Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.     

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19–405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni–NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 μg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 μg/ml.     

肺炎克雷伯氏菌特异性抗原的筛选与鉴定

目的寻找肺炎克雷伯氏菌（Klebsiella pneumoniae）血清学检测用特异性抗原。方法双向电泳分离K.pneumoniae总蛋白,通过免疫印迹（Western blotting）与常见病原菌的多抗反应筛选特异性抗原蛋白,原核表达该蛋白并用ELISA法验证。结果获得K.pneumoniae的双向电泳图谱。寻找Western blotting中与K.pneumoniae自身多抗反应而不与与其它病原菌多抗反应的蛋白,质谱鉴定为酸性磷酸酶（Acid phosphatase,GI：238894261）。经表达纯化并以酶联免疫吸附试验（ELISA）验证,证明该蛋白作为包被抗原的灵敏度高,特异性强。结论 K.pneumoniae酸性磷酸酶是适用于该菌感染检测特异性抗原蛋白。     

Production of Polyclonal Antibodies Using Recombinant Coat Protein of Papaya ringspot virus and their Use in Immunodiagnosis

Production of polyclonal antibodies requires large amount of purified virus that can be avoided by the use of recombinant coat protein (CP). Recombinant CP of Papaya ringspot virus (PRSV) was thus used for the production of polyclonal antibodies as the virus purification from papaya tissues provides low virus yields. CP was expressed as a fusion protein (～72 kD) containing a fragment of E. coli maltose binding protein. Polyclonal antibodies from rabbits immunized with the fusion protein, successfully detected natural infection of PRSV in papaya and cucurbits samples collected from different locations at 1:4000 dilution in direct antigen-coated enzyme-linked immunosorbent assay.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Arginine decarboxylase of oats is clipped from a precursor into two polypeptides found in the soluble enzyme

We have examined soluble oat (Avena sativa) arginine decarboxylase by probing its structure with polyclonal antibodies that separately recognize amino-terminal and carboxyl-terminal antigens and with a monoclonal antibody that immunoprecipitates enzyme activity. These experiments indicated that oat arginine decarboxylase is clipped from a 66,000-D precursor polypeptide into 42,000- and 24,000-D produce polypeptides. Both of these are found in the enzyme and may be held together by disulfide bonds. A full-length precursor protein could not be detected in plants but could be produced by expression of the cDNA in Escherichia coli. Analysis of the expression of the cDNA in E. coli, with antibodies and using pulse labeling with [³⁵S]methionine, indicated that the bulk of the expressed protein was the full-length 66,000-D form. Small amounts of 42,000- and 24,000-D polypeptides could also be detected. A reconstruction experiment, adding a radioactively labeled full-length protein isolated from E. coli to powdered oat leaves, supported the idea that the protein extraction method used for western blots was not likely to result in artifactual proteolytic degradation.     

Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.     

Production of polyclonal antibodies to a recombinant coat protein of potato virus Y

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.     

Cloning and expression of FimA-c3d recombinant protein

Recombinant protein consisting of an antigen fused to C3d may elicit a more robust immune response than the antigen alone. The objective of the present study was to clone FimA-c3d recombinant DNA into pCold-TF vector and successfully express in prokaryotic expression vector. FimA subunit of type I fimbriae from Salmonella enterica serovar Enteritidis was conjugated to mouse complement fragment molecule C3d. FimA and FimA-mC3d, FimA-mC3d₂ and FimA-mC3d₃ were cloned into the expression vector pCold-TF. Constructions of the recombinants pCold-TF-fimA with different copies of C3d were confirmed by digestion with restriction enzymes and sequencing. Soluble fusion proteins of FimA with different copies of C3d were induced by IPTG and were expressed in Escherichia coli BL21 (DE3) under optimal conditions. The results showed that the proteins induced from recombinants pCold-TF-fimA, pCold-TF-fimA-mC3d, pCold-TF-fimA-mC3d₂ and pCold-TF-fimA-mC3d₃ were 70, 100, 130 and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC). Finally we conclude that antigen conjugated with mC3d can be functionally expressed in prokaryotic vectors.     

Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required.     

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M_r 21 000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.     

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.     

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

The coat protein (CP) coding regions of two Czech Potato mop‐top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV‐CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV‐specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme‐linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA‐ ELISA (indirect plate‐trapped antigen (PTA)‐ELISA).     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

Cloning,expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody

Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni⁺-NTA affinity chromatography (95–98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.     

Expression, purification, and immunogenic characterization of Epstein–Barr virus recombinant EBNA1 protein in Pichia pastoris

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390–641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4⁺ and CD8⁺ T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.     

Anti-attacin polyclonal antibody from an in vitro derived antigen used for immunoblot to quantify attacin expressed in transgenic apple

A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l^–1) by Ni²⁺-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody.     

Oligomeric Structure and Functional Characterization of the Urea Transporter from Actinobacillus pleuropneumoniae

Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family.     

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)₆-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni²⁺-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.     

A novel xylanase from Streptomyces sp. FA1: Purification,characterization, identification,and heterologous expression

A xylanase (XynA) was purified from the culture medium of Streptomyces sp. FA1, which was previously isolated from a bamboo retting system. XynA had a molecular mass of 43 kDa, displayed maximal activity at pH 5.5, retained 41% of its maximal activity at pH 11.0, and was stable over a wide pH range (3.0 ～ 11.0). Purified XynA was subjected to peptide mass fingerprinting, which led to the cloning of the xynA gene. The xynA gene, which encodes a mature protein of 436 amino acids, was heterologously expressed in E. coli BL21(DE3). The activity in the culture medium could reach 213.5 U/mL, which was 11.2-fold higher than that produced by Streptomyces sp. FA1. BLAST searching revealed that full-length XynA shares less than 90% identity with most of its homologues, whereas amino acids 48-436 of the enzyme share 97% identity with an open reading frame encoding a putative full-length mature xylanase from Streptomyces tendae. The truncated xynA gene, xynA ^48-436 , was cloned and expressed in E. coli, however, no xylanase activity could be detected in the culture medium. Based on these results, it is suggested that XynA is a new member of glycoside hydrolases family10 with exceptional catalytic efficiency at alkaline pH.