Use of a Sec-dependent secretion system for secreting proteins that are usually secreted by a Sec-independent secretion system, bacteria containing it and their use

• US 5,939,317 A
• Filed: 06/06/1997
• Issued: 08/17/1999
• Est. Priority Date: 10/06/1995
• Status: Expired due to Fees

• Current Assignee: SKW Biosystems Co.
• Original Assignee: SKW Biosystems (Cargill Incorporated)
• Inventors: Bigret, Marc, Prevots, Fabien, Venema, Gerhardus, Fayard, Blandine, Kok, Jan
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): McGarry, Sean
• Application Number:
  US08/849,373
• Time in Patent Office: 802 Days
• Field of Search: 435/6, 435/69.1, 435/172.1, 435/172.3, 435/252.9, 435/243, 435/320.1, 536/23.1, 536/23.4, 536/23.7, 536/24.1
• US Class Current: 435/320.1
• CPC Class Codes: C07K 14/315 from Streptococcus (G), e.g. Enterococci
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea

Abstract

The invention relates to the use of a Sec-dependent secretion system for secreting proteins normally secreted by a Sec-independent secretion system.

Claims

1. A DNA construct for the secretion, by a Sec-dependent system, of a protein normally secreted by a Sec-independent system, characterized in that it comprises a promoter, a signal sequence recognized by the Sec-dependent secretion system, a DNA sequence coding for a mature protein normally secreted by a Sec-independent system, and a terminator wherein said construct does not contain a multiple cloning site.

2. The construct according to claim 1, characterized in that the signal sequence recognized by the Sec-dependent secretion system is the sp signal sequence of the protease of lactococcus.

3. The construct according to claim 1, characterized in that the DNA sequence coding for the protein is the DNA sequence coding for a bacteriocin.

4. The construct according to claim 3, characterized in that the bacteriocin is the bacteriocin LcnB.

5. The construct according to claim 1, characterized in that the promoter is the P32 promoter of lactococcus and the terminator is that of the LcnB immunity gene (lciB).

6. The construct according to claim 2, characterized in that the DNA sequence coding for the protein is the DNA sequence coding for a bacteriocin.

7. The construct according to claim 6, characterized in that the bacteriocin is the bacteriocin LcnB.

8. The construct according to claim 2, characterized in that the promoter is the P32 promoter of lactococcus and the terminator is that of the LcnB immunity gene (lciB).

9. The construct according to claim 3, characterized in that the promoter is the P32 promoter of lactococcus and the terminator is that of the LcnB immunity gene (lciB).

10. The construct according to claim 4, characterized in that the promoter is the P32 promoter of lactococcus and the terminator is that of the LcnB immunity gene (lciB).

11. The construct according to claim 6, characterized in that the promoter is the P32 promoter of lactococcus and the terminator is that of the LcnB immunity gene (lciB) .

12. The construct according to claim 7, characterized in that the promoter is the P32 promoter of lactococcus and the terminator is that of the LcnB immunity gene (lciB) .

13. A process for secreting a protein that is normally secreted by a Sec-independent secretion system, using a Sec-dependent secretion system, comprising

a) providing a DNA sequence coding for said protein, a promoter, a signal sequence recognized by the Sec-dependent secretion system and a terminator;

b) constructing a DNA construct comprising said DNA sequence, said promoter, said signal sequence and said terminator;

c) introducing said construct into a lactic acid bacterium by gene transfer; and

d) secreting said protein by the lactic acid bacterium containing said construct.

14. The process according to claim 13, characterized in that the signal sequence is the sp signal sequence of the protease of lactococcus.