Rapid Purification of Recombinant Anthrax-Protective Antigen under Nondenaturing Conditions (original) (raw)
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Constitutive expression of anthrax protective antigen
The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins-protective antigen (PA), lethal factor (LF), and edema factor (EF)-by the bacterium. PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA. PA is the major component of vaccines against anthrax since it confers protective immunity. The large-scale production of recombinant proteinbased anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5␣ strain. We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells. We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor. The protein was purified using metal-chelate affinity chromatography. Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture. It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis. This is the first report of constitutive overexpression of protective antigen gene in E. coli.
Expression and Purification of Anthrax Toxin Protective Antigen fromEscherichia coli
Protein Expression and Purification, 1996
LF is believed to be a zinc-dependent metalloprotease Anthrax toxin consists of three separate proteins, (7). Lethal toxin has been found to cause an early influx protective antigen (PA), lethal factor (LF), and edema of sodium and efflux of potassium from J774A.1 cells, factor (EF). PA binds to the receptor on mammalian leading to ATP depletion resulting in lysis of cells (8). cells and facilitates translocation of EF or LF into the PA alone is nontoxic, an essential immunogen, and the cytosol. PA is the primary component of several anprimary component of the human vaccine against anthrax vaccines. In this study we expressed and purified thrax (9,10).
Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo
Biochemical and Biophysical Research Communications, 2003
The anthrax toxin complex is primarily responsible for most of the symptoms of anthrax. This complex is composed of three proteins, anthrax protective antigen, anthrax edema factor, and anthrax lethal factor. The three proteins act in binary combination of protective antigen plus edema factor (edema toxin) and protective antigen plus lethal factor (lethal toxin) that paralyze the host defenses and eventually kill the host. Both edema factor and lethal factor are intracellularly acting proteins that require protective antigen for their delivery into the host cell. In this study, we show that deletion of certain residues of protective antigen results in variants of protective antigen that inhibit the action of anthrax toxin both in vitro and in vivo. These mutants protected mice against both lethal toxin and edema toxin challenge, even when injected at a 1:8 ratio relative to the wild-type protein. Thus, these mutant proteins are promising candidates that may be used to neutralize the action of anthrax toxin.
Vaccine, 2010
Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.
Expression and purification of the recombinant Protective Antigen of B anthracis
Protective antigen (PA) is a major component of the vaccine against anthrax. The structural gene for the 83-kDa PA was expressed as fusion protein with 6؋ Histidine residues in Escherichia coli. Expression of PA in E. coli under the transcriptional regulation of the T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine-HCl and the protein was purified under denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the denaturant while immobilized on the Ni-NTA column. The protein was then purified using Mono-Q column on FPLC. The yield of the purified recombinant PA (rPA) from this procedure was 2 mg/ liter of the culture. The rPA had an apparent molecular mass of 83 kDa as determined by SDS-PAGE. Antisera to native PA recognized the fusion protein. The rPA was biologically as well as functionally active. Thus, the recombinant PA may be used to develop an effective recombinant vaccine against anthrax.
Journal of Industrial Microbiology & Biotechnology, 2002
The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-Á Á ÁFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-Á Á ÁFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-Á Á ÁFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14-18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg / l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities.
Vaccine, 2006
The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyltransferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD 50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine. Published by Elsevier Ltd. (W.J. Ribot). rPA purified by a research and development (R&D) process is known to contain differentially charged forms (isoforms) of pure rPA protein with equivalent relative mass . Micro-heterogeneity in PA structure was first described for protein purified from fermentor cultures of the Sterne strain grown in R-medium . In an effort to optimize the yield of PA for vaccine production, the gene encoding PA (pag) was cloned into a B. subtilis strain [15] and, later, into the non-virulent, asporogenic Delta-Sterne-1 CR4 strain . Although the Delta-Sterne-1 pPA102-CR4 strain produced rPA in higher yields, micro-heterogeneity was still observed . Although the basis for the micro-heterogeneity in rPA is under investigation, the presence of a negatively charged 0264-410X/$ -see front matter. Published by Elsevier Ltd.
The Open Vaccine Journal, 2009
We describe a novel hybrid anthrax toxin approach that incorporates multiple components into a single vaccine product. The key domains of protective antigen (PA) and lethal factor (LF) that may be critical for inducing protective immunity are combined into one recombinant molecule. Two LF N-terminal domain-PA hybrids, one with wild-type PA and another with furin cleavageminus PA, were expressed in E. coli and purified in a native form. Both the hybrids bind to the extracellular domain of the host receptor, CMG2; the wild-type hybrid can be cleaved by furin exposing the LF interacting domain, allowing it to oligomerize into lethal toxin as well as translocation pore-like complexes. The hybrid antigens are immunogenic in Dutch-belted rabbits, eliciting strong PA-specific and LF-specific antibodies. However, the lethal toxin neutralizing antibody titers are 3-7 times lower than those elicited by PA-alum. The hybrid antigens conferred 100% (6/6) protection in rabbits challenged intranasally with a 100 LD 50 dose of Bacillus anthracis Ames strain spores.
Expression and Purification of the Recombinant Protective Antigen of Bacillus anthracis
Protein Expression and Purification, 1999
Protective antigen (PA) is a major component of the vaccine against anthrax. The structural gene for the 83-kDa PA was expressed as fusion protein with 6؋ Histidine residues in Escherichia coli. Expression of PA in E. coli under the transcriptional regulation of the T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine-HCl and the protein was purified under denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the denaturant while immobilized on the Ni-NTA column. The protein was then purified using Mono-Q column on FPLC. The yield of the purified recombinant PA (rPA) from this procedure was 2 mg/ liter of the culture. The rPA had an apparent molecular mass of 83 kDa as determined by SDS-PAGE. Antisera to native PA recognized the fusion protein. The rPA was biologically as well as functionally active. Thus, the recombinant PA may be used to develop an effective recombinant vaccine against anthrax.