Protein Expression and Purification (Protein Expr Purif)

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: 1.43

Impact Factor Rankings

2015 Impact Factor Available summer 2015
2011 Impact Factor 1.587

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


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    • NIH Authors articles will be submitted to PubMed Central after 12 months
    • Publisher last contacted on 18/10/2013
  • Classification
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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in Escherichia coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. Escherichia coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in Escherichia coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; 109. DOI:10.1016/j.pep.2015.02.010
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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: 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 05/2015; 109:99-105. DOI:10.1016/j.pep.2014.11.015
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    ABSTRACT: Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.03.007
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    ABSTRACT: MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.03.008
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    ABSTRACT: Recombinant human erythropoietin receptor (rhEPOR) has applicability as an affinity ligand for purification of recombinant human erythropoietin (rHuEPO) because of its specific binding to rHuEPO. For application of rhEPOR as a ligand for purification of rHuEPO, soluble rhEPOR was expressed in the periplasm of Escherichia coli and engineered by directed evolution through random mutagenesis and integration of mutations. From the screening of random mutagenesis, we identified an amino acid mutation (H114Y) contributing to rHuEPO binding and four amino acid mutations (R76S, A132D, A162D, and C181Y) contributing to expression of soluble rhEPOR. However, the rHuEPO that binds to engineered rhEPOR having H114Y mutation is difficult to dissociate from the engineered rhEPOR. Therefore, H114Y mutation was not suitable for the construction of the rhEPOR ligand. As a rhEPOR ligand, engineered rhEPOR containing four amino acid mutations (EPORm4L) was constructed by integration of mutations except for H114Y. The expression of EPORm4L (127 mg l(-1) of culture medium) was markedly increased in comparison with wild-type rhEPOR (2 mg l(-1) of culture medium). Small-scale affinity chromatography demonstrated that EPORm4L worked as an affinity ligand for purification of rHuEPO. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.03.005
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    ABSTRACT: Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16°C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.03.006
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    ABSTRACT: Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that 1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and 2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.03.004
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    ABSTRACT: Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The enzyme can hydrolyze and inactivate variety of organophosphate (OP)-compounds, including chemical warfare nerve agents. Thus, OPH is a strong candidate for the development of therapeutic intervention against OP-poisoning in humans and other animals. It is also a promising bio-decontaminating agent for clean-up of OP-contaminated objects and areas. For successful commercial application, long-term storage stability of purified OPH enzyme is important. In this study we have cloned and expressed recombinant OPH (r-OPH) in Escherichiacoli and the effect of different excipients on the long-term storage stability of purified enzyme was analyzed. The enzyme was stored in either aqueous solution or in lyophilized form at 25°C for 60days in the presence or absence of different excipients and the stability of the enzyme was determined by monitoring the paraoxon-hydrolyzing activity. Our results suggest that, (a) maltose, trehalose, arginine and proline were most effective in stabilizing the enzyme when stored in aqueous buffer at 25°C, and (b) maltose, trehalose, and mannose exerted maximum stabilization effect when the enzyme was stored in lyophilized form at 25°C for 60days. The study shows that common excipients can be used to stabilize purified OPH enzyme in order to store it for long period of time under different storage conditions. The results of this study can be used to develop formulation(s) of OPH enzyme for commercial use. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.01.012
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    ABSTRACT: Voltage-gated sodium channels participate in the propagation of action potentials in excitable cells. Eukaryotic Navs are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1-S6). The first four segments form the voltage-sensing domain and S5 and S6 create the pore domain with the selectivity filter. Prokaryotic Navs resemble these characteristics, but are truly tetrameric. They can typically be efficiently synthesized in bacteria, but production in-vitro with cell-free synthesis has not been demonstrated. Here we report the cell-free expression and purification of a prokaryotic tetrameric pore-only sodium channel. We produced milligram quantities of the functional channel protein as characterized by size-exclusion chromatography, infrared spectroscopy and electrophysiological recordings. Cell-free expression enables advanced site-directed labelling, post-translational modifications, and special solubilization schemes. This enables next-generation biophysical experiments to study the principle of sodium ion selectivity and transport in sodium channels. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; 26. DOI:10.1016/j.pep.2015.03.002
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    ABSTRACT: The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.03.001
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    ABSTRACT: Salivary α-glucosidases (MalI) have been much less characterized when compared with midgut α-glucosidases, which have been studied in depth. Few studies have been reported on the partial characterization of MalI, but no clear function has been ascribed. The aim of this study is to purify and characterize the recombinant Culex quinquefasciatus (CQ) α-glucosidase expressed in Pichia pastoris. The cDNA encoding mature Cx. quinquefasciatus α-glucosidase gene with polyhistidine tag (rCQMalIHis) was successfully cloned into the expression vector, pPICZαB, designated as pPICZαB/CQMalIHis. The activity of recombinant rCQMalIHis expressed in P. pastoris could be detected at 3.75U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69kDa band corresponds to the Cx. quinquefasciatus α-glucosidase. Thus, rCQMalIHis is a glycoprotein. The rCQMalIHis exhibited optimum pH and temperature at 5.5 and 35°C, respectively. The catalytic efficiency (kcat/Km) of the purified rCQMalIHis for maltotriose is higher than those for sucrose, maltotetraose, maltose and p-nitrophenyl-α-glucoside, indicating that the enzyme prefers maltotriose. Additionally, the rCQMalIHis is significantly inhibited by d-gluconic acid δ-lactone, but not by Mg(2+), Ca(2+) and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8±5.6nM, but weakly inhibited by glucose with IC50 115.9±7.3mM. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.02.018
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    ABSTRACT: Peptidyl-prolyl cis/trans isomerase (PPIase) catalyzes the isomerization of peptide bonds to achieve conformational changes in native folded proteins. An FKBP-type PPIase with an approximate molecular weight of 17 kDa was isolated from Vibrio anguillarumO1 and named VaFKBP17. To investigate its biochemical properties, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli. A protease-coupled assay for isomerization activity, using Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate, indicated that the activity of VaFKBP17 was highest at low temperature (5 °C) and alkaline conditions (pH 10). The immunosuppressant FK506 inhibited the isomerization activity of VaFKBP17. The chaperone activity of VaFKBP17 was assessed using a citrate synthase thermal aggregation activity assay. To evaluate its ability to catalyze protein refolding, the effect of VaFKBP17 on inclusion bodies was investigated during a dilution process. In this assay, VaFKBP17 was able to assist protein refolding. These results provide evidence that VaFKBP17 possesses chaperone-like activity. The structural homology of VaFKBP17 relative to other known bacterial FKBPs was also examined. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 03/2015; DOI:10.1016/j.pep.2015.02.019
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    ABSTRACT: Transforming growth factor beta 3 (TGFβ3) is an important cytokine, functioning in cell proliferation and differentiation, and has been considered to have therapeutic potential for treating various diseases and for scar reduction in adult wound healing. In the current study, a Chinese hamster ovary (CHO) cell line overexpressing recombinant human TGFβ3 (rhTGFβ3) was established. Through a 15-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 133 mg/l of rhTGFβ3 protein. The rhTGFβ3 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 65%, with protein purity greater than 97%. The N-terminal amino acid sequences of the purified rhTGFβ3 were confirmed by N-terminal sequencing analysis. The purified rhTGFβ3 was further demonstrated to be functionally active by measuring the inhibition of growth of HT-2 cells, revealing a half-maximal effective concentration of 42.11 pg/ml and specific activity of 1.84 X 10(7) U/mg. Copyright © 2015 Elsevier Inc. All rights reserved.
    Protein Expression and Purification 02/2015; 110. DOI:10.1016/j.pep.2015.02.014
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    ABSTRACT: Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 02/2015; DOI:10.1016/j.pep.2015.02.016
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    ABSTRACT: The second α-galactosidase gene (designated as RmgalB) was cloned from the thermophilic fungus Rhizomucor miehei and expressed in Pichia pastoris. The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241bp encoding 746 amino acids with two introns. The recombinant α-galactosidase (RmgalB) was secreted at high levels of 1953.9Uml(-1) in high cell density fermentor, which is the highest yield obtained for a α-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1kDa in SDS-PAGE. The enzyme exhibited a very high specific activity of 505.5Umg(-1). The optimum temperature and pH of RmgalB were determined to be 55°C and pH 5.5, respectively. It was stable within pH 5.5-9.5 and up to 55°C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 02/2015; 110. DOI:10.1016/j.pep.2015.02.015
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    ABSTRACT: Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe(2+) in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe(3+), is then stored inside the protein shell in the form of non-reactive insoluble Fe2O3. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characherisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected. Copyright © 2015 Elsevier Inc. All rights reserved.
    Protein Expression and Purification 02/2015; DOI:10.1016/j.pep.2014.12.011
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    ABSTRACT: The large scale screening of cytokine mutants is a component of binding and activity mapping and requires an efficient method of cytokine protein expression. Here, we compared recombinant IL-7 expression with and purification from Escherichia coli and Pichia pastoris. The IL-7 cytokine contains three disulfide bonds that are essential for its biological activity, and which are formed upon secretion through P. pastoris, but not in the reducing cytoplasm of E. coli. In contrast to a previous report we found that P. pastoris secretes active but N-linked hyperglycosylated IL-7. Enzymatic deglycosylation was incompatible with activity measurements in a cell based assay. E. coli expressed IL-7 was refolded from solubilized inclusion bodies. A chromatographic purification step between inclusion body solubilization and refolding increased the yield of biologically active monomeric IL-7, and decreased the amount of inactive soluble aggregates. Cation exchange chromatography of untagged IL-7, and IMAC of His-tagged IL-7 improved refolding yields to a similar extend, indicating that the removal of contaminating components in the solubilized inclusion bodies improves refolding efficiency. We conclude that a chromatographic purification step of IL-7 solubilized from E. coli inclusion bodies increases refolding yield, and may be a suitable general rescue strategy for obtaining folded and biologically active proteins from inclusion bodies. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 02/2015; 110. DOI:10.1016/j.pep.2015.02.013