Preparation of protective antigen
View Patent ↗A polynucleotide sequence is provided comprising a nucleic acid sequence encoding recombinant Protective Antigen (rPA). Also provided are expression vectors and host cells comprising the polynucleotide sequence of the invention, and methods for producing rPA.
1. A method for producing rPA comprising expressing a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1,
wherein said polynucleotide encodes a recombinant Bacillus anthracis Protective Antigen (rPA).
2. A method according to claim 1 , wherein said polynucleotide is expressed in a host cell.
3. A method according to claim 1 , wherein said polynucleotide is DNA, and is optionally first transcribed into RNA in vitro and the RNA is then translated in a host cell.
4. A method according to claim 2 , wherein the host cell is an E. coli host cell.
5. A method according to claim 4 , wherein the E. coli host cell is E. coli RV308.
6. A method according to claim 1 , comprising expressing rPA from an expression vector comprising said polynucleotide.
7. A method according to claim 6 , wherein the expression vector comprises the cpg leader sequence encoded by SEQ ID NO: 3.
8. A method according to claim 6 , wherein the vector is plasmid pMTL1015, encoded by SEQ ID NO: 4.
9. A method according to claim 6 , wherein the vector is expressed in E. coli RV308.
10. A method according to claim 6 , further comprising initial steps of transforming the expression vector into a host cell, and culturing the transformed host cell in a growth medium.
11. A method according to claim 10 , wherein said growth medium is free of animal products.
12. A method according to claim 10 , comprising culturing the transformed host cells at a temperature of less than 40° C.
13. A method according to claim 10 , further comprising harvesting the host cell.
14. A method according to claim 13 , further comprising extracting rPA from the host cell.
15. A method according to claim 14 , further comprising a separation step.
16. A method according to claim 15 , wherein the separation step is selected from one or more chromatography steps, and one or more filtration steps.
17. A method according to claim 16 , wherein at least one of said filtration step(s) is a diafiltration step.
18. A method according to claim 16 , wherein at least one of said chromatography step(s) is selected from an ion-exchange chromatography step and a hydrophobic charge chromatography step.
19. A method according to claim 2 , comprising:
(a) obtaining host cells that express the polynucleotide or an expression vector comprising said polynucleotide;
(b) extracting the expressed rPA from the host cells;
(c) subjecting the extracted rPA to a diafiltration step;
(d) subjecting the diafiltered rPA to at least one chromatography step selected from anion exchange and hydrophobic charge chromatography; and
(e) carrying out a further diafiltration step.
20. A method according to claim 1 , wherein said polynucleotide further comprises a polynucleotide encoding a secretion sequence.
21. A method according to claim 1 , wherein said polynucleotide further comprises a polynucleotide encoding a secretion sequence for periplasmic translocation of said recombinant Bacillus anthracis Protective Antigen (rPA).
22. A method according to claim 1 , wherein said polynucleotide further comprises a polynucleotide encoding a secretion sequence for extracellular translocation of said recombinant Bacillus anthracis Protective Antigen (rPA).
23. A method according to claim 21 , wherein said secretion sequence is cleavable from said recombinant Bacillus anthracis Protective Antigen (rPA) during periplasmic translocation.
24. A method according to claim 22 , wherein said secretion sequence is cleavable from said recombinant Bacillus anthracis Protective Antigen (rPA) during extracellular translocation.
25. A method according to claim 20 , wherein said secretion sequence is encoded by the nucleic acid sequence of SEQ ID NO:3.
26. A method according to claim 1 , wherein said polynucleotide further comprises a codon encoding a methionine residue at the 5′ end.
27. A method according to claim 26 , wherein said polynucleotide comprises the nucleic acid sequence of SEQ ID NO:7.
28. A method according to claim 6 , wherein said expression vector further comprises a promoter that is selected so as to ensure that said rPA polypeptide is highly expressed.
29. A method according to claim 28 , wherein said promoter is a malate dehydrogenase (mdh) promoter.
30. A method according to claim 6 , wherein said expression vector further comprises a selectable marker.
31. A method according to claim 6 , wherein said expression vector expresses said polynucleotide in the absence of a chemical inducer.
32. A method according to claim 28 , wherein said expression vector is plasmid pMTL1015, comprising the nucleic acid sequence of SEQ ID NO:4.
33. A method according to claim 21 , wherein said expression vector is deposited with the ECACC under the accession number 04061401.
34. A method according to claim 6 , wherein the expression vector further comprises the nucleic acid sequence of SEQ ID NO:4.
35. A method according to claim 6 , wherein the expression vector is deposited with the ECACC under the accession number 04052501.