be responsible. Analysis by X-ray crystallography indicates that the TBRII-ECD possesses a binding pocket for NDSB-201. The pyridinium group of the additive stacks with a phenylalanine side chain in the binding site. The ability of NDSB-201 to occupy a pocket on the protein provides a molecular mechanism for the additive's ability to minimize TBRII-ECD aggregation and stabilize the folded state. NDSB-201 also accelerates TBRII-ECD crystallization, suggesting it may serve as a useful crystallization additive for proteins refolded with it. Our results also suggest there is a site on TBRII-ECD that could be targeted by small-molecule modulators. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.06.001
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    ABSTRACT: Cyclin H (CycH), a member of the large cyclin family, participates in every process of cell division. Its biological functions and importance have received wide attention in mammalians, but not in higher plants. This work reports a protein purification protocol for obtaining Arabidopsis CycH;1 (AtCycH;1) from prokaryotic expression system, followed by characterization of its biophysical properties. The protein was constructed with a His-tag at its N-terminus. One-step nickel-affinity purification yielded high pure target protein, which behaved as a monomer in the testing condition. Circular Dichroism spectrum revealed that AtCycH;1 is a helical protein containing a significant amount of disordered structures. Further assays indicated that AtCycH;1 exhibits poor heat-resistance and can be easily degraded in room temperature, suggesting low stability for the protein. The flexible and unstable properties may be intrinsic to the protein in vivo as it has to bind with different partners during the cell cycle and be promptly degraded to meet the phase transition. The instability, however, can be improved by adding SO4(2-) ion in the protein buffer. The presence of a high concentration of SO4(2-) is capable of increasing the thermal stability and inhibiting the degradation. Irrespective of whether the association of SO4(2-) with AtCycH;1 drives the protein into more compact form or not, the current results may provide clues for a successful crystallization of AtCycH;1 and its subsequent structural analysis in the future. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 06/2015; 114. DOI:10.1016/j.pep.2015.06.003
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    ABSTRACT: The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB. Copyright © 2015 Elsevier Inc. All rights reserved.
    Protein Expression and Purification 06/2015; DOI:10.1016/j.pep.2015.05.014
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    ABSTRACT: Streptomyces coelicolor is a soil-dwelling bacterium that undergoes an intricate, saprophytic lifecycle. The bacterium takes up exogenous nucleosides for nucleic acid synthesis or use as carbon and energy sources. However, nucleosides must pass through the membrane with the help of transporters. In the present work, the SCO4884 and SCO4885 genes were cloned into pCOLADuet-1 and overexpressed in Escherichia coli BL21. Each protein was monomeric. Using isothermal titration calorimetry, we determined that SCO4884 and SCO4885 are likely nucleoside receptors with affinity for adenosine and pyrimidine nucleosides. On the basis of bioinformatics analysis and the transporter classification system, we speculate that SCO4884–SCO4888 is an ABC-like transporter responsible for the uptake of adenosine and pyrimidine nucleosides.
    Protein Expression and Purification 05/2015; 109. DOI:10.1016/j.pep.2015.02.004
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    ABSTRACT: The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; DOI:10.1016/j.pep.2015.05.013
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    ABSTRACT: Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an E. coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; DOI:10.1016/j.pep.2015.05.011
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    ABSTRACT: Glutamate oxaloacetate transaminase (GOT) catalyzes the reversible reaction of ʟ-aspartate and α-ketoglutarate into oxaloacetate and ʟ-glutamate and plays a key role in carbon and nitrogen metabolism in all organisms. In human tissues, GOTs are pyridoxal 5'-phosphate-dependent (PLP) enzymes which exist in cytoplasm and mitochondrial forms, GOT1 and GOT2, respectively. GOT1 expression correlates with the growth of several tumors because cancer cells can utilize the amino acid glutamine to fuel anabolic processes, and therefore, GOT1 represents a new therapeutic target in cancer. In this work, human GOT1 was expressed in Escherichia coli periplasmic space, and purified by a combination of His-tag immobilized metal-ion affinity chromatography and anion exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 37 °C and a pH of 7.5. Cations such as Na(+), K(+) and Mg(2+) slightly inhibited the activity of recombinant human GOT1, while Zn(2+), Mn(2+), Cu(2+), Ni(2+), Co(2+) and Ca(2+) had stronger inhibitory effects. Crystals of human GOT1 were grown using the hanging-drop vapor diffusion method at 4 °C with 0.1 M Bis-Tris pH 6.0 and 21% (w/v) PEG 3350. The crystals diffracted to 2.99 Å resolution and belonged to space group P43212 with the unit cell parameters a = b = 93.4, c = 107.4 Å, α = β = γ = 90°. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; 113. DOI:10.1016/j.pep.2015.05.010
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    ABSTRACT: Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; 113. DOI:10.1016/j.pep.2015.05.009
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    ABSTRACT: Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8μg/mL solubilized rTOP protein and the refolding medium containing 0.3mM oxidized glutathione, 0.05mM dithiothreitol, 5mM CaCl2, 5% glycerol in 50mM Tris-HCl buffer, pH 9.6, with 1μM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6mg per L of E. coli culture). Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; 113. DOI:10.1016/j.pep.2015.05.007