Use of the RTX secretion system to achieve heterologous polypeptide secretion by
Compositions and methods for secreting large heterologous polypeptides are described.
1. A nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a fragment of a V. cholerae RTX protein comprising the RTX secretion signal sequence; and
(b) a heterologous polypeptide.
2. The nucleic acid of claim 1 , wherein the fragment comprises at least 40 carboxyl terminal amino acids of the RTX protein.
3. The nucleic acid of claim 1 , wherein the fragment comprises at least 100 carboxyl terminal amino acids of the RTX protein.
4. The nucleic acid of claim 1 , wherein the fragment comprises at least 105 carboxyl terminal amino acids of the RTX protein.
5. The nucleic acid of claim 1 , wherein the fragment consists of between 40 and 100 carboxyl terminal amino acids of the RTX protein.
6. The nucleic acid of claim 1 , wherein the fragment consists of 100 carboxyl terminal amino acids of the RTX protein.
7. The nucleic acid of claim 1 , wherein the fragment consists of 105 carboxyl terminal amino acids of the RTX protein.
8. The nucleic acid of claim 1 , wherein the fusion protein further comprises an amino terminal fragment of the RTX protein.
9. The nucleic acid of claim 8 , wherein the amino terminal fragment comprises 50 amino acids.
10. The nucleic acid of claim 9 , wherein the amino terminal fragment consists of 50 amino acids.
11. The nucleic acid of claim 8 , wherein the heterologous polypeptide is located between the amino terminal fragment and the fragment comprising the RTX secretion signal sequence.
12. The nucleic acid of claim 1 , wherein the heterologous polypeptide is C. difficile toxin A.
13. The nucleic acid of claim 1 , wherein the heterologous polypeptide comprises an antigenic portion of C. difficile toxin A.
14. The nucleic acid of claim 13 , wherein the antigenic portion comprises a carboxyl terminal fragment of C. difficile toxin A.
15. The nucleic acid of claim 1 , wherein the heterologous polypeptide comprises 300 carboxyl terminal amino acids of C. difficile toxin A.
16. An expression vector comprising the nucleic acid of claim 1 .
17. A V. cholerae cell comprising the vector of claim 16 .
18. A V. cholerae cell transfected with the vector of claim 16 , wherein the cell expresses the fusion protein.
19. The V. cholerae cell of claim 18 , wherein the cell secretes the fusion protein.
20. The V. cholerae cell of claim 18 , wherein the V. cholerae cell is an El Tor V. cholerae cell.
21. The V. cholerae cell of claim 20 , wherein the V. cholerae cell does not express cholera toxin A subunit.
22. The V. cholerae cell of claim 21 , wherein the V. cholerae cell is a Peru2 V. cholerae cell.
23. A method for producing a fusion protein, the method comprising maintaining the cell of claim 17 under conditions sufficient to allow expression of the fusion protein.