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: 1.70

Impact Factor Rankings

2016 Impact Factor Available summer 2017
2014 / 2015 Impact Factor 1.695
2013 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

Additional details

5-year impact 1.62
Cited half-life 8.50
Immediacy index 0.30
Eigenfactor 0.01
Article influence 0.45
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


  • Pre-print
    • Author can archive a pre-print version
  • Post-print
    • Author can archive a post-print version
  • Conditions
    • Authors pre-print on any website, including arXiv and RePEC
    • Author's post-print on author's personal website immediately
    • Author's post-print on open access repository after an embargo period of between 12 months and 48 months
    • 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
    • Author's post-print may be used to update arXiv and RepEC
    • Publisher's version/PDF cannot be used
    • Must link to publisher version with DOI
    • Author's post-print must be released with a Creative Commons Attribution Non-Commercial No Derivatives License
    • Publisher last reviewed on 03/06/2015
  • Classification

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel pullulanase gene (PbPulA) from Paenibacillus barengoltzii was cloned. PbPulA has an open reading frame of 2028 bp encoding 675 amino acids. It was heterologously expressed in Escherichia coli as an intracellular soluble protein. The recombinant pullulanase (PbPulA) was purified to homogeneity with a molecular mass of about 75 kDa on SDS-PAGE. PbPulA was optimally active at pH 5.5 and 50 °C. It was stable within pH 5.5–10.5. The enzyme exhibited strict substrate specificity towards pullulan, but showed relatively low activity towards amylopectin and no activity towards other tested polysaccharides. The Km and Vmax values of the enzyme on pullulan were 2.94 mg/mL and 280.5 μmol/min/mg, respectively. The addition of PbPulA in gelatinized rice and maize starches significantly increased the resistant starch type 3 (RS3) yields. Final yields from rice and maize starches were 10.82 g/100 g and 11.41 g/100 g, respectively. These properties make PbPulA useful in starch industries.
    No preview · Article · May 2016 · Protein Expression and Purification
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    ABSTRACT: The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins.
    No preview · Article · Apr 2016 · Protein Expression and Purification
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    ABSTRACT: Der p 2, a major allergen derived from the house dust mite Dermatophagoides pteronyssinus, is one of the most clinically relevant allergens worldwide. Recombinant Der p 2 (rDer p 2) is useful in clinical diagnosis and disease-specific immunotherapy. However, previous studies showed that Der p 2 can only be expressed in Escherichia coli (E. coli) cells as inclusion bodies, thus protein refolding is required to obtain functional products. Here we report a new method to produce biologically active Der p 2 protein in E. coli. N-terminal hexahistidine- and trigger factor (TF)-tagged Der p 2 was expressed in soluble form in E. coli and purified using a combination of chromatography processes. This procedure produced milligram-level high purity Der p 2 per liter of bacterial culture. Moreover, far-UV region circular dichroism (CD) analysis and serum specific IgE reactivity test demonstrated that the secondary structure and IgE reactivity properties of rDer p 2 produced in our study were almost identical to those of natural Der p 2 (nDer p 2). In conclusion, the method developed in this work provides a useful tool for the production of immunologically active recombinant Der p 2 for clinical applications.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca+2 and Mg+2 ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca+2 and 1 mM Mg+2 resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. We report herein the recombinant expression of three hydrophobic ELPs (VPGIG)n with variable lengths (n = 20, 40, 60) and sub-ambient transition temperatures. These ELPs were purified from the cytoplasmic soluble fraction of Escherichia coli by inverse transition cycling, and their exact molecular weight was confirmed by various mass spectrometry techniques. Transition temperatures of ELP20, ELP40, and ELP60 were measured at 18.6 °C, 12.4 °C and 11.7 °C, respectively.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: The present paper reports the production, purification and biochemical characterization of an extracellular alkaline protease from Bacillus sp. SB12. The enzyme has been used as an alternative to conventional chemicals treatement for dehairing of goat skins. The protease was optimally active at 37 °C and pH 9. Starch at 2% (w/v) was used as the best carbon source and the addition of yeast extract and peptone at 1% each supported the maximum level of protease production in the presence of 5 mM Ca(2+). Protease purification was performed with ammonium sulphate precipitation at 70% saturated fraction followed by dialysis and gel filtration chromatography using Sephadex G-100. The purified enzyme was homogeneous on non-denaturing PAGE and appeared as a single band with an apparent molecular weight of 41 kDa. This enzyme was moderately thermostable and has a wide pH stability range extending from pH 7 to 11. It showed high tolerance toward surfactants agents and organic solvents while it was completely inhibited by PMSF indicating the serine protease type. Purified protease was used to remove hair from goat skin proving its potential application in leather processing industry. The results revealed that the protease has enhanced the quality and physico-chemical properties of the skins while reducing the pollution.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250±84 μg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Ferredoxins are small, acidic proteins containing iron-sulfur clusters that are widespread in living organisms. They play key roles as electron carriers in various metabolic processes, including respiration, photosynthesis, fermentation, nitrogen fixation, carbon dioxide fixation, and hydrogen production. However, only several kinds of ferredoxins are commercially available now, greatly limiting the investigation of ferredoxin-related enzymes and metabolic processes. Here we describe the heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin. Adding extra iron and sulfur sources to the medium in combination with using Escherichia coli C41(DE3) harboring pCodonplus and pRKISC plasmids as host greatly enhanced iron-sulfur cluster synthesis in the three ferredoxins. After induction for 12 h in terrific broth and purification by affinity chromatography and anion exchange chromatography, approximately 3.4 mg of streptavidin (Strep)-tagged and 3.7 mg of polyhistidine (His)-tagged clostridial 2[4Fe4S] ferredoxins were obtained from 1 l of culture. Excitingly, after induction for 24 h in terrific broth, around 40 mg of His-tagged clostridial [2Fe2S] and 23 mg of His-tagged agrobacterial [2Fe2S] ferredoxins were purified from 1 l of culture. The recombinant ferredoxins apparently exhibited identical properties and physiological function to native ferredoxins. No negative impact of two different affinity tags on ferredoxin activity was found. In conclusion, we successfully developed a convenient method for heterologous overproduction of the three kinds of ferredoxins with satisfactory yields and activities, which would be very helpful for the ferredoxin-related researches.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Thioredoxins (Trxs) regulate the activity of target proteins in the chloroplast redox regulatory system. In vivo, a disulfide bond within Trxs is reduced by photochemically generated electrons via ferredoxin (Fd) and ferredoxin-thioredoxin reductase (FTR: EC FTR is an αβ-heterodimer, and the β-subunit has a 4Fe-4S cluster that is indispensable for the electron transfer from Fd to Trxs. Reconstitution of the light-dependent Fd/Trx system, including FTR, is required for the biochemical characterization of the Trx-dependent reduction pathway in the chloroplasts. In this study, we generated functional FTR by simultaneously expressing FTR-α and -β subunits under the control of tandem T7 promoters in Escherichia coli, and purifying the resulting FTR complex protein. The purified FTR complex exhibited spectroscopic absorption at 410 nm, indicating that it contained the Fe-S cluster. Modification of the expression system and simplification of the purification steps resulted in improved FTR complex yields compared to those obtained in previous studies. Furthermore, the light-dependent Trx-reduction system was reconstituted by using Fd, the purified FTR, and intact thylakoids.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Kynurenine aminotransferase (KAT) is a pyridoxal-5’-phosphate (PLP) dependent enzyme that catalyses kynurenine (KYN) to kynurenic acid (KYNA), a neuroactive product in the tryptophan metabolic pathway. Evidence suggests that abnormal levels of KYNA are involved in many neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and schizophrenia. Reducing KYNA production through inhibiting kynurenine aminotransferase 2 (KAT2) would be a promising approach to understanding and treating the related neurological and mental disorders. In this study we used an optimized codon sequence to overexpress histidine-tagged human KAT2 (hKAT2) using an Escherichia coli expression system. After a single step of Ni-NTA based purification the purified protein (>95%) was confirmed to be active by an HPLC based activity assay and was crystallized using the hanging-drop vapour diffusion method. The crystal system represents a novel spacegroup, and a complete X-ray diffraction data set was collected to 1.83 Å resolution, and higher resolution data than for any reported native human KAT2 structure. The optimised method of protein production provides a fast and reliable technique to generate large quantities of active human KAT2 suitable for future small-molecule lead compound screening and structural design work.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: The Norovirus (NoV) P particle (PP) is a subviral particle formed by 24 copies of the protruding (P) domain of the capsid protein. Each P domain has three surface loops that can be used for foreign antigen presentation. Hence, PPs have been demonstrated to be an excellent platform for vaccine development against many pathogens. However, current processes for preparing those chimeric PP vaccines vary and would change the original sequence of the PP. A detailed strategy also has not been reported for inserting a foreign antigen into all three loops. In order to develop a novel method for preparing distinct types of PP-based protein vaccines, we created two restriction enzyme sites (EagI and KpnI) in the P domain by site-directed mutagenesis without changing its original sequence. A synthesized gene with three copies of the Alzheimer’s disease (AD) immunogen Aβ1-6 was then incorporated in loop2 of the P domain. Additionally, a synthesized gene with one copy of Aβ1-6 was inserted into each loop of the P domain. Furthermore, two recombinant proteins PP-3 copy-Aβ1-6-loop2 and PP-1 copy-Aβ1-6-loop123 were successfully purified without affecting PP formation. Particle size analysis and TEM observations demonstrated that the two chimeric P particles were still able to form 24-mer nanoparticles. Moreover, the two chimeric PP-based AD vaccines could both efficiently elicit strong immune responses in the mouse model. In conclusion, we have successfully established a novel method for preparing vaccines based on the NoV PP which would not affect PP sequence and function.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant E. coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.
    No preview · Article · Jan 2016 · Protein Expression and Purification
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    ABSTRACT: The sequence and structure of the target protein exert a marked effect on its soluble expression in E. coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen for mutations that resulted in enhanced soluble expression in E. coli. Two interesting mutations (A390I and D401V) were identified, which are located at the interaction surface between the A and C domains of BLA. The A390I mutation enhanced soluble BLA expression by 2.0-fold compared to wild type, while D401V decreased soluble expression 160-fold. Structural analysis revealed that A390 and D401 residues could affect the interaction between the A and C domains of BLA. Therefore, soluble expression of the target protein in E. coli could be affected by introduction of a mutation in the protein sequence.
    No preview · Article · Dec 2015 · Protein Expression and Purification
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    ABSTRACT: Mixtures of neutral salts and polyethylene glycol are used for various purposes in biological studies. Although the effects of each component of the mixtures are theoretically well investigated, comprehension of their integrated effects remains insufficient. In this work, their roles and effects as a precipitant were clarified by studying dependence of precipitation curves on salt concentration for integral membrane protein/detergent particles of different physicochemical properties. The dependence of precipitation curves was reasonably related to intermolecular interactions among relevant molecules such as protein, detergent and polyethylene glycol by considering their physicochemical properties. The obtained relationships are useful as basic information to learn the early stage of biological macromolecular associations.
    No preview · Article · Dec 2015 · Protein Expression and Purification
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    ABSTRACT: We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.
    No preview · Article · Dec 2015 · Protein Expression and Purification