Title of Invention	"A RECOMBINANT PROTEIN USEFUL FOR INHIBITING ANTHRAX TOXIN"
Abstract	The present invention relates to a novel recombinant molecule useful for anthrax toxin inhibition and also provides a method for inhibition of anthrax toxin action using the new molecule.

Full Text

Field of invention The present invention relates to a recombinant protein useful for inhibiting anthrax toxin. The invention also provides a method for inhibition of anthrax toxin action using the new molecule. The main utility of the invention is to develop a candidate molecule for anthrax toxin inhibition and for providing a method for inactivation of toxic activity of a toxin of the nature of anthrax toxin. This molecule has potential for use as a therapeutic agent in neutralizing anthrax toxin action in individuals infected with Bacillus anthracis Related Prior art Anthrax is a bacterial disease caused by Bacillus anthracis. The disease primarily affects herbivores but humans can also get infected while dealing with such animals. B. anthracis is a potential agent of bio-terrorism. Main symptoms comprise dizziness, fever, edema followed by death. The toxic action of anthrax has been attributed to anthrax toxin produced by the bacterium. The toxin can be resolved into three distinct protein components protective antigen (PA), lethal factor (LF) and edema factor (EF). The combination of EF and PA (edema toxin) produces skin edema, while LF and PA (Lethal toxin) are lethal to animals. The three proteins are individually non-toxic. EF is a calcium and calmodulin dependent adenylate cyclase that acts by increasing the intracellular cAMP levels in eukaryotic cells and LF is a Zn.sup.2+ dependent metalloprotease that leads to increase in IL-1 and TNF-.alpha, production by susceptible cells and cleaves several MAP Kinase Kinases (MKK 1, 2 and 3) (Leppla, 1999). According to the current model of anthrax toxin action, PA binds to anthrax toxin receptor present on cell surface and gets proteolytically activated by cell surface proteases to PA63. This allows oligomerization and binding of LF/EF. The toxin complex is internalized by receptor mediated endocytosis and is exposed to acidic pH inside the endosome. This change in pH triggers both membrane insertion by PA63 and translocation of LF/EF into the cytosol (Leppla, 1999).The main object of the invention is to provide a novel molecule for anthrax toxin inhibition. Another object is to provide a method for inactivation of toxic activity of a toxin of the nature similar to that of anthrax toxin. Yet another object of the invention is to provide a therapeutic agent for use in neutralizing anthrax toxin action in individuals infected with Bacillus anthracis. Summary of the Invention Accordingly, the present invention provides a recombinant protein useful for inhibiting anthrax toxin, the recombinant protein comprising SEQ ID NO 1, wherein the amino acid residues in the 2beta2 — 2beta3 loop region of the native PA represented by residues 302 to 324 of SEQ ID No. 1 are mutated, wherein the amino acid sequence at 2beta2 — 2beta 3 loop in the native PA being 302EVHGNAEVHASFFDIGGSVSAGF324 and the mutated amino acid sequence at 2beta2 - 2beta3 loop in the recombinant PA-I being 302VGVSISAGYQNGFTGNITTSAGF324. The invention also provides an isolated polynucleotide encoding the said recombinant protein. Further provided is an isolated DNA vector comprising the said polynucleotide. In addition is provided an isolated transformed host cell comprising the said polynucleotide. The said recombinant protein is produced by the expression of the polynucleotide of claim 2. Brief Description of the Figures FIG. 1: PA and PA-I were purified from the cell supernatants of B. anthracis and analyzed on 10% SDS-PAGE. Lane 1: Molecular Weight Marker (kDa); Lane 2: Native PA; Lane 3: PA-I. FIG. 2: J774A.1 cells were cultured in 96 well plates in DMEM containing 10% fetal bovine serum and incubated with LF (1 .mu.g/ml) in combination with varying concentrations of PA and PA-I for 3 h at 37.degree. C. At the end of the experiment, toxicity was determined by MTT assay. FIG. 3: CHO-K1 cells were incubated with PA-I or PA-D mixed with varying concentrations of wild type PA at 37.degree. C. for 3 h in combination with LF.sup.l-254.TR.PE.sup.398-613. At the end of 3 h, cells were incubated with medium containing .sup.3H-leucine (1 .mu.Ci/ml) for 1 h at 37.degree. C. At the end of the experiment, amount of .sup.3H-leucine incorporation was measured. Results are expressed as percentage of .sup.3H-leucine incorporated by viable cells in the absence of added proteins. FIG. 4: CHO-K1 cells, preloaded with .sup.86Rb.sup.+, were incubated with trypsin cleaved PA and PA-I mixed in equimolar ratios at neutral pH for 2 h at 4.degree. C. After washing twice with cold phosphate buffered saline, the cells were subjected to acidic pH shock. The leakage of .sup.86Rb.sup.+ into the medium was then determined. Results are expressed as percentage of .sup.86Rb.sup.+ associated with cells in the absence of added proteins. Detailed Description of the Invention The present invention provides a novel molecule, said molecule being a recombinant protective antigen and useful for anthrax toxin inhibition. In an embodiment of the present invention the recombinant protein designated as PA-I of SEQ ID NO:l, useful for inhibiting anthrax toxin. In another embodiment of the present invention recombinant protein is non toxic to host cells. In still another embodiment of the present invention the recombinant protein inhibits native protein Protective Antigen (PA) mediated cellular intoxication. In an embodiment of the present invention the recombinant protein inhibits the channel forming ability of PA protein. Yet in another embodiment of the present invention the recombinant protein when applied with PA in the ratio of about 1:1, completely inhibits the anthrax lethal toxin. Yet in another embodiment of the present invention the recombinant protein PA-I has oligopeptide of SEQ ID NO:2 instead of oligopeptide of SEQ ID NO:3 of native PA. Yet in another embodiment of the present invention the gene encoding the recombinant protein (PA-I), having sequence SEQ ID NO:4. Still in another embodiment of the present invention the oligonucleotide primers of SEQ ID NO:5 and SEQ ID NO:6. In another embodiment of the present invention the site of mutation itself is of 69 bp and some flanking region on both sides of this has been taken into consideration to prepare the Primer of SEQ ID NO:5. In one more embodiment of the present invention the SEQ ID NO:5 is reverse primer while SEQ ID NO:6 is forward primer. Further in another embodiment of the present invention, wherein process for constructing a recombinant protein PA-I comprising steps: i) amplifying a region of PA gene encoding 2.beta.2 2 .beta.3 loop using the primers of SEQ ID NO:5 and SEQ ID NO:6; ii) mutating the amplified PA gene by replacing SEQ ID NO:3 of native PA with SEQ ID NO:2, iii) cloning the amplified mutated PA gene of step (ii) into a vector, and iv) expressing the clone in a host to obtain the recombinant protein PA-I. In another embodiment of the present invention, wherein the host used is selected from a group comprising E. coli, Bacillus anthracis etc. Still in another embodiment of the present invention, wherein the vector for cloning the mutant gene is selected from a group of expression vector comprising plasmid pYS5 & pMSl. Yet in another embodiment of the present invention, wherein the concentration of PA-I used for testing anthrax toxin inhibition is in the range of 0.01 .mu.g/ml to 0.1 .mu.g/ml. In another embodiment of the present invention a composition useful in inhibiting anthrax toxin, said composition comprising a recombinant protein PA-I of SEQ ID NO:l and pharmacologically acceptable additive(s). Still in another embodiment of the present invention, a method of treating anthrax infection in a subject in need thereof, said method comprising step of administering an effective amount of PA-I in pharmacologically acceptable additive(s). Yet in another embodiment of the present invention a method of treatment, wherein the fluid is glucose or PBS. Further in another embodiment of the present invention, wherein the PA-I is administered intravenously. Yet in another embodiment of the present invention, wherein the subject is mammals, preferably human. Yet in another embodiment of the present invention, wherein the recombinant protein PA- I completely inhibits the toxicity of anthrax lethal toxin. Still in another embodiment of the present invention, wherein recombinant protein PA-I results in 100% survival of rats even after 72 hours of injecting the toxin. In one more embodiment of the present invention, wherein recombinant protein PA-I inhibits the pore formation by native PA in cells. The changes in the amino-acid sequence in this loop have rendered it non-toxic and imparted a dominant negative phenotype consequently inhibiting the anthrax toxin action. The mutagenesis of the PA gene in this region has caused inhibition of pore-forming ability of wild-type PA by PA-I by defective channel formation. In yet another embodiment of the invention, in vivo system used to test the in vivo anthrax toxin inhibitory effect can be Fischer 344 rats, guinea pigs, mice and the like. The vector for cloning the mutant gene may be any expression vector such as plasmid pYS5, pMSl, and the like. In still another embodiment of the invention, mammalian cell lines used can be CHO-K1, J774A. 1, RAW 264.7 and the like. Method for inactivation of toxic activity of a toxin of the nature similar to that of anthrax toxin. Thus, the present invention provides a novel candidate molecule useful for anthrax toxin inhibition in vivo comprising the recombinant protective antigen [PA] protein. Native PA plus the recombinant protein in equimolar ratio in combination with lethal factor [LF] thereby leading to complete inhibition of native toxin activity. Main utility of the invention si to develop a candidate molecule for anthrax toxin inhibition and for providing a method for inactivation of toxic activity of a toxin of the nature of anthrax toxin. This molecule can be useful as therapeutic agent for use in neutralizing anthrax toxin action in individuals infected with Bacillus anthracis. Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosures. EXAMPLE 1: Reagents: Biochemicals and reagents were purchased from Sigma Chemical Co., St. Louis, USA. Bacterial culture media was purchased from Difco Laboratories, Becton Dickinson, Delhi, India. The enzymes and chemicals for DNA manipulations were obtained from New England BioLabs, USA. 3H-Leucine were obtained from Amersham Pharmacia Biotech, Piscataway, NJ, USA. The Chinese Hamster Ovary cell line (CHO-K1) and J774A.1 macrophage cell line were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% calf serum and 50 |J.g/ml gentamicin sulfate (Life Technologies, Inc., USA) at 37 °C in a CO2 incubator. EXAMPLE 2: Construction of the mutant PA gene: Mutation in the PA gene was constructed in the plasmid pYS5 (Singh et al., 1989). A non-mutagenic oligonucleotide primer corresponding to nucleotides 2169-2200 and spanning the unique HindIII site was used for PCR with a mutagenic primer corresponding to nucleotides 2785-2860 encompassing the unique PstI site and containing the desired mutations at nucleotides 2792-2851 (nucleotide numbering is according to Welkos et al., 1988). PCR was performed in a 100 ul tube at the following conditions: 94 °C: 1 min. 94 °C: 30 sec 55 °C: 1 min 72 °C: 1 min 72 °C: 10 min 4°C:1h The constituents of the reaction were: 1 OX PCR buffer: IX Template DNA: 0.5 µg Forward primer: 0.5 µM Reverse Primer: 0.5 µM dNTPs: 20 µM Taq DNA polymerase: 2.5 U/µl The amplified PCR product was digested with PstI and Hindlll as describer below: 1 OX Buffer:1X Template: 10 µg PstI: 10 U HindIII:10U and purified on a 1% low melting point agarose gel. The DNA sample was dissolved in 6X sample buffer (final concentration 1X), loaded on low melting point agarose gel and run at 50V. The plasmid pYS5 was digested with the same enzymes, purified on agarose gel and ligated to the mutant fragment. The DNA sequence of the mutant PA gene was verified by DNA sequencing of at least 200 base pairs spanning the mutated region. EXAMPLE 3: Expression and purification of recombinant protein PA-I: The plasmid carrying the desired sequence was transformed into E. coli dam dcm strain SCS110. Unmethylated plasmid DNA was purified and used to transform B. anthracis BH441. B. anthracis was transformed by adding 2 µg of DNA into electrocompetent cells and exposing them to a voltage of 1.5 kV and resistance of 200 Ω The transformed culture was grown overnight and the cell supernatant was concentrated using concentrator and the protein analyzed using SDS-PAGE. EXAMPLE 4: Molecular weight determination: The molecular weight of PA-I was determined by SDS-PAGE (Laemlli, 1970). The protein sample (2 µg) was dissolved in 5X SDS dye (final concentration 1X) and run on the 10 % gel. The molecular weight of PA-I was found to be equal to that of native PA (83 kDa) as determined by SDS-PAGE using appropriate molecular weight standards (Figure 1). EXAMPLE 5: Cytotoxicity assay : To study the cytotoxicity, varying concentrations of PA and PA-I were added to J774A.1 cells together with LF (1.0 µg/ml) and incubated for 3 h at 37°C. At the end of the experiment, cell viability was determined using MTT assay (Singh et al., 1994). The result showed that the mutant PA protein PA-I is completely non-toxic to J774A.1 cells (Figure 2). EXAMPLE 6: Inhibition of the activity of native PA by PA-I Inhibition of activity of native PA by PA-I was investigated by mixing of the mutant PA protein and native type PA at varying ratios resulted in alterations in the cytotoxic activity of the toxin containing the native protein (PA plus LF). When the mutant and native PA were present at equimolar concentrations, complete inhibition in protein synthesis of CHO-Kl cells was observed. A significant inhibition could be detected when the ratio of PA-I to PA was 1:4. These data suggest that the PA-I inhibits native PA mediated cellular intoxication (Figure 3). EXAMPLE 7: Inhibition of pore forming ability of native PA by PA I: Recombinant protein (PA-I) and the native protein (PA) were mixed together (2 µg/ml each) at the neutral pH and incubated with CHO-Kl cells preloaded with 86Rb+ at 4 °C. After 2 h, the cells were washed to remove unbound proteins and incubated with isotonic buffer of pH 5.0 or 7.0 for 30 min. at 37 °C. Whereas native PA released 62% of the radiolabel from cells, equimolar mixture containing PA and PA-I showed insignificant release of 86Rb+. The results suggest that there is complete inhibition of channel forming ability of PA by PA-I (Figure 4). The capacity of PA-I to dramatically alter the channel forming ability of native PA provides evidence that these two species can interact to form dysfunctional hetero-oligomeric structures EXAMPLE 8: In vivo inhibition of anthrax toxin activity: Animal experiments were performed to test the efficacy of PA-I to act as a dominant negative inhibitor of lethal toxin action in vivo (that is in equimolar concentration with respect to native PA. Native lethal toxin (40 ug PA + 8 jag LF) resulted in the death of male Fischer 344 rats in approximately 60 min. (Table 1), whereas a 1:1 mix containing native PA and PA-I (40 ug PA + 40 ug PA-I + 8 ug LF) protected rats and no symptoms were evident even after 48 h. Equimolar ratio of native PA and PA-D resulted in the death of rats within 70 minutes. Table 1. Inhibitory action of PA-I on Fischer 344 rats: (Table Removed) a TTD is the time to death of Fischer 344 rats after administration of proteins. Advantages Main advantage of the invention is in providing a novel recombinant candidate molecule which is a dominant negative inhibitor which inhibitis anthrax toxin action, can be valuable for treatment of anthrax toxin action. Use of Bacillus anthracis as a bioweapon has become the bane of the defence establishments in various countries. The recombinant protein also has good potential for use as therapeutic agent for neutralizing anthrax toxin action in individuals infected with Bacillus anthracis. The invention also provides a method for inactivation of toxic activity of a toxin of the nature similar to that of anthrax toxin. Thus, the present invention provides a novel candidate molecule useful for anthrax toxin inhibition in vivo comprising the recombinant protective antigen (PA) protein. Native PA plus the recombinant protein in equimolar ratio in combination with lethal factor (LF) thereby leading to complete inhibition of native toxin activity. Main utility of the invention is to develop a candidate molecule for anthrax toxin inhibition and for providing a method for inactivation of toxic activity of a toxin of the nature of anthrax toxin. This molecule can be useful as therapeutic agent for use in neutralizing anthrax toxin action in individuals infected with Bacillus anthracis. SEQUENCE LISTINGS (Sequence Removed) We claim: 1. A recombinant protein useful for inhibiting anthrax toxin, the recombinant protein comprising SEQ ID NO 1, wherein the amino acid residues in the 2beta2 - 2beta3 loop region of the native PA represented by residues 302 to 324 of SEQ ID No. 1 are mutated, wherein the amino acid sequence at 2beta2 - 2beta 3 loop in the native PA being 302EVHGNAEVHASFFDIGGSVSAGF324 and the mutated amino acid sequence at 2beta2 - 2beta3 loop in the recombinant PA-I being 302VGVSISAGYQNGFTGNITTSAGF324. 2. An isolated polynucleotide encoding the recombinant protein as claimed in claim 1. 3. An isolated DNA vector comprising the polynucleotide of claim 2. 4. A recombinant protein as claimed in claim 1, wherein it is produced by the expression of the polynucleotide of claim 2. 5. A recombinant protein useful for inhibiting anthrax toxin substantially as herein described with reference to the foregoing examples.

Documents:

165-DEL-2003-Abstract-(03-09-2008).pdf

165-del-2003-abstract.pdf

165-DEL-2003-Claims-(03-09-2008).pdf

165-DEL-2003-Claims-(19-09-2008).pdf

165-del-2003-claims.pdf

165-DEL-2003-Correspondence-Others-(03-09-2008).pdf

165-del-2003-correspondence-others-(15-10-2008).pdf

165-DEL-2003-Correspondence-Others-(19-09-2008).pdf

165-DEL-2003-Correspondence-Others-(24-09-2008).pdf

165-del-2003-correspondence-others.pdf

165-del-2003-correspondence-po.pdf

165-DEL-2003-Description (Complete)-(03-09-2008).pdf

165-DEL-2003-Description (Complete)-(19-09-2008).pdf

165-del-2003-description (complete).pdf

165-DEL-2003-Drawings-(03-09-2008).pdf

165-del-2003-form-1-(15-10-2008).pdf

165-DEL-2003-Form-1-(19-09-2008).pdf

165-DEL-2003-Form-1-(24-09-2008).pdf

165-del-2003-form-1.pdf

165-del-2003-form-18.pdf

165-DEL-2003-Form-2-(03-09-2008).pdf

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165-DEL-2003-Form-3-(03-09-2008).pdf

165-del-2003-form-3.pdf

165-DEL-2003-Form-5-(03-09-2008).pdf

165-DEL-2003-Petition-137-(03-09-2008).pdf

165-DEL-2003-Petition-137-(19-09-2008).pdf