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.     

The expression of the protective antigen of Bacillusanthracis in Bacillus subtilis

The expression of Bacillus anthracis protective antigen (PA) in B. subtilis from the pag gene in pPA101–Colour RGB 0,0,1281 was explored in different genetic backgrounds in an attempt to identify opportunities to maximize expression. Introduction of AtxA, which positively regulates PA expression in B. anthracis did not improve expression levels in the protease-deficient strain WB600. Plasmid pPA101–1 was found to carry a deletion which created a new fusion point between vector and insert sequence, and which removed part of the AtxA binding site. The deletion may have occurred as a consequence of recombination between TCTAT sequences within both the vector and insert. Host mutations could influence expression; PA levels from pPA101–1 are threefold higher in a ccpA mutant than in an otherwise isogenic parent, and eightfold higher in an abrB mutant. These data demonstrate that the introduction of mutations affecting catabolite repression and growth phase regulation results in an increase in the yield of PA in this host–vector system. Combining these mutations with a multiply protease-negative background could potentially allow further improvements in PA yield.     

炭疽杆菌保护性抗原在大肠杆菌中的表达及其纯化

利用基因重组技术获取炭疽杆菌保护性抗原(PA)。将炭疽杆菌保护性抗原编码基因pag与pET载体连接构建重组质粒,转化大肠杆菌DE3株,诱导表达炭疽杆菌保护性抗原,并经亲和层析及凝胶过滤纯化此抗原。实验成功构建了表达炭疽杆菌保护性抗原的重组菌株,纯化后PA纯度达90%,且经检测纯化产物具有天然PA的生物学活性。同时表明从大肠杆菌中纯化PA较以往从炭疽杆菌中获取PA简便易行。     

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.     

炭疽杆菌保护性抗原基因的克隆与序列测定

采用聚合酶链反应从炭疽芽孢杆菌减毒株ＹＢ１中扩增其保护性抗原（ＰＡ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２２０５ｂｐ,编码７３５个氨基酸残基,与献报道的标准菌株Ｓｔｅｒｎｅ株的ＰＡ序列只有４个碱基的差异。     

Unfolding transitions of Bacillus anthracis protective antigen

Protective antigen (PA) is an 83kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 degrees C. We show for the first time circular dichroism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a beta II to a beta I CD spectrum.     

Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis

Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.     

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.     

Location of receptor-binding region of protective antigen from Bacillus anthracis

The pag gene, which codes for protective antigen (PA), a common component of the lethal and edema toxins of Bacillus anthracis, was cloned and expressed in Escherichia coli. Nested deletions of pag were generated into the C-terminus coding region. Recombinant proteins were analyzed by Western blot with either an anti-PA polyclonal antisera or two monoclonal antibodies that neutralized lethal toxin and edema toxin activities by inhibiting the binding of PA to cell receptors. Localization of the receptor binding domain within the C-terminal region of PA was suggested by the inability of the monoclonal antibodies 3B6 and 14B7 to recognize the recombinant proteins expressed by C-terminal deletions of the pag gene.     

炭疽芽孢杆菌保护性抗原在大肠杆菌中的表达及纯化

按照炭疽芽孢杆菌保护性抗原（PA）基因成熟肽编码序列设计引物,从炭疽杆菌pOX1质粒中扩增出PA基因片段,将该片段定向插入到原核表达载体pET-28a中,获得了pET-PA原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为PA基因的成熟呔编码序列。将该重组质粒转化大肠杆菌BL21（DE3）,经IPTG诱导,重组蛋白在大肠杆菌表达系统中获得了高效表达;Western印迹分析表明表达产物具有良好的免疫学活性。     

The effect of the protective antigen of Bacillus anthracis on the formation of immunity under the action of live anthrax vaccines

The preparation of low-molecular protective antigen (PA) isolated from strain 34F2 (Sterne) and having a molecular weight of 34 and 51 kD, unlike the preparation of high-molecular PA with a molecular weight of 87 kD, suppressed the formation of acquired resistance to anthrax when introduced into guinea pigs in mixture with live spores of strains of STI, 34F2 and new vaccine strain 228/8; this phenomenon was mainly accompanied by a decrease in the level of antibodies to lethal factor (LF) nad in the antitoxic activity of blood serum. The immunosuppressing action of low-molecular PA depended on the kind of vaccine strain introduced together with this antigen, which suggested the existence of differences in the ligand determinants of strains 34F2 and STI. In contrast to high-molecular PA, low-molecular PA blocked the action of the lethal mixture of PA and LF on the culture of peritoneal mononuclear phagocytes of CBA mice. The competitive relationships between low-molecular PA and high-molecular PA are discussed.     

Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.     

Construction and characterization of a protective antigen-deficient Bacillus anthracis strain

The pag gene coding for protective antigen (PA), one of the three toxin components of Bacillus anthracis, has been cloned into the mobilizable shuttle vector pAT187 and transferred by conjugation from Escherichia coli to B. anthracis. Using this strategy, an insertionally mutated pag gene constructed and characterized in E. coli, was introduced into B. anthracis Sterne strain. This transconjugant was used to select a recombinant clone (RP8) carrying the inactivated pag gene on the toxin-encoding plasmid, pXO1. Strain RP8 was deficient for PA while still producing the two other toxin components, i.e. lethal factor (LF) and edema factor (EF). In contrast to spores from the wild-type Sterne strain, spores prepared from RP8 were totally non-lethal in mice. These results clearly establish the central role played by PA in B. anthracis pathogenicity.     

Protective and other biological properties of Bacillus anthracis soluble antigens

The soluble antigens were explored of the culture filtrate (CF) derived during static growth of B. anthracis vaccine strain 34F2 on a medium containing casein hydrolysate. Electrofocusing of CF preparations revealed that the protective activity was distributed over a wide range of pH 3-7. The most pronounced and stable protective activity was observed at pH 4.6-4.8. Following toxin factors were isolated and identified: protective antigen (87 kD), oedema factor (87 kD) and lethal factor (78-81 kD). The greatest protective activity was associated with antigens characterized by a molecular weight of 78-87 kD and toxic activity. Preparations of the oedema and lethal factors had the same protective activity as protective antigen (PA) preparations. Other CF soluble antigens protected about 30% of immunized guinea pigs. A protein was isolated with a molecular weight of 80 kD and isoelectric point at pH 5.3-5.7 which was not toxic and did not form toxic mixtures in association with other toxin factors; this protein featured a high immunogenic activity, however, it protected only 31% of immunized animals. Factors are analyzed which determine differences in the protective effects of live and chemical vaccines.     

Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis

Anthrax toxin is a complex of protective antigen (PA, 735 aa), lethal factor (LF, 776 aa), and edema factor (EF, 767 aa). PA binds to cell surface receptors and is cleaved by cell surface proteases into PA63, while LF and EF compete for binding to PA63. The PA63-LF/EF complex is internalized into the cytosol and causes different pathogenic responses in animals and cultured cells. 1-300 amino acid residues of LF have been viewed as the region responsible for the high affinity binding of LF to PA. Amino acid analysis of LF and EF revealed a common stretch of 7 amino acids (147VYYEIGK153). In the present study, each amino acid of this stretch was replaced by alanine at a time. Y148A, Y149A, I151A, and K153A mutants were found to be deficient in their ability to lyse J774A.1 cells and their binding ability to PA63 was drastically reduced. We propose that these four amino acids play a crucial role in the process of binding of LF to PA63.     

Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes

Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T(1/2) value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 degrees C was observed. The half-life (t(1/2)) of PA thermal inactivation at 40 degrees C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax.     

A study of the physiology of Bacillus anthracis Sterne during manufacture of the UK acellular anthrax vaccine

AIM: To analyse the growth of Bacillus anthracis during simulations of the UK anthrax vaccine manufacturing process. METHODS AND RESULTS: Simulated vaccine production runs were performed using the toxigenic, acapsulate Sterne 34F(2) strain of B. anthracis in semi-defined medium. After rising during the logarithmic growth phase, the pH of the culture starts to fall at about 18 h from pH 8.7 to reach <7.6 at 26 h, coincident with consumption of glucose and optimal production of protective antigen (PA; 7.89 g ml(-1), SD 1.0) and lethal factor (LF; 1.85 g ml(-1), SD 0.29). No increased breakdown of toxin antigens was seen over the 26-32 h period. When glucose was exhausted, amino acids (principally serine) were utilized as an alternative carbon source. Sporulation was not observed during the 32 h. CONCLUSIONS: PA and LF, the principal constituents in the UK anthrax vaccine, undergo little degradation during vaccine fermentation. The vaccine manufacturing process is robust and reproducible. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed analysis of the manufacturing process used for the UK acellular anthrax vaccine; insight gained into the process will support continued and safe vaccine manufacture.