Protein Expression and Purification (Protein Expr Purif )

Publisher: Elsevier

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.

  • Impact factor
    1.43
  • 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

  • Pre-print
    • Author can archive a pre-print version
  • Post-print
    • Author can archive a post-print version
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    • Voluntary deposit by author of authors post-print allowed on authors' personal website, arXiv.org or institutions open scholarly website including Institutional Repository, without embargo, where there is not a policy or mandate
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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: A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4 mg enzyme per L of growth culture with a specific activity of 159 U mg−1. By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 68.5 kDa. The pH optimum for the oxidation of galactose was in the range of pH 6–7.5. Optimum temperature for the enzyme activity was 35 °C, with a half-life of 11.2 min, 5.3 min, and 2.7 min for incubation at 40 °C, 50 °C, and 60 °C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226 U mg−1) and the highest catalytic efficiency (kcat/Km) for melibiose (2700 mM−1 s−1). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.
    Protein Expression and Purification 12/2014; 14.
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    ABSTRACT: AarF domain containing kinase 3 (ADCK3) is a mitochondrial protein known to have a role in the electron transport chain. Despite being required for the biosynthesis of coenzyme Q10, a lipid-soluble electron transporter found to be essential for aerobic cellular respiration, the precise biological function of ADCK3 remains unknown. Patients with mutations in ADCK3 experience an onset of neurological disorders from childhood, including cerebellar ataxia and exercise intolerance. After extensive screening for soluble recombinant protein expression, an N-terminal fusion of maltose-binding protein was found to facilitate the overexpression of the human ADCK3 kinase domain in Escherichia coli as a soluble and biologically active entity. For the first time our work reveals Mg(2+)-dependent ATPase activity of ADCK3, providing strong support for the theoretical prediction of this protein being a functional atypical kinase. Copyright © 2014. Published by Elsevier Inc.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac(TM)-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis. Copyright © 2014. Published by Elsevier Inc.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrixschenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomycescerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: Penicillin-binding protein 3 (PBP3) of P. aeruginosa is the primary target of β-lactams used to treat pseudomonas infections. Meanwhile, structure change and overproduction of PBP3 play important roles in the drug resistance of P. aeruginosa. Therefore, studies on the gene and structure of PBP3 are urgently needed. P. aeruginosa CMCC 10104 is a type culture strain common used in China. However, there is no report on its genomic and proteomic profiles. In this study, based on ftsI of P. aeruginosa PAO1, the gene encoding PBP3 was cloned from CMCC 10104. A truncated version of the ftsI gene, omitting the bases encoding the hydrophobic leader peptide (amino acids 1-34), was amplified by PCR. The cloned DNA shared 99.76% identity with ftsI from PAO1. Only four bases were different (66 C-A, 1020 T-C, 1233 T-C, and 1527 T-C). However, there were no differences between their deduced amino acid sequences. The recombinant PBP3 (rPBP3), containing a 6-histidine tag, was expressed in E. coli BL21 (DE3). Immobilized metal affinity chromatography (IMAC) with Ni(2+)-NTA agarose was used for its purification. The purified rPBP3 was identified by SDS-PAGE and western blot analysis, and showed a single band at about 60 kDa with purity higher than 95%. The penicillin-binding assay indicated that the obtained rPBP3 was functional and not hindered by the presence of the C-terminal His-tag. The protocol described in this study offers a method for obtaining purified recombinant PBP3 from P. aeruginosa CMCC 10104. Copyright © 2014. Published by Elsevier Inc.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: Lipid modification of proteins plays key roles in cellular signaling pathways. We describe the development of myristoylated preS1-nanocages (myr-preS1-nanocages) that specifically target human hepatocyte-like HepaRG cells in which a specific receptor-binding peptide (preS1) is joined to the surface of naturally occurring ferritin cages. Using a genetic engineering approach, the preS1 peptide was joined to the N-terminal regions of the ferritin cage via flexible linker moieties. Myristoylation of the preS1 peptide was achieved by co-expression with yeast N-myristoyltransferase-1 in the presence of myristic acid in Escherichia coli cells. The myristoylated preS1-nanocages exhibited significantly greater specificity for human hepatocyte-like HepaRG cells than the unmyristoylated preS1-nanocages. These results suggest that the lipid-modified nanocages have great potential for effective targeted delivery to specific cells. Copyright © 2014. Published by Elsevier Inc.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: Mercury resistance is the most widespread of all anti-microbial resistance occurring in a wide variety of Gram-negative and Gram-positive bacterial genera. The systems that are most studied and best understood are those encoded in mercury resistance (Mer) operons in Gram-negative bacteria. The mercury detoxification functions by the importation of highly toxic Hg(2+) into cytoplasm and enzymic reduction to volatile Hg(0). MerT is a small (13kDa) inner membrane protein involved in mercuric ion transport system. We have overexpressed recombinant 6His-tagged MerT from Escherichia coli in a native folded form and purified it to homogeneity in n-dodecyl-β-d-maltopyranoside (DDM) by immobilized metal affinity chromatography (IMAC). Circular dichroism showed that the protein is largely α-helical. Size-exclusion chromatography (SEC) in a variety of detergents showed that the protein exists in a multiple of oligomeric states as also confirmed by SEC coupled with multiple-angle light scattering. Copyright © 2014. Published by Elsevier Inc.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 5' sequence coding for its secretion signal peptide (chiA74Δsp) into a cold shock expression vector (pCold I) for production of the enzyme in Escherichia coli BL21-Rosetta2. As a result, the N-terminal end of ChiA74Δsp ORF was fused to an artificial sequence of 28 amino acid, including a 6x histidine tag for purification of recombinant 6xHis tagged-ChiA74Δsp (rChiA74, ∼74 kDa). Along with a protein of ∼74 kDa, we co-purified its ∼55 kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40°C. Most divalent cations (e.g. Ba(+2) and Ca(+2), Mn(+2), Mg(+2), Zn(+2), Cu(+2)) at concentration of 10 mM reduced chitinase activity by ∼30%, and Hg(+2) (10 mM) drastically inhibited ChiA74 activity by ∼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11 ± 0.01 nmol/min, 2.15 μM ± 0.45 and 3.81 s(-1), respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected. Copyright © 2014. Published by Elsevier Inc.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6 mg of a recombinant peptide (RVG29-9R-6His) from 0.4 g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: The autoantigen U1-68/70 K is the dominant diagnostic marker in Mixed Connective Tissue Disease (MCTD) that until recently could not be expressed in its full-length form [Northemann et al., 1995]. Using cell-free expression screening, we successfully produced the snRNP protein U1-68/70 K in a soluble full-length form in Escherichia coli cells. The protein length and identity was determined by Western Blot and MS/MS analysis. Additionally, its reactivity in the autoimmune diagnostic was confirmed. Establishment of a cell-free expression system for this protein was important for further elucidation of protein expression properties such as the cDNA construct, expression temperature and folding properties; these parameters can now be determined in a fast and resource-conserving manner.
    Protein Expression and Purification 12/2014;
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    ABSTRACT: GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding.
    Protein Expression and Purification 11/2014;
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    ABSTRACT: A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in Pichia pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10 kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75 °C, while recombinant protein encoded by the “wild type” xylanase gene xyn2, also expressed in Pichia, was 20 °C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80 °C for 10 min. The effects of the optimal pH and temperature for higher expression levels in Pichia pastoris were also determined, 6.0 and 22 °C, respectively. The maximum xylanase activity of Mxyn2p was 13,000 nkat/mg (9.88 g/l) in fed-batch cultivation after 168 h induction with methanol in a 50 l bioreactor.
    Protein Expression and Purification 11/2014;
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    ABSTRACT: House dust mite (Dermatophagoides farinae) allergen Der f1 is one of the most important indoor allergens associated with asthma, eczema and allergic rhinitis in humans. Therefore, sufficient quantities of Der f1 cysteine protease to be used for both experimental and therapeutic purposes are very much needed. Using recombinant DNA technology, high expression rates of cysteine proteases were obtained. The cDNA sequence encoding pro-Der f1 was cloned and expressed in Escherichia coli using the T7 based expression vector pET-44a and induced by isopropyl-β-D-thiogalactoside at a final concentration of 0.2 mM. Recombinant pro-Der f1 (pro-rDer f1) was expressed as an inclusion body and the isolated protease was solubilized, refolded and purified. The protease activities and IgE reactivities of pro-rDer f1 that were refolded by size-exclusion chromatography (SEC) were higher than those obtained by dilution. The pair of pro-rDer f1 polypeptides produced by this method could be used for more effective and safer allergen-specific immunotherapy or to produce enzymatically and immunologically active Der f1 for diagnostic testing and deciphering of immunotherapy mechanisms.
    Protein Expression and Purification 11/2014;
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    ABSTRACT: The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.
    Protein Expression and Purification 11/2014;
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    ABSTRACT: VWA domains are the predominant independent folding units within matrilins and mediate protein-protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in E. coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.
    Protein Expression and Purification 11/2014;
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    ABSTRACT: Integrins are a family of transmembrane receptors and amongst their members, integrin β1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin β1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin β1. The recombinant plasmid was transformed into E. coli BL21 (DE3) cells and after induction by isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8 M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg per liter of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by western blot using monoclonal antibodies against his-tag and integrin β. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn2+ stimulation.
    Protein Expression and Purification 11/2014;
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    ABSTRACT: Helicobacter pylori infections are associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinoma. Bacterial chemotaxis, mediated by four different chemoreceptors (also termed transducer-like proteins (Tlp)), plays an important role in initial colonization and development of disease. Chemoreceptor sensory domains of H. pylori share no significant sequence similarity with those of E. coli or any other non-Epsilonproteobacteria. The structural basis of how chemical signals are recognized by chemoreceptors of H. pylori is poorly understood mainly due to the lack of a robust procedure to purify their sensory domains in a soluble form. This study reports a method for extraction of the periplasmic sensory domain of transducer-like protein C (TlpC) from inclusion bodies and refolding to yield 5 mg pure crystallizable protein per 1 l of bacterial culture. Purified protein was monomeric in solution by size-exclusion chromatography and folded according to the circular dichroism spectrum. Crystals have been grown by the hanging-drop vapour-diffusion method using PEG 4,000 as a precipitating agent. The crystals belonged to space group C2, with unit-cell parameters a = 189.3, b = 103.2, c = 61.8 Å, β = 98.3. A complete X-ray diffraction data set has been collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains three monomers.
    Protein Expression and Purification 11/2014;