Compositions and methods relating to a mutant
In one aspect, the invention relates to an immunogenic composition that includes a mutant Clostridium difficile toxin A and/or a mutant Clostridium difficile toxin B. The mutant toxin may include a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type C. difficile toxin. The mutant toxins may include at least one amino acid that is chemically crosslinked. In another aspect, the invention relates to methods and compositions for use in culturing Clostridium difficile and in producing C. difficile toxins.
1. A culture medium comprising soy hydrolysate, yeast extract, and glucose, and a recombinant Clostridium difficile cell; wherein the cell comprises a Clostridium sporogenes feredoxin promoter and a nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195.
2. The medium according to claim 1 , further comprising polyethylene glycol 2000.
3. The medium according to claim 1 , further comprising thiamphenicol.
4. The medium according to claim 1 , wherein the cell is derived from C. difficile VPI 11186.
5. The medium according to claim 1 , wherein the cell is derived from a Clostridium difficile cell selected from the group consisting of Clostridium difficile 1351, Clostridium difficile 3232, Clostridium difficile 7322, Clostridium difficile 5036, Clostridium difficile 4811, and Clostridium difficile VPI 11186.
6. The medium according to claim 1 , wherein the cell is a Clostridium difficile VPI 11186 cell.
7. The medium according to claim 1 , wherein a sporulation gene of the Clostridium difficile cell is inactivated.
8. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 188.
9. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 189.
10. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 190.
11. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 191.
12. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 192.
13. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 193.
14. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 194.
15. The medium according to claim 1 , wherein the cell comprises a polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 195.