High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli

The entire encoding region for Aspergillus flavus uricase was cloned into pET-32a and expressed in Escherichia coli BL21 (DE3). The uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form. A scalable process aimed to produce and purify multi-gram quantities of highly pure, recombinant urate oxidase (rUox) from E. coli was developed. The rUox protein was produced in a 30 L fermentor containing 25 L of 2x YT medium and purified to >99% purity using hydrophobic interaction, anion-exchange, and gel filtered chromatography. The final yield of purified rUox from fermentation resulted in approximately 27 g of highly pure, biologically active rUox per kg of cell paste (approximately 238 mg/8.8 g cell paste/L). The results presented here exhibit the ability to generate multi-gram quantities of rUox from E. coli that may be used for the development of pharmaceutics of reducing the hyperuricemia.     

重组炭疽保护性抗原的表达、纯化与生物活性分析

构建分泌型表达质粒 ,在大肠杆菌中实现了重组炭疽保护性抗原 (rPA)的分泌型表达。重组蛋白位于细菌外周质 ,表达量约占菌体总蛋白的 10 %。以离子交换、疏水层析和凝胶过滤为基础 ,建立了rPA的纯化工艺 ,每升培养物可获得约 15mgrPA ,纯度可达 95 %以上。体外细胞毒性试验显示rPA具有较好的生物学活性。用rPA免疫家兔产生的抗血清在体外可抑制炭疽致死毒素的活性 ,表明rPA可诱导机体产生保护性免疫。以上结果为今后发展新一代炭疽疫苗打下基础     

Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli

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 protein-based anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5alpha 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.     

Production of biologically active Bacillus anthracis edema factor in Escherichia coli

Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.     

Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.     

Optimized production and purification of Bacillus anthracis lethal factor

Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis. The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli-Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl-Sepharose, Q-Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20-30 mg/L) were higher than those obtained in other expression systems (1-5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed.     

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.     

Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone,PrsA

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.     

Nasal immunization with the mixture of PA63, LF, and a PGA conjugate induced strong antibody responses against all three antigens

A new generation anthrax vaccine is expected to target not only the anthrax protective antigen (PA) protein, but also other virulent factors of Bacillus anthracis. It is also expected to be amenable for rapid mass immunization of a large number of people. This study aimed to address these needs by designing a prototypic triantigen nasal anthrax vaccine candidate that contained a truncated PA (rPA63), the anthrax lethal factor (LF), and the capsular poly-gamma-D-glutamic acid (gammaDPGA) as the antigens and a synthetic double-stranded RNA (dsRNA), polyriboinosinic-polyribocytodylic acid (poly(I:C)) as the adjuvant. This study identified the optimal dose of nasal poly(I:C) in mice, demonstrated that nasal immunization of mice with the LF was capable of inducing functional anti-LF antibodies (Abs), and showed that nasal immunization of mice with the prototypic triantigen vaccine candidate induced strong immune responses against all three antigens. The immune responses protected macrophages against an anthrax lethal toxin challenge in vitro and enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge in vivo. The anti-PGA Abs were shown to have complement-mediated bacteriolytic activity. After further optimization, this triantigen nasal vaccine candidate is expected to become one of the newer generation anthrax vaccines.     

Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main component of all the vaccines against anthrax. The currently available vaccines have traces of other proteins that contribute to its reactogenicity. Thus, purified PA is recommended for human vaccination. PA loses its biological activity within 48h at 37 degrees C and its thermolability has been a cause of concern as accidental exposure to higher temperatures during transportation or storage could decrease its efficacy. In the present study, we have used protein engineering approach to increase the thermostability of PA by mutating amino acid residues on the surface as well as the interior of the protein. After screening several mutants, the mutants Gln277Ala and Phe554Ala have been found to be more thermostable than the wild-type PA. Gln277Ala retains approximately 45% and Phe554Ala retains approximately 90% activity, even after incubation at 37 degrees C for 48h while in the same period wild-type PA loses its biological activity completely. It is the first report of increasing thermostability of PA using site-directed mutagenesis. Generation of such mutants could pave the way for better anthrax vaccines with longer shelf life.     

Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C

The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28°C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Production of extracellular domain of human tissue factor using maltose-binding protein fusion system

Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.     

Cloning of the protective antigen gene of Bacillus anthracis

The tripartite protein toxin of Bacillus anthracis consists of protective antigen (PA), edema factor (EF), and lethal factor (LF). As a first step in developing a more efficacious anthrax vaccine, recombinant plasmids containing the PA gene have been isolated. A library was constructed in the E. coli vector pBR322 from Bam HI-generated fragments of the anthrax plasmid, pBA1. Two clones producing PA were identified by screening lysates with ELISA (enzyme-linked immunosorbent assay). Western blots revealed a full-size PA protein in the recombinant E. coli, and a cell elongation assay demonstrated biological activity. Both positive clones had a 6 kb insert of DNA, which mapped in the Bam HI site of the vector. The two inserts are the same except that they lie in opposite orientations with respect to the vector. Thus PA is encoded by the plasmid pBA1.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1?, pXO2?), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1? A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture.     

Expression of anthrax lethal factor gene by osmolyte induction

The anthrax toxin consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA mediates the entry of LF and EF to the cytosol where they exert their effects. Although PA is the major component of the vaccines against anthrax, LF has also been found to play an important role in enhancing protective immunity. We have developed an osmolyte-inducible LF expression system. The protein expression system contributed no additional amino acids to the recombinant LF making it suitable for the human vaccine trials.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

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-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-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-ΔFF-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. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001