Protein Expression and Purification Journal Impact Factor & Information

Publisher: Elsevier

Journal description

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

Current impact factor: 1.70

Impact Factor Rankings

2015 Impact Factor Available summer 2016
2014 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
    ​ green

Publications in this journal

  • Joane K Rustiguel · Patricia S Kumagai · Marcelo Dias-Baruffi · Antonio J Costa-Filho · Maria Cristina Nonato
    [Show abstract] [Hide abstract]
    ABSTRACT: Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities.
    Protein Expression and Purification 10/2015; DOI:10.1016/j.pep.2015.09.026
  • Timo Stressler · Thomas Eisele · Susanne Meyer · Julia Wangler · Thomas Hug · Sabine Lutz-Wahl · Lutz Fischer
    [Show abstract] [Hide abstract]
    ABSTRACT: The high specific lysyl endopeptidase (Lys-C; EC is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functional properties. Up to now, the high price makes this application nearly impossible. In this work, the increased expression for Escherichia coli optimized Lys-C was investigated. The cloned sequence had a short artificial N-terminal pro-peptide (MGSK). The expression of MGSK-Lys-C was tested using three expression vectors and five E. coli host strains. The highest expression rate was obtained for the expression system consisting of the host strain E. coli JM109 and the rhamnose inducible expression vector pJOE. A Lys-C activity of 9,340 ± 555 nkatTos-GPK-pNA/Lculture could be achieved under optimized cultivation conditions after chemical refolding. Furthermore, the influence of the native pre-N-pro peptide of Lys-C from Lysobacter enzymogenes ssp. enzymogenes ATCC 27796 on Lys-C refolding was investigated. The pre-N-pro peptide was expressed recombinantly in E. coli JM109 using the pJOE expression vector. The optimal concentration of the pre-N-pro peptide in the refolding procedure was 100 μg/mLrefolding buffer and the Lys-C activity could be increased to 541,720 nkatTos-GPK-pNA/Lculture. With the results presented, the expensive lysyl endopeptidase can be produced in high activity and high amounts and the potential of Lys-C for tailor-made protein hydrolysates with bioactive (e.g. antihypertensive) and/or techno functional (e.g. foaming, emulsifying) properties can be investigated in future time studies.
    Protein Expression and Purification 10/2015; DOI:10.1016/j.pep.2015.09.024
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human cystatin C (cysC) is a soluble basic protein belonging to the cysteine protease inhibitor family. CysC is a potent inhibitor of cathepsins - proteolytic enzymes that degrade intracellular and endocytosed proteins, remodel extracellular matrix, and trigger apoptosis. Inhibition is via tight reversible binding involving the N-terminus as well as two β-hairpin loops of cysC. As a significant component of cerebrospinal fluid, cysC has numerous other functions, including support of neural stem cell growth and differentiation. Several studies suggest that cysC may bind to the Alzheimer-related protein beta-amyloid (Aβ), and inhibit its aggregation and toxicity. Because of an increasing recognition of its important biological roles, there is considerable interest in methods to produce full-length recombinant human cysC. Several researchers have reported success, but with processes that require multiple purification steps. Here we report successful production of human cysC using an intein-based expression system and a simple one-column purification scheme. The recombinant protein so obtained was natively folded and active as an enzyme inhibitor. Unexpectedly, even mild concentration by ultrafiltration caused significant oligomerization. The oligomers are noncovalent and retain the native secondary structure and inhibitory activity of the monomer. The oligomers, but not the monomers, were highly effective at inhibiting aggregation of Aβ. These results demonstrate the critical importance of careful physicochemical characterization of recombinant cysC protein prior to evaluation of its biological functions.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.023
  • [Show abstract] [Hide abstract]
    ABSTRACT: The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αβγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.014
  • [Show abstract] [Hide abstract]
    ABSTRACT: S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54 fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.020
  • [Show abstract] [Hide abstract]
    ABSTRACT: Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.016
  • [Show abstract] [Hide abstract]
    ABSTRACT: The extracellular protease APSm1 was purified to homogeneity from Stenocarpella maydis that was grown in acidic minimal media with glucose and ammonium sulfate. The purification procedure consisted of ion exchange chromatography coupled to an FPLC (Fast Protein Liquid Chromatography) system, resulting in a 15.3% recovery and a 2.3-fold increase in specific activity. The molecular weight of the purified enzyme was estimated to be 56.8 kDa by SDS-PAGE. Enzymatic activity toward hemoglobin was optimal at pH 2.0 and at 25° C. The effects of six protease inhibitors on APsm1 activity were tested. Pepstatin A inhibited APSm1 activity, as the protein is in fact an aspartyl protease. The pure enzyme degraded hemoglobin, albumin and proteins obtained from corn germ at pH 3 but did not have any milk-clotting activities. The Km and Vmax values obtained were 0.514 mg/mL and 0.222 μmol/min, respectively, using hemoglobin as the substrate. This work is the first to report the purification of a secreted aspartyl protease from S. maydis.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.017
  • [Show abstract] [Hide abstract]
    ABSTRACT: Single domain antibody (sdAb) is often expressed as inclusion bodies in Escherichia coli cytoplasm. Establishing an effective in vitro refolding method for sdAb obtained from inclusion bodies would be important for sdAb research. In this study, dilution refolding condition for a camelid sdAb specific against human beta-2-microglobulin was optimized for the sdAb purified from the inclusion bodies of Escherichia coli BL21 (DE3). Single factor methods based on protein concentration, velocity of dilution, incubation time and refolding buffer composition were first investigated. Then the key refolding buffer compositions were selected for further optimization by means of the Box-Behnken design of response surface methodology (RSM). The activity of the refolded sdAb was determined by measuring its specific antigen-binding ability using indirect ELISA. The optimized refolding condition of sdAb consisted of a 10-fold dilution in 10 mM Tris-HCl (pH 8.0) containing 1.24 mM GSH, 1 mM GSSG, 352 mM L-Arg, 0.65% PEG-2000, and a 16 h incubation at 4 °C. Further comparison of the activities between the refolded sdAb and purified soluble sdAb expressed in E. coli Rosetta-gami (DE3) pLysS indicated that the sdAb was correctly refolded, as assayed by isothermal titration calorimetry. This work could provide an important strategy for the recombinant production and application of sdAb.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.019
  • [Show abstract] [Hide abstract]
    ABSTRACT: Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 μg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.013
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase A2 (PLA2) and protease (P) are enzymes responsible of myotoxic, edematogenic and hemostasis disorder effects observed in the envenomation by Bothrops alternatus pitviper. Their partitioning coefficient (Kp) in different polyethyleneglycol/potassium phosphate aqueous two-phase systems (ATPSs) was determined in order to both achieve a better understanding of the partitioning mechanism and define optimal conditions for toxin isolation. Polyethyleneglycols (PEGs) of molecular weights 1,000; 3,350; 6000 and 8,000; different temperatures (5, 20 and 37 °C) and phase volume ratios of 0.5; 1 and 2 were assayed. PLA2 partitioned preferentially to the top phase while P mainly distributed to the bottom phase. Either entropically- or enthalpically-driven mechanisms were involved in each case (PLA2 and P). The aqueous two-phase system formed by PEG of MW 3,350 (12.20 % wt/wt) and KPi pH 7.0 (11.82 % wt/wt) with a volume ratio of one and a load of 1.25 mg of venom/g of system showed to be the most efficient to recover both enzymes. It allowed obtaining the 72 % of PLA2 in the top phase with a purification factor of 2 and the 82 % of P at the bottom phase simultaneously. A further adsorption batch step with DEAE-cellulose was used to remove satisfactorily the PEG from the top phase and recover the active PLA2. The proposed methodology is simple, inexpensive, and only requires professionals trained in handling basic laboratory equipment. It could be easily adoptable by developing countries in which the snakebite accidents cause considerable morbidity and mortality.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.012
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn(2+) and Ni(2+) provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn(2+) was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn(2+)-purified HbA and LtEc (2.4 ± 1.3% and 5.3 ± 2.1%, respectively), but slightly higher for Ni(2+)-purified LtEc (8.4 ± 1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn(2+)) precipitation and tangential flow filtration.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.006
  • [Show abstract] [Hide abstract]
    ABSTRACT: In a previous study the full-length open reading frame of the Arabian camel, Camelus dromedarius liver cytosolic glucose-6-phosphate dehydrogenase (G6PD) cDNA was determined using reverse transcription polymerase chain reaction. The C. dromedarius cDNA was found to be 1545 nucleotides (accession number JN098421) that encodes a protein of 515 amino acids residues. In the present study, Camelus dromedarius recombinant G6PD was heterologously overexpressed in Eschericia coli BL21 (DE3) pLysS and purified by immobilized metal affinity fast protein liquid chromatography (FPLC) in a single step. The purity and molecular weight of the enzyme were analysed on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity was determined to be 2000 EU/mg protein. The optimum temperature and pH were found to be 60 °C and 7.4 respectively. The isoelectric point (pI) for the purified G6PD was determined to be 6.4. The apparent Km values for the two substrates NADP+ and G6P were found to be 23.2 μM and 66.7 μM respectively. The far-UV Circular dichroism (CD) spectra of G6PD showed that it has two minima at 208 and 222 nm as well as maxima at 193 nm which is characteristic of high content of α -helix. Moreover, the far-UV CD spectra of the G6PD in the presence or absence of NADP(+) were nearly identical.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.002
  • [Show abstract] [Hide abstract]
    ABSTRACT: Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active β-galactosidase from Planococcus sp-L4 (bgal) was optimized for expression of recombinant "BGalP" in Pichia pastoris. As a result of codon optimization, the codon adaptation index was improved from 0.58 to 0.85 after replacing rare codons. After transformation of two Pichia pastoris strains (KM71H and GS115), the activity of BGalP enzyme was measured in the culture supernatants using ortho-Nitrophenyl-β-galactoside (ONPG). Maximal activity was recorded as 3.7 U/ml on day 11 in KM71H clone #2 which was 20% higher than the best GS115 clone. Activity measurements under different conditions indicated optimal activity at pH 6.5. It was active at temperatures ranging from 0 to 55°C with deactivation occurring at or above 60°C. Protein analysis of the crude ultra-filtrate showed the enzyme was ∼75 kDa and was the major constituent (85%) of the sample. This enzyme have the potential to find utility for the breakdown of lactose in chilled milk and subsequently can be deactivated by pasteurization. The use of BGalP would minimize energy consumption thus decreasing cost and also help to preserve the nutritional elements of the milk.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.008
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in P.pastoris. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90°C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p<0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.010
  • [Show abstract] [Hide abstract]
    ABSTRACT: The arginine deiminase (ADI, E.C - a key enzyme of ADI pathway of Enterococcus faecium GR7 was purified to homogeneity. A sequential purification strategy involving ammonium sulfate fractionation, molecular sieve followed by Sephadex G-100 gel filtration was applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified with a fold of 16.92 and showed a final specific activity of 76.65 IU/mg with a 49.17% yield. The dimeric ADI has a molecular mass of about 94364.929 Da, and comprises of hetrodimers of 49.1 kDa and 46.5 kDa as determined by MALDI-TOF and PAGE analysis. To assess anti-cancerous activity of ADI by MTT assay was carried out against cancer cell lines (MCF-7, Sp2/0-Ag14 and Hep-G2). Purified ADI exhibited the most profound antiproliferative activity against Hep-G2 cells; with half-maximal inhibitory concentration (IC50) of 1.95 μg/ml. Purified ADI from E. faecium GR7 was observed to induce apoptosis in the Hep-G2 cells by DNA fragmentation assay. Our findings suggest the possibility of a future use of ADI from E. faecium GR7 as a potential anticancer drug.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.011
  • [Show abstract] [Hide abstract]
    ABSTRACT: Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains - I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using western blot, mass spectrometry and circular dichroism (CD) spectroscopy.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.001
  • [Show abstract] [Hide abstract]
    ABSTRACT: The emergence of antibiotic resistant pathogenic strains of bacteria has necessitated the development of novel antimicrobial agents. The puroindoline A and B (PINA and PINB) proteins of wheat, well-known for their roles in determining the important phenotype of grain texture, are also antimicrobial, making them attractive as natural bio-control agents. However, the biochemical basis of PIN functionality remains unclear due to limitations in expressing them at the required yield and purity and lack of accurate tertiary structure. This study focussed on rapid transient expression of PINs targeted to different subcellular compartments (chloroplast, apoplast, endoplasmic reticulum and cytosol) of Nicotiana benthamiana leaf cells using the deconstructed tobacco mosaic virus-based 'magnICON(®)' system. The expressed recombinant PINs were characterised by western blot using the Durotest anti-friabilin antibody, enzyme-linked immunosorbent assays (ELISA) and antimicrobial activity tests. Maximum yield of the His-tagged PINs occurred when targeted to the chloroplast. Both PINs exhibited oligomeric and monomeric forms on gels, but western blots with the widely used Durotest anti-friabilin antibody identified only oligomeric forms. Only the PINs purified by a hydrophobic interaction method exhibited monomeric forms with the anti-His tag antibody, indicating correct folding. Interestingly, the Durotest antibody did not bind to monomers, suggesting their epitope may be obscured. PINs purified by His-tag affinity purification under native conditions or by the hydrophobic method exhibited antimicrobial activities. The successful in planta expression and optimisation of purification will enable future studies to examine the detailed structure of the PINs and explore novel bio-control applications in health, food and/or agriculture.
    Protein Expression and Purification 09/2015; DOI:10.1016/j.pep.2015.09.009