Abstract:
Anthrax toxin, comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF) is a major virulent factor of  B. anthracis . Protective antigen, PA is the main component of all the vaccines against anthrax. The protective efficacy of PA is greatly increased if small quantities of LF of EF are incorporated into the vaccines. An ideal vaccine against anthrax should contain PA, LF and EF together, but this combination would be toxic. Therefore, the biologically inactive mutant preparations of PA, LF and EF may be used together for better immunoprotection. The present invention describes the method for generation of recombinant vaccine against anthrax, comprising of non-toxic, mutant anthrax toxin proteins. The procedure involves site-directed mutagenesis of the native genes of the toxin proteins, the expression and purification of the mutant proteins and finally characterization of these proteins.

Description:
FIELD OF THE INVENTION  
       [0001]     This invention relates to a Recombinant DNA construct and a process for the preparation of a nontoxic anthrax vaccine.  
       BACKGROUND OF THE INVENTION  
       [0002]     Anthrax, a zoonotic disease is caused by gram-positive, sporulating bacteria,  Bacillus anthracis . Humans are accidental hosts through food of animal origin, animal products and contamination of the environment with  Bacillus anthracis  (Brachman P. S., 1970, Aninals. N.Y. Acad. Sci. 174, 577-582). Anthrax is one of the oldest known bacterial diseases and occurs in most parts of the world including India. The major virulent factors of  B. anthracis  include poly-D-glutamic acid capsule and a three-component anthrax toxin complex. Anthrax toxin (Leppla S. H., 1991, In Source Book of Bacterial protein toxins, pp 277-302.), comprising of protective antigen PA(83 kDa), lethal factor (LF-(90 kDa) and edema factor (EF-(89 kDa) is a major virulent factor of  B. anthracis . LF/EF, the catalytic moieties of this complex require PA to enter the cell cytosol. PA in combination with LF (called the lethal toxin), causes death in experimental animals (Smith H. and Keppie J., 1954, Nature, 173, 869-870). PA in combination with EF (called the edema toxin) causes edema in the skin of the experimental animals (Stanley J. L. and Smith H., 1961, J. Gen Microbiol., 26, 49-66). PA is the receptor-binding moiety that facilitates the translocation of the catalytic moieties, LF and EF, into the target cells. After translocation into the cell, LF, a metalloprotease causes cleavage of certain Mitogen so activated protein kinase kinases (MAPKKs) resulting in inactivation of signal transduction pathways (Duesbery N. S., et. al., 1998, Science, 280. 734-737). On the other hand, EF, upon entering the cells, gets activated by calmodulin to cause massive increase in intracellular cAMP levels (Leppla S. H., 1982, Proc. Natl. Acad. Sci. USA., 79, 3162-3166).  
         [0003]     The first step of the intoxication process is the binding of PA to the cell surface receptors (Bradley K. A. et al, 2001, Nature, 414, 225-229). After binding to the receptors on the cell surface, PA gets nicked by cell surface proteases to yield a 63-kDa fragment (Klimpel el R. K., et. al., 1992, Proc. Natl. Acad. Sci. USA., 89, 10277-10281) which oligomerises and binds to LF/EF (Milne J. C., et. al., 1994, J. Biol. Chem., 269, 20607-20612). Binding of LF/EF is competitive. The whole complex then undergoes receptor-mediated endocytosis. Acidification of the endosonie (Friedlander A. M., 1986, J. Biol. Chests. 261, 7123-7126) results in the insertion of the PA-oligomer into the endosomal membrane to form pores (Milne J. C. and Collier R. J., 1993 Mol. Microbiol., 10, 647-653) through which LF/EF are translocated into the cell cytosol.  
         [0004]     PA has four domains that are organized primarily into antiparallel-beta sheets with only a few short helices of less than four turns (Petosa C., et. al., 1997, Nature, 385, 833-838). Domain 1 is responsible for binding to LF/EF during the anthrax intoxication process. Domain 2 is dominated by a beta barrel and plays a role in membrane insertion and translocation. Domain 3 is the smallest domain and is important for oligomerization of PA and possibly also in the binding of PA to LF/EF. Domain 4 is the receptor-binding domain.  
         [0005]     Crystal structure of LF, determined recently, shows that LF has 4 domains (Pannifer A. D., et al, 2001, Nature, 414, 229-233). Domain 1 is involved in binding to PA. This domain has significant homology to the N-terminal 1-250 residues of EF. In fact, most of the residues in this region are absolutely conserved.  
         [0006]     Of all the three toxin proteins, —PA is the most immunogenic and is an essential component of the vaccine against anthrax (Gladstone G. P., 1946, Br. J. Exp. Pathol, 97, 349-418). It has been observed that the protective efficacy of PA is greatly increased if small quantities of LF or EF are incorporated into the vaccine (Pezard et. al., 1995, infect. Immun., 63, 1369-1372). However, this also happens to be the primary reason of toxigenicity and reactogenicity of the vaccines. Anthrax toxin (Leppla S. H., 1991, In Source Book (of Bacterial protein toxins, pp 277-302.), comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF) is a major virulent factor of  B. anthracis.    
         [0007]     The currently used anthrax vaccine is derived from a non-capsulated, avirulent strain of the bacterium known as Sterne&#39;s strain (Sterne M., 1939, J. Vet. Sci. Anim. Ind, 13, 307-312). In Russia and China, the live spore vaccines based on Sterne strain are used. In UK the vaccine is alum precipitated culture filtrate of the Sterne strain while the US vaccine consists of an alhydrogel-adsorbed cell free culture filterates of a non-capsulating, non proteolytic derived strain V770 isolated from bovine anthrax (Turnbull P. C. B, 1991, Vaccine, 9, 533-539). All these currently used anthrax vaccines, apart from being crude have undefined composition. They are reactogenic and do not provide protection against all natural strains of  B. anthracis.    
         [0008]     U.S. Pat. No. 2,017,606 describes the preparation of anthrax antigen by growing the bacilli with a suitable culture medium, separating the bacilli from the culture medium.  
         [0009]     U.S. Pat. No. 2,151,364 describes a method of producing an anthrax vaccine which comprises preparing the suspension of anthrax spores, adding to the suspension a sterile solution containing alum.  
         [0010]     RU patent 2,115,433 describes the method of production of anthrax vaccine, which comprises of living spores of non-capsulated strain of  B. anthracis  and protective antigen of  B. anthracis.    
         [0011]     WO patent 000252 describes a method of production of anthrax vaccine using non-toxic protective antigen from  B. anthracis  for use in inducing immune response, which is protective against anthrax.  
         [0012]     The drawbacks in the above-mentioned patents are that all of them use  Bacillus anthracis  cultures/spores.  Bacillus anthracis  is an infectious organism and can not be handled without containment facilities. The vaccine prepared is contaminated with other toxic and non-toxic proteins from  Bacillus anthracis  resulting in a number of side effects and reactogenicity.  
         [0013]     These vaccines also have a certain degree of residual virulence for certain species of domesticated and laboratory animals. The Sterne strain is toxigenic and is pathogenic at high doses. As a result it is considered unsafe and unsuitable for human use. This vaccine can cause undesirable side effects including necrosis at the site of inoculation.  
         [0014]     Therefore there is a need to develop a second-generation anthrax vaccine which does not have side effects and has a well-defined composition.  
         [0015]     The object of the present invention is to render the anthrax toxin non-toxic without affecting its immunogenicity, in order to develop a safe and effective anthrax vaccine.  
         [0016]     To achieve said object, the present invention provides a recombinant DNA construct comprising an expression vector and a DNA fragment including genes for wild type Protective Antigen (PA) or wild type Lethal Factor (LF) or wild type Edema Factor (EF)  
         [0017]     The present invention also provides a recombinant DNA construct comprising: 
        an expression vector and a DNA fragment including genes for mutant type Protective Antigen (PA) or mutant type Lethal Factor (LF) or mutant type Edema Factor (EF)        
 
         [0019]     Said vector is a prokaryotic vector such as vector is PQE 30 and said expression vector contains T5 promoter and 6X histidine tag.  
         [0020]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe202.  
         [0021]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Leu203.  
         [0022]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Pro205.  
         [0023]     The said DNA fragment is the gene for protective antigen with Alanine-substitution at residue Ile207.  
         [0024]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residues Pro205, Trp226 and Phe236.  
         [0025]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe552.  
         [0026]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Ile574.  
         [0027]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe552 and Phe554.  
         [0028]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Ile562 and Ile574.  
         [0029]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Leu566 and Ile574.  
         [0030]     The DNA fragment is the gene for protective antigen ith Alanine-substitution at residue Phe552 and Phe554, Ile562, Leu566 and Ile574.  
         [0031]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe427.  
         [0032]     The DNA fragment is the gene for protective antigen with deletion of residue Asp 425.  
         [0033]     The DNA fragment is the gene for protective antigen with deletion of residue Phe 427.  
         [0034]     The DNA fragment is the gene for protective antigen With Alanine-substitution at residue Trp346.  
         [0035]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Leu352.  
         [0036]     The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Trp346, Met350 and Leu352.  
         [0037]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Tyr148.  
         [0038]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Tyr149.  
         [0039]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Ile151.  
         [0040]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Lys153.  
         [0041]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Asp187.  
         [0042]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Phe190.  
         [0043]     The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Asp 187, Leu188, Leu 189 and Phe 190.  
         [0044]     The DNA fragment is the gene for edema factor with Alanine-substitution at residue Tyr137.  
         [0045]     The DNA fragment is the gene for edema factor with Alanine-substitution at residue Tyr138.  
         [0046]     The DNA fragment is the gene for edema factor with Alanine-substitution at residue Ile140.  
         [0047]     The DNA fragment is the gene for edema factor with Alanine-substitution at residue Lys142.  
         [0048]     The protein encoded by said DNA fragment is expressed in a prokaryotic host. The said prokaryotic host is an  E. coli  strain.  
         [0049]     A protein expressed by gene DNA fragment is wild type PA wild type LF, wild type EF and their mutagenised variants.  
         [0050]     This invention further discloses a method for producing mutagenised anthrax toxin protein comprising: 
        mutagenizing PA LF &amp; EF genes using different mutagenic primers of the kind as herein defined for PCR reaction;     treating said mutant PCR product along with the native template with a n enzyme to cleave the native template of said PCR product;     transforming said mutant product in  E. coli  strain;     isolating the recombinant construct from transformed  E. coli  strain and confirming the desired mutation;     transforming the confirmed mutant construct in appropriate  E. coli  expression strain to express the mutant protein and     purifying the said expressed mutant protein.        
 
         [0057]     The purification is carried Out using Ni-NTA chromatography and/or other chromatographic techniques.  
         [0058]     The genes are cloned in PQE expression vector containing T5 promoter and 6X histidine tag.  
         [0059]     The mutations were affected in the first domain of PA at residues 202, 203, 205. The mutations were affected in the third domain of PA at residues 552, 574 552+554, 562+574, 566+574, 552+554+562+566+574 resulting in mutant proteins that were defective in oligomerization. The mutations were affected in the second domain of PA at residues 425 &amp; 427 of loop 4 of domain 2. These mutations impaired the translocation-ability of PA The mutations were affected in the second domain of PA at residues 346, 352 and 346+350+352 in loop 3 of domain 2 such that PA becomes biologically inactive. The mutations were affected in the 1 st  domain of LF at residues 148, 149, 151, 153, 187, 190 and 187+188+189+190 impaired the binding of LF to PA. The mutations were affected in the 1 st  250 residues of EF.  
         [0060]     An anthrax vaccine comprising an anthrax toxin protein selected from wild type PA or wild type LF or wild type EF.  
         [0061]     An anthrax vaccine comprising an anthrax toxin protein selected from mutant type PA or mutant type LF or mutant type EF or a combination thereof.  
         [0062]     An anthrax vaccine comprising an anthrax toxin protein selected is a combination of anyone selected from wild type PA or wild type LF or wild type EF with any one or more selected from mutant type PA or mutant type LF or mutant type EF.  
         [0063]     A pharmaceutical composition comprising an effective amount of a anthrax toxin protein as claimed by the present invention.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0064]     An ideal vaccine against anthrax should contain PA, LF, EF together, but at the same time it should be non-toxic and safe. Purified recombinant proteins with defined composition may be used in the vaccine to minimize reactogenicity of the vaccine. Further, these anthrax toxin proteins maybe rendered non-toxic by introducing mutations that affect the biological activity of the proteins without affecting their structure or immunogenicity. These non-toxic, mutant anthrax toxin proteins may be used together to create a safe, non-reactogenic and effective recombinant vaccine against anthrax. Thus, the prime objective of this invention was to create a process for making a safe and effective, second-generation vaccine against anthrax comprising of non-toxic anthrax toxin proteins that have been produced by site-directed mutagenesis of the different functionally important domains of the toxin proteins.  
         [0065]     The inventors of this application have PCR amplified the genes for PA, LF and EF. They have cloned these genes in pQE30 expression vector (Gupta P., et. al., 1998, Infect. Immun., 66, 862-865; Gupta P., et. al., 1999 Protein Expr. Purif. 16, 369-376; Kumar P., et. al. 2001, Infect. Immun., 69, 6532-6536). The vector contains T5 promoter and a 6X -Histidine tag, which allows convenient purification of the recombinant proteins ( FIG. 1 ).  
         [0066]     Conditions for overexpression of the said genes using the above mentioned recombinant plasmids, from  E. coli  strains have been optimised by the inventors (Chauhan V., et. al., 2001, Biochem. Biophys. Res. Commun., 283, 308-315).  
         [0067]     Using the above mentioned recombinant plasmid, inventors of the present process, introduced mutations in the said genes to make the expressed recombinant proteins defective in their biological function, thereby rendering them non-toxic. The invention involves the expression and purification of the said mutant proteins from  E. coli  strains. It further involves full characterization of the purified mutant proteins to pinpoint the defect that renders them non-toxic.  
         [heading-0068]     Mutations Introduced in Protective Antigen as Part of the Invention  
         [0069]     1. Mutations that make PA defective in binding to LF/EF. The inventors introduced series of mutations in the 1 st  domain of PA. Among the mutations introduced, the mutations at residues 202, 203, 205, 207 and 205+226+236 were found to be defective in binding to LF.  
         [0070]     2. Mutations that make PA defective in oligomerization. The authors of this invention introduced mutations in the 3 rd  domain of PA. The mutation at the residues 552, 574, 552+554, 562+574 566+574, 552+554+562+566+574 resulted in mutant proteins that were defective in oligomerization.  
         [0071]     3. Mutations that make PA translocation-defective. Inventors have introduced mutations at residues 425 and 427 of loop 4 of domain 2. These mutations impaired the translocation-ability of PA.  
         [0072]     4. Mutations that make PA defective in insertion/translocation. Authors have discovered that when mutations are introduced at the residues 346, 352 and 346+350+352 in loop 3 of domain 2, PA becomes biologically inactive. The mutant proteins were able to bind to the cell-surface receptors, get proteolytically activated to form oligomers and bind to LF. The biological inactivity of these mutant proteins may pertain to a defect in insertion/translocation.  
         [heading-0073]     Mutations Introduced in Lethal Factor as Part of the Invention  
         [0074]     Mutations that make LF defective in binding to PA. The inventors of this process have introduced mutations in the 1 st  domain of LF. They found that mutation at residues 148, 149, 151, 153, 187, 190 and 187+188+189+190 impaired the binding of LF to PA.  
         [heading-0075]     Mutations Introduced in Edema Factor as Part of the Invention  
         [0076]     Mutations that make EF defective in binding to PA. The inventors of this process have introduced series of mutations in the 1 st  250 residues of EF. It was found that mutation at residues 137, 138, 140 and 142 drastically impaired the binding of EF to PA.  
         [0077]     After the expression and purification of the mutant proteins the proteins were evaluated for their biological activity.  
         [0078]     Inventors have found that the above-mentioned mutants of PA when added along with wild-type LF, were nontoxic to J774A.1 cells. Likewise mutants of LF when added along with wild-type PA were non-toxic to J774A.1 cells. Similarly, mutants of EF when added along with wild-type PA were unable to produce cAMP-toxicity in CHO cells (Table 2).  
         [heading-0079]     The purified mutant protein was analyzed for their biological activity by assaying:  
         [none]    
       
         
           
              Ability of PA to bind to cell surface receptors,  
              Ability of PA to bind to LF or EF,  
              Ability of PA to oligomerize,  
              Membrane insertion ability of PA oligomer,  
              Ability of PA to translocate LF or EF to the cytosol,  
              Ability of lethal toxin to kill macrophage cell lines like RAW264.7 and J774A.1  
              Ability of edema toxin to elongate CHO cells. 
 
 Immunization Studies 
 
           
         
       
     
         [0088]     Protective antigen, as the name suggests is a highly immunogenic protein. In fact it is a necessary component of the vaccine against anthrax. Immunization with wild-type recombinant PA elicits high anti-PA titers and provides protection against anthrax lethal challenge in guinea pigs. It was further observed that mutant PA was as immunogenic as the wild-type PA and could easily substitute the wild-type PA in vaccine (Singh et. al. 1998, Infect. Immun. 66, 3447-3448). Immunization studies also indicate a significant contribution of LF/EF to immunoprotection. On basis of these results the inventors have is developed a recombinant vaccine against anthrax, which comprises of mutants of all the three anthrax toxin components.  
         [0089]     The anthrax toxin based recombinant vaccine developed by the inventors has the following advantages:  
         [0090]     1. The process described here does not involve handling of  B. anthracis  cultures (at any stage). This process is therefore safe, cost-effective and does not require the sophisticated containment facilities.  
         [0091]     2. The vaccine developed by the inventors has well-defined composition and will therefore not have any batch to batch variation.  
         [0092]     3. The invention described here utilizes purified mutant anthrax toxin protein. As a result, this second-generation anthrax vaccine will not be reactogenic and will not cause any side-effects unlike the previous vaccine.  
         [0093]     4. Additionally, this invention comprises of non-toxic mutant proteins, which when administered (either alone or in combination) do not cause any toxigenicity or pathogenicity as associated with the currently used vaccine.  
         [0094]     5. The invention described here is therefore safe and suitable for animal/human use.  
         [heading-0095]     Details of the Experimental Procedures  
         [0096]     Site-Directed Mutagenesis of Anthrax Toxin Proteins  
         [0097]     To introduce the desired mutations in the anthrax toxin proteins, complementary mutagenic primers were used (refer Table 1) to amplify the wild type anthrax toxin genes (for PA or LF or EF). High fidelity Pfu DNA polymerase was used for the PCR reaction. Entire lengths of both the strands of the plasmid DNA were amplified in a linear fashion during several rounds of thermal cycling, generating a mutant plasmid with staggered nicks on the opposite strands ( FIG. 2 ). The amplification was checked by agarose gel electrophoresis of the PCR product. The product of the amplification was treated with DpnI that specifically cleaves fully methylated G me6  ATC sequences. The digestion reaction was carried out in 20 μl reaction volume with 10 ng of the amplified product, 2 μl of 10X DpnI reaction buffer and 0.1 U of DpnI. After DpnI digestion, DpnI resistant molecules that are rich in desired mutants were recovered by transformation of the DNA into the appropriate  E. coli  strain. The mutations were confirmed by sequencing of the above constructs using Perlcin Elmer cycle DNA sequencing kit.  
         [heading-0098]     Expression and Purification of the Mutant Anthrax Toxin Proteins  
         [0099]     The confined constructs were transformed into  E. coli  expression strains expressing T5 RNA polymerase. Transformed cells were grown in Luria broth medium (LB) containing 100 μg/ml of ampicillin and 25 μg/ml of kanamycin, at 37° C., to an OD 600  of 0.8. Induction vas then done with 0.5 mM IPTG and the incubation was continued at 37° C. for 3 to 4 hours. Cells were then harvested by centrifugation at 6000 rpm for 10 minutes. The cells then lysed. The protein profile was analysed by SDS-PAGE and western blotting. The mutant PA proteins were purified using Ni-NTA metal-chelate affinity chromatography and other chromatographic techniques (Kumar P., et. al. 2001, Infect. Immun., 69, 6532-6536; Gupta P., et. al., 1998, Infect. Immun., 66, 862-865; Gupta P., et. al., 1999 Protein Expr. Purif. 16, 369-376). The purified mutant proteins were analysed by SDS-PAGE and western blotting and were estimated using Bradford&#39;s method. For storage the purified proteins were dialysed against 50 mM HEPES and stored as aliquotes at −70° C.  
         [heading-0100]     Cell Culture  
         [0101]     Macrophage like cell line J774A.1 was maintained in RPMI 1640 medium containing 10% heat inactivated FCS, 25 mM HEPES, 100U/ml penicillin and 200 μg/ml streptomycin in a humidified 5% CO 2  environment at 37° C.  
         [0102]     CHO cells were maintained in EMEM medium containing 10% heat inactivated FCS, 25 mM HEPES, 100U/ml penicillin and 200 μg/ml streptomycin in a humidified 5% CO 2  environment at 37° C.  
         [0103]     To study the biological activity of the wild-type PA or its mutant proteins, varying concentrations of these proteins were added along with LF (1 μg/ml) to J774A.1 cells plated in 96-wells plates. Incubation was allowed for 3 hrs. at 37° C. and then cell viability (Bhatnagar et. al. 1989, Infect. Immun., 57, 2107-2114) was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) dye (Bhatnagar R., et. al., 1999, Cell Signal., 11, 111-116). MT-T dissolved in RPMI was added to each well at a final concentration of 0.5 mg/ml and incubated for another 45 min at 37° C. to allow uptake and oxidation of the dye by viable cells. The medium was replaced by 0.5% (w/v) sodium dodecyl sulphate (SDS), 25 mM HCl in 90% isopropyl alcohol and the plate vortexed. The absorption was read at 540 nm using microplate reader (BIORAD).  
         [0104]     Similarly, to study the biological activity of wild-type LF or its mutant proteins, varying concentrations of these proteins were added along with PA (1 μg/ml) to J774A.1 cells plated in 96-wells plates. Incubation was allowed for 3 hrs. at 37° C. and then cell viability as determined using MTT dye, as detailed above.  
         [0105]     To study the biological activity of wild-type EF or its mutant proteins, varying concentrations of these proteins were added along with PA (1 μg/ml) to CHO cells that were plated in 96-wells plates. Incubation was allowed for 3 hrs. at 37° C. and then the cells were microscopically examined for elongation. Rise in intracellular cAMP levels of the cells upon toxin treatment was determined (Kumar P., et. al., 2001, Infect. Immun., 69, 6532-6536) with cAMP EIA kit of Amersham Pharmacia.  
         [0106]     Further experiments were then done to understand how mutations were affecting the biological activity of the anthrax toxin mutant proteins.  
         [heading-0107]     Binding of PA to Cell Surface Receptors  
         [0108]     J774A.1 cells were allowed to grow to confluence in 24-well plates before incubating with 1 μg/ml of wild-type PA or its mutant protein at 4° C. for 2 hrs. The cells were then washed with cold RPMI, dissolved in SDS lysis buffer and subjected to SDS-PAGE for electroblotting. The blot was developed with anti-PA antibodies to study the binding of wild-type PA or its mutant protein with the cell surface receptors  
         [heading-0109]     Proteolytic Cleavage of PA and Mutant Proteins in Solution  
         [0110]     Wild-type PA and its mutant proteins were tested for susceptibility to cleavage by trypsin. The proteins (1.0 mg/ml) were incubated with 1 μg/ml of trypsin for 30 minutes at room temperature in 25 mM HEPES, 1 mM CaCl 2 , 0.5 mM EDTA pH 7.5. The digestion reaction was stopped by adding PMSF to a concentration of 1 mM. For SDS-PAGE, the samples were boiled in SDS sample buffer for 5 minutes and resolved on 12% SDS-PAGE.  
         [0111]     Binding of PA to LF on the Surface of Cells  
         [0112]     J774A. 1 cells were washed twice with RPMI and then incubated with 1 μg/ml of wild-type PA or its mutant protein at 4° C. for 3 hrs. The cells were then washed with cold RPMI to remove unbound protein. The cells were further incubated with LF (1.0 μg/ml) for 3 hours and then washed with cold RPMI to remove unbound LF. The cells were dissolved in SDS lysis buffer and subjected to SDS-PAGE for electroblotting. The blot was developed with anti-LF antibodies to study the binding of the wild-type PA or its mutant protein to LF.  
         [heading-0113]     Oligomerization of PA in Solution  
         [0114]     PA upon proteolytic cleavage oligomerises to form heptamers. To study the ability of the wild-type PA and its mutant proteins to form oligomers, the proteins (1 mg/ml) were digested with trypsin for 30 minutes at 25° C. The samples were brought to pH 5.0 by addition of 1M Tris pH5.0 to a final concentration of 100 mM. and were boiled for 5 minutes in SDS sample buffer (0.0625M Tris-Cl, 1.25% SDS, 2.5%, β-mercaptoethanol and 5% glycerol, pH6.8) before loading on a 3-12% gradient gel. Silver staining was done to detect the formation of oligomers.  
         [heading-0115]     Binding of LF/EF to Cell-Surface Bound PA.  
         [0116]     J774A.1 cells were washed with cold RPMI and then incubated with 1 μg/ml of wild-type PA at 4° C. for 3 hrs. The cells were washed again with cold RPMI to remove unbound protein. Wild-type LF/EF or the mutant proteins (1.0 μg/ml) were then added and incubation was continued for 3 hours. The cells were then washed with cold RPMI to remove unbound LF/EF. Later, the cells were dissolved in SDS lysis buffer and subjected to SDS-PAGE for electroblotting. The blot was developed with anti-LF/EF antibodies to study the binding of LF/EF to cell-surface bound.  
                                                   RESIDUE   CHANGE   PRIMERS   DOMAIN   DEFECT                   PA mutants:                           Phe202   To alanine   5′CTTTTCATGAATATTAGAAATCCATGCTGAAAG   I   Defective in                       binding to Lethal                       factor               Leu203   To alanine   CTTTTCATGAATATTAGAAATCCATGGTGAAGCAAAAGT   I   Defective in                       binding to Lethal                       factor               Pro205   To alanine   CTTTTCATGAATATTAGAAATCCATGGTGAAAGAGCAGTTCT   I   Defective in                       binding to Lethal                       factor               Ile207   To alanine   TTTGGTTAACCCTTTCTTTTCATGAATATTAGAAATCCATGGT   I   Defective in               GAAAGAAAAGTTCTTTTATTTTTGACATCAACCGTATATCCTT       binding to Lethal               CTACCTCTAATGAATCAGCGATTCC       factor               Pro205 + Trp226 +   To alanine   CTTTTCATGAATATTAGAAATCCATGGTGAAAGAGCAGTTCT and   I   Defective in       Phe236       GGATTTCTAATATTCATGAAAAGAAAGGATTAACCAAATATA       binding to Lethal               AATCATCTCCTGAAAAAGCGAGCACGGCTTCTGATCCGTACA       factor               GTGATGCCGAAAAGGTT               Phe552   To alanine   CAAGGGAAAGATATCACCGAATTTGATGCTAATTTCGATC   III   Oligomerization                       defective               Ile574   To alanine   GAATTAAACGCGTCTAACGCATATACTG   III   Oligomerization                       defective               Phe552 + Phe554   To alanine   ATTTTGAGATGTTTGTTGATCGGCATTAGCATCAAATTC   III   Oligomerization                       defective               Ile562 + Ile574   To alanine   CAGTATATGCGTTAGACGCGTTTAATTCCGCTTAACTGATTCT   III   Oligomerization               TGGCATTTTGAGATG       defective               Leu566 + Ile574   To alanine   ATCAGGCAGCGGAATTAAACGCGTCTAACGCATATACTG   III   Oligomerization                       defective               Phe552 + Phe554 +   To alanine   CAGTATATGCGTTAGACGCGTTTAATTCCGCTGCCTGATTCTT   III   Oligomerization       Ile562 + Leu566 +       GGCATTTTGAGATG and       defective       Ile574       ATTTTGAGATGTTTGTTGATCGGCATTAGCATCAAATT               Phe427   To alanine   GTAATTGGAGTAGAACTGGCATCGTCTTGTGC   II   Translocation                       defective               Asp425   Residue   GTAATTGGAGTAGAACTGAAATCTTGTTCATTTAATGCG   II   Translocation           deleted           defective               Phe427   Residue   GCACAAGACGATAGTTCTACTCCAATTAC   II   Translocation           deleted           defective               Trp346   To alanine   CGGTCGCAATTGATCATTCACTATCTCTAGCAGGGGAAAGAA   II   Membrane               CTGCGGCTGAAACAATG       insertion/                       translocation                       defective               Leu352   To alanine   CGGTCGCAATTGATCATTCACTATCTCTAGCAGGGGAAAGAA   II   Membrane               CTTGGGCTGAAACAATGGGTGCAAATACCGCTGAT       insertion/                       translocation                       defective               Trp346, Met350   To alanine   CGGTCGCAATTGATCATTCACTATCTCTAGCAGGGGAAAGAA   II   Membrane       and Leu352       CTGCGGCTGAAACAGCGGGTGCAAATACCGCTGAT       insertion/                       translocation                       defective               LF mutants:       Tyr148   To alanine   GTAGAAGGTACCGAAAAGGCACTGAACGTTGCTTAT   I   Defective in                       binding to                       Protective Antigen               Tyr149   To alanine   GTAGAAGGTACCGAAAAGGCACTGAACGTTTATGCTGAA   I   Defective in                       binding to                       Protective Antigen               Ile151   To alanine   GTAGAAGGTACCGAAAAGGCACTGAACGTTTATGAAGCAGGT   I   Defective in                       binding to                       Protective Antigen               Lys153   To alanine   GTAGAAGGTACCGAAAAGGCACTGAACGTTTATGAAATAGGT   I   Defective in               GCAATA       binding to                       Protective Antigen               Asp187   To alanine   TGTGGGATGTTCCTTAAGCTGATTAGTAAATAAAAGAGCTTGT   I   Defective in               TCATCTGA       binding to                       Protective Antigen               Phe190   To alanine   TGTGGGATGTTCCTTAAGCTGATTAGTAGCTAAAAGATCTTG   I   Defective in                       binding to                       Protective Antigen               Asp187, Leu188,   To alanine   TGTGGGATGTTCCTTAAGCTGATTAGTAGCTGGAGCAGCTTGT   I   Defective in       Leu189, Phe190       TCATCTGA       binding to                       Protective Antigen               EF mutants:       Tyr137   To alanine   CCTTACTTATGATATCAAGAGAAATCCCC TTT CC AAT TTC   Defective in binding               AGC ATA TAC TTC TTT ACT TTG TTC AC   to Protective Antigen               Tyr138   To alanine   CCTTACTTATGATATCAAGAGAAATCCCC TTT CC AAT TTC   Defective in binding               ATA AGCTAC TTC TTT ACT TTG TTC AC   to Protective Antigen               Ile140   To alanine   CCTTACTTATGATATCAAGAGAAATCCCC TTT CCAGCTTC   Defective in binding               ATA ATATAC TTC TTT ACT TTG TTC AC   to Protective Antigen               Lys142   To alanine   CCTTACTTATGATATCAAGAGAAATCCCC GCT CC AAT TTC   Defective in binding               ATA ATATAC TTC TTT ACT TTG TTC AC   to Protective Antigen                  
 
         [0117]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
               
               
                 CHARACTERISTICS OF MUTANTS 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                   
                 OLIGOMER 
                 LF/EF 
                   
               
               
                 MUTATION 
                 RECEPTOR 
                 TRYPSIN 
                 FOR- 
                 BIND- 
                 TOX- 
               
               
                 IN PA 
                 BINDING 
                 NICKING 
                 MATION 
                 ING 
                 ICITY 
               
               
                   
               
               
                 Phe202Ala 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 Leu203Ala 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 Pro205Ala 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 Ile207Ala 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 Pro205Ala + 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 Trp226Ala + 
               
               
                 Phe236Ala 
               
               
                 Phe552Ala 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Ile574Ala 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Phe552Ala + 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Phe554Ala 
               
               
                 Ile562Ala + 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Ile574Ala 
               
               
                 Leu566Ala + 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Ile574Ala 
               
               
                 Phe552Ala + 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Phe554Ala + 
               
               
                 Ile562Ala + 
               
               
                 Leu566ala + 
               
               
                 Ile574Ala 
               
               
                 Phe427Ala 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                 Asp425del 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                 Phe427del 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                 Trp346Ala 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                 Leu352Ala 
                 + 
                   
                 + 
                 + 
                 − 
               
               
                 Trp346Ala + 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                 Met350Ala + 
               
               
                 Leu352Ala 
               
               
                   
               
             
          
           
               
                   
                 MUTATION 
                   
                   
               
               
                   
                 IN LF 
                 BINDING TO PA 
                 TOXICITY 
               
               
                   
                   
               
               
                   
                 Tyr148Ala 
                 − 
                 − 
               
               
                   
                 Tyr149Ala 
                 − 
                 − 
               
               
                   
                 Ile151Ala 
                 − 
                 − 
               
               
                   
                 Lys153Ala 
                 − 
                 − 
               
               
                   
                 Asp187Ala 
                 − 
                 − 
               
               
                   
                 Phe190Ala 
                 − 
                 − 
               
               
                   
                 Asp187Ala + Leu188Ala + 
                 − 
                 − 
               
               
                   
                 Phe190Leu189Ala 
               
               
                   
                   
               
             
          
           
               
                   
                 MUTATION 
                   
                   
               
               
                   
                 IN EF 
                 BINDING TO PA 
                 TOXICITY 
               
               
                   
                   
               
               
                   
                 Tyr137Ala 
                 − 
                 − 
               
               
                   
                 Tyr138Ala 
                 − 
                 − 
               
               
                   
                 Ile140Ala 
                 − 
                 − 
               
               
                   
                 Lys142Ala 
                 − 
                 −