Non-hemolytic ClyA for excretion of proteins
The disclosure below provides a protein export system utilizing non-hemolytic variants of HlyE family member proteins for efficiently producing recombinant protein from a host cell. In a preferred embodiment, the protein export system utilizes protein export machinery endogenous to the host bacterium into which the protein export system vector is introduced.
1. A method for producing a fusion protein, comprising:
(a) transforming a population of bacteria with an expression vector encoding a fusion protein, wherein said fusion protein comprises a protein of interest linked to the carboxy terminus of an export protein, wherein said export protein is a Salmonella enterica serovar Typhi ( S. Typhi ) cytolysin A (ClyA) protein having substantially reduced hemolytic activity in comparison to the ClyA protein of SEQ ID NO:2, said export protein having the amino acid sequence set forth in SEQ ID NO:2 and having one or more mutations selected from the group consisting of an S195N mutation, an I198N mutation, an A199D mutation, an E204K mutation and a C285W mutation, and
(b) culturing transformed bacteria of (a) in a culture medium under conditions such that said fusion protein is expressed and exported into the culture medium.
2. The method of claim 1 , wherein said bacteria is selected from the group consisting of Salmonella spp., Vibrio spp., Escherichia spp., and Shigella spp.
3. The method of claim 1 , wherein said bacteria is S. Typhi.
4. The method of claim 1 , wherein said bacteria is E. coli , enterotoxigenic E. coli (ETEC) or enteroaggregative E. coli (EAEC).
5. The method of claim 1 , wherein said bacteria is Shigella flexneri 2a.
6. The method of claim 1 , wherein the protein of interest is an antigen.
7. The method of claim 1 , further comprising collecting said fusion protein from the culture medium.
8. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an S195N mutation.
9. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an I198N mutation.
10. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an A199D mutation.
11. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an E204K mutation.
12. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an C285W mutation.
13. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has a C285W mutation, and one additional mutation selected from the group consisting of an I198N mutation, an A199D mutation, and an E204K mutation.
14. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an I198N mutation, an A199D mutation and an E204K mutation.
15. The method of claim 1 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an I198N mutation and a C285W mutation.
16. The method of claim 1 , wherein the protein of interest is anthrax toxin PA83 protein.
17. An expression vector comprising an expression cassette, wherein the expression cassette comprises an export protein coding sequence linked to a protein of interest coding sequence in a 5′ to 3′ arrangement, wherein said export protein is a Salmonella enterica serovar Typhi ( S. Typhi ) cytolysin A (ClyA) protein having substantially reduced hemolytic activity in comparison to the ClyA protein of SEQ ID NO:2, said export protein having the amino acid sequence set forth in SEQ ID NO:2 and having one or more mutations selected from the group consisting of an S195N mutation, an I198N mutation, an A199D mutation, an E204K mutation and a C285W mutation.
18. The expression vector of claim 17 , wherein the protein of interest is an antigen.
19. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an S195N mutation.
20. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an I198N mutation.
21. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an A199D mutation.
22. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an E204K mutation.
23. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an C285W mutation.
24. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has a C285W mutation, and one additional mutation selected from the group consisting of an I198N mutation, an A199D mutation, and an E204K mutation.
25. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an I198N mutation, an A199D mutation and an E204K mutation.
26. The expression vector of claim 17 , wherein said S. Typhi cytolysin A (ClyA) protein has the amino acid sequence set forth in SEQ ID NO:2 and has an I198N mutation and a C285W mutation.
27. The expression vector of claim 17 , wherein the protein of interest is anthrax toxin PA83 protein.