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
  • Conditions
    • Voluntary deposit by author of pre-print allowed on Institutions open scholarly website and pre-print servers
    • Voluntary deposit by author of authors post-print allowed on institutions open scholarly website including Institutional Repository
    • Deposit due to Funding Body, Institutional and Governmental mandate only allowed where separate agreement between repository and publisher exists
    • Set statement to accompany deposit
    • Published source must be acknowledged
    • Must link to journal home page or articles' DOI
    • 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 PMC after 12 months
    • Authors who are required to deposit in subject repositories may also use Sponsorship Option
    • Pre-print can not be deposited for The Lancet
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Thirteen human interferon-α (IFNα) subtypes were expressed in E. coli and purified using an N-terminal affinity tag from the prodomain of subtilisn. IFNα subtypes were expressed in soluble form and purified from cell lysates or refolded and purified from inclusion bodies. Proteins produced by either protocol exhibited biological activities equal to or greater than commercially prepared IFNα preparations. The IFNαs were used to produce an anti-IFNα16 antibody (MAb-1B12) that specifically neutralized the biological activity of IFNα16, but not the 12 other IFNαs. Using MAb-1B12, and a previously generated IFNAR1/IFNAR2-FChk heterodimer, an assay was developed to determine total type I IFN biological activity and IFNα16-derived biological activity in an unknown sample.
    Protein Expression and Purification 08/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: An efficient heterologous expression system for Auricularia auricula-judae dye-decolorizing peroxidase (DyP) has been constructed. DNA coding for the mature protein sequence was cloned into the pET23a vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant DyP was obtained in high yield as inclusion bodies, and different parameters for its in vitro activation were optimized with a refolding yield of ∼8.5% of the E. coli-expressed DyP. Then, a single chromatographic step allowed the recovery of 17% of the refolded DyP as pure enzyme (1.5 mg per litre of culture). The thermal stabilities of wild DyP from A. auricula-judae and recombinant DyP from E. coli expression were similar up to 60 °C, but the former was more stable in the 62-70 °C range. Stabilities against pH and H2O2 were also measured, and a remarkably high stability at extreme pH values (from pH 2 to pH 12) was observed. The kinetic constants of recombinant DyP for the oxidation of different substrates were determined and, when compared with those of wild DyP, no important differences were ascertained. Both enzymes showed high affinity for Reactive Blue 19 (anthraquinone dye), Reactive Black 5 (azo dye), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,6-dimethoxyphenol, with similar acidic pH optima and oxidative stabilities. Oxidation of veratryl alcohol and a nonphenolic lignin model dimer were confirmed, although as minor enzymatic activities. Interestingly, two sets of kinetic constants could be obtained for the oxidation of Reactive Blue 19 and other substrates, suggesting the existence of more than one oxidation site in this new peroxidase family.
    Protein Expression and Purification 08/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: A new endoglucanase gene cel124 was cloned from a metagenomic library and expressed in E. coli. Catalytic triad analysis showed that the catalytic triad sites were different from the known endoglucanases. Cel124, a 34 kDa protein, exhibited a specific activity (29.08 U mg(-1)) toward 1% of sodium carboxymethyl cellulose and was stable at 50°C for 30 min. The optimal temperature and pH for its catalytic activity were 50°C and pH 5.5 respectively. Cel124 could hydrolyze soluble cellulose, but not insoluble cellulose or other polysaccharides. The kinetic parameters (5.63 mg ml(-1) for Km and 0.0397 mmol min(-1) mg(-1) for Vmax) were measured. 3 M NaCl in the system could increase its activity by 2 fold. Site-directed mutation and circular dichroism spectra test suggested that the residue (Glu41) was essential for its activity, might be a potential active site. Based on our data, we proposed that Cel124 might represent a new type of endoglucanase.
    Protein Expression and Purification 08/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: CD20 is a B cell lineage specific surface antigen involved in various B cell malignancies. So far, several murine and chimeric antibodies have been produced against this antigen among which Rituximab is a commercially approved antibody widely used in treatment of cancers associated with CD20 overexpression. The current study reports the production and characterization of a humanized single chain version of Rituximab through CDR grafting method. For either heavy or light chain variable domains, a human antibody with the highest sequence homology to Rituximab was selected from human germline sequences and used as framework donors. Vernier zone residues in framework regions were replaced with those of Rituximab to retain the antigen binding affinity of parental antibody. The reactivity of humanized single chain antibody with CD20 was examined by ELISA and dot blot assays. The ability of antibody to suppress the growth of CD20 overexpressing Raji cells was tested by MTT assay. Analysis of reactivity with CD20 antigen revealed that the humanized single chain antibody reacted to the target antigen with high affinity. Proliferation inhibition assay showed that humanized scFv could suppress the proliferation of Raji cells efficiently in a dose-dependent manner. This successful production of a humanized scFv with the ability to inhibit growth of CD20-expressing cancer cell may provide a promising alternative strategy for CD20 targeted therapy.
    Protein Expression and Purification 07/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: The csdazl gene is a sex related gene of half-smooth tongue sole (Cynoglossus semilaevis). Our research group have cloned full length cDNA of csdazl, and studied its expression pattern. To get the further information of csdazl, we constructed a prokaryotic expression plasmid, pET-32a-CSDAZL, expressed in E.coli BL21 and purified the fusion protein by His Trap. In order to detect the biological activity of the fusion protein, we injected the protein with liposome into fish, and detected other sex-related genes' mRNA expression. The results showed that the expression levels of half-smooth tongue sole female-related genes Cyp19a and Foxl2 significantly decreased between 6 to 24 h; however, both genes' expressions returned to their normal levels 72h after injection, indicating that recombinant CSDAZL protein could down-regulated the expression of female-related genes, Foxl2 and Cyp19a genes, implying that the fusion protein has biological activity and csdazl plays a role in sex differentiation by regulating sex related genes' expression.
    Protein Expression and Purification 07/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.
    Protein Expression and Purification 07/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of E. coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.
    Protein Expression and Purification 07/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: 3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) [EC 4.1.2.16] is the first and rate-limiting enzyme in the 3-deoxy-D-manno-octulosonate (KDO) biosynthetic pathway. The enzyme is widely expressed in bacteria and plants. Their well conserved protein sequences imply a similar oligomeric arrangement. However, the reported size exclusion chromatrographic analysis suggested a species-dependent self-assembling. To clarify the discrepancy and explore the self-assembling property of KDO8PS, we expressed and purified the Arabidopsis enzyme in E. coli system. The enzyme was highly purified using a two-step purification strategy including nickel affinity and size exclusion chromatography with an expected pH activity profile. The identity of the purified enzyme was confirmed by Western-blot and mass fingerprints. Further analysis by analytical ultracentrifugation indicated that both bacteria and Arabidopsis enzymes are homotetramer. Furthermore, the purified enzyme from the plant has been crystallized and a complete set of X-ray data to 2.1Å resolution has been collected.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Rhoptry protein 6 (ROP6) from Toxoplasma gondii is a 480-amino acid protein with no homology to any reported excretory or secretory protein. Especially, unlike the many other rhoptry protein types, ROP6 does not have a kinase domain. The biochemical and biophysical properties of ROP6 are unknown. Here, we investigated its structure using an in silico analysis method and overexpression and purification using an Escherichia coli system. The protein was purified to more than 85% homogeneity using immobilized metal affinity chromatography in denaturing conditions. After purification, ROP6 showed slow migration in SDS-PAGE, including fast proteolysis. This implies that ROP6 has a high percentage of flexible regions or extended loop structures. Secondary structure prediction and prediction of intrinsically disordered regions by using various bioinformatics tools, indicated that approximately 60% of ROP6 is predicted to be intrinsically disordered or random coil regions. These observations indicate that ROP6 is an intrinsically disordered protein.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sapium sebiferum (L.) Roxb is one of the most important woody oil trees in China. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays the principal role in the transformation of saturated fatty acids into unsaturated fatty acids in oil plants. In this study, the full-length cDNA encoding S.sebiferum (L.) Roxb SAD (SsSAD) was amplified by RT-PCR and cloned into the cold-shock-inducible expression vector, pCold TF. The recombinant prokaryotic expression plasmid was then transformed into the BL21 star (DE3) strain of Escherichia coli (E. coli). Expression of the fusion protein was induced at low temperature in the transformed bacteria using isopropylthio-d-galactoside (IPTG). A recombinant protein with a molecular mass of 101kDa was highly expressed in transformed E. coli, and present in both the supernatant and pellet fractions of bacterial lysates. The supernatant was further purified by affinity chromatography and subjected to Western blot analysis. Ninety percent of the recombinant protein was present in the soluble fraction, confirming that the expression and purification strategy was successful in producing high-quality SsSAD protein. An enzyme activity assay was used to verify that the recombinant SsSAD had stearoyl-ACP desaturase activity. These data lay a foundation for further investigation of the structure and function of SsSAD.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Metallothionein 3 (MT3) is an important biochemical mediator regulating many physiological and pathophysiological processes including neuron cell protection, privation of reactive oxygen species-induced DNA damage, and protection against light induced retinal damage. In this study, a human gene encoding for MT3 with c-terminal extension of His6-tag was inserted into vector pPICZaA, and overexpressed in Pichia pastoris strain X-33. The rhMT3 was purified by one step Ni(+)-NTA affinity chromatography yielding 270 mg/L of over 90% purity. Functional analysis of the purified rhMT3 using inductively coupled plasma mass spectrometry demonstrated that it has biological function, binding with metal ions Cd(2+), Cu(2+) and Zn(2+). In summary, the experimental procedure we have developed facilitates production of large amounts of an active rhMT3 for further research and drug development.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5 mg/L, higher than the system in E. coli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26 kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: When used as an N-terminal fusion expression partner, the Escherichia coli stress-responsive protein, CysQ dramatically increased the cytoplasmic solubility of various aggregation-prone heterologous proteins: Pseudomonas putida cutinase (CUT), human granulocyte colony-stimulating factor (hG-CSF), human ferritin light chain (hFTN-L), arginine deiminase (ADI), human interleukin-2 (IL2), human activation induced cytidine deaminase (AID), and deletion mutant of human glutamate decarboxylase (GAD448-585). As compared with well-known fusion tags such as glutathione-S-transferase (GST) and maltose-binding protein (MBP), the performance of CysQ as solubility enhancer was evidently better than GST and was similar to or better than MBP for the seven heterologous proteins above. This is likely due to the intrinsic ability of CysQ to form its native conformation, probably promoting the binding of molecular chaperones during the folding of CysQ-fusion protein. When used as a substrate, p-nitrophenyl butyrate (PNB) was successfully hydrolyzed to p-nitrophenol by CysQ-CUT fusion mutant. Even after CysQ was removed, the solubility of hFTN-L and hG-CSF, the secondary structure of hG-CSF, and self-assembly activity of hFTN-L were successfully maintained. Conclusively, it seems that CysQ is a highly effective solubility enhancer and fusion expression partner for the production of a variety of bio-active recombinant proteins.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Streptokinase, a plasminogen activator which converts plasminogen to plasmin and consequently promotes fibrinolysis, is the leading drug for treating acute myocardial infarction in developing countries and its production is industrially demanded. In this work, the substantial influence of inclusion body (IB) post-solubilization condition on the performance of a sequential chromatography method for large-scale purification of recombinant streptokinase was demonstrated. In the preliminary experiments, various post-solubilization pH conditions were studied, and it was shown that the pH value of solubilized inclusion bodies (i.e., in 4 M urea) had a marked impact on the purity of streptokinase obtained at the end of post-solubilization process. When the pH value of the solution containing solubilized IBs was decreased from 7.5 to 6.5 and 6.0, the greatest increases (10% and 27%, respectively) in streptokinase purity occurred. The influence of different post-solubilization pH conditions on the efficiency and yield of large-scale chromatographic purification methods was next investigated. When the solubilized IBs solution with pH adjusted to 6.0 was utilized for subsequent sequential chromatography process, the complete elution peak with high overall yield (91.3%) and purity (98%) was achieved. In comparison to this, while the sequential chromatography procedure was instigated by using the solubilized IBs solution with pH 4.2, four elution fractions (EF1 to EF4) with disparate target protein purities (i.e., 57%, 77.3%, 91.4% and 86.7%, respectively) were attained, the process was incompletely effective, and the highest recovery and purity figures (81.8% and 91.4%, respectively, belonging to EF3) were much lower than those for the earlier process.
    Protein Expression and Purification 06/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12 hours induction with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 μM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 μM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.
    Protein Expression and Purification 05/2014;

Related Journals