Enhanced secretion of a polypeptide by a microorganism

• US 6,846,653 B2
• Filed: 10/09/2001
• Issued: 01/25/2005
• Est. Priority Date: 10/10/2000
• Status: Expired due to Term

• Current Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Kolkman, Marc
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): KATCHEVES, KONSTANTINA T
• Application Number:
  US09/975,132
• Publication Number:
  US 20020182672A1
• Time in Patent Office: 1,204 Days
• Field of Search: 435/69.1, 435/69.7, 435/69.8, 435/71.1
• US Class Current: 435/69.8
• CPC Class Codes: C07K 14/5403 IL-3
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/75 for Bacillus

Abstract

Described herein are methods for the enhanced production of secreted proteins. The secretion of a protein of interest having a substantially non-polar carboxy tail is enhanced by the placement of charged amino acid residues at the carboxy terminus either by adding to the native peptide or by replacing, i.e., substituting, the terminal residues of the native peptide.

Claims

1. A process for enhanced secretion of a polypeptide by bacteria, comprising:

(a) culturing bacterial cells that contain a recombinant expression vector comprising a first DNA sequence encoding a polypeptide that can be secreted by the bacteria and a second DNA sequence encoding a charged, amino-acid tag covalently bonded at the carboxy-terminus of said polypeptide, such that the polypeptide is produced by the cells, and wherein said tag comprises one or two negatively charged amino acid residues; and

(b) optionally, recovering the polypeptide from the culture medium.

2. The process of claim 1, wherein said tag comprises two negatively charged amino acid residues or two positively charged amino acid residues.

3. The process of claim 2, wherein said tag comprises two negatively charged amino acid residues, selected from the group consisting of D and E.

4. The process of claim 3, wherein said tag comprises two D residues.

5. The process of claim 2, wherein said tag comprises two positively charged amino acid residues, wherein said two positively charged amino acid residues are lysines (Ks).

6. The process of claim 1, wherein said bacteria is a Bacillus species.

7. The process of claim 6, wherein said bacteria is B. subtilis.

8. The process of claim 1, wherein said expression vector further includes a DNA sequence encoding a signal peptide operatively linked to said first DNA sequence.

9. The process of claim 8, wherein said signal peptide is B. licheniformis α

-amylase (AmyL) signal peptide.

10. The process of claim 1, wherein said polypeptide is a heterologous protein selected from the group consisting of hormones, enzymes, and growth factors.

11. The process of claim 10, wherein said protein is human interleukin.

12. A method for enhancing the secretion of a heterologous polypeptide by a Bacillus species, comprising:

substituting one or more of the C-terminal amino acids residues of said polypeptide with at least one charged amino acid residue, or adding one or more charged amino acid residues to the C-terminus of said polypeptide, wherein the number of amino acid residues substituted or added to the C-terminus of said polypetide comprises two amino add residues.

13. The method of claim 12, wherein the last two amino acid residues of said polypeptide are substituted with a D.

14. The method of claim 12, wherein the last two amino acid residues of said polypeptide are substituted with a E.

15. The method of claim 12, wherein the last two amino acid residues of said polypeptide are substituted with a K.

16. The method of claim 12, wherein two D residues are added at the C-terminus of said polypeptide.

17. The method of claim 12, wherein two E residues are added at the C-terminus of sad polypeptide.

18. The method of claim 12, wherein two K residues are added at the C-terminus of said polypeptide.