Export and modification of (poly)peptides in the lantibiotic way

• US 6,861,236 B2
• Filed: 02/07/2003
• Issued: 03/01/2005
• Est. Priority Date: 05/24/2002
• Status: Active Grant

• Current Assignee: LanthioPep BV (Morphosys Ag)
• Original Assignee: Applied Nanosystems B.V.
• Inventors: Leenhouts, Cornelis Johannes, Moll, Gert Nikolaas
• Primary Examiner(s): Carlson, Karen Cochrane
• Assistant Examiner(s): Desai, Anand U
• Application Number:
  US10/360,101
• Publication Number:
  US 20040009550A1
• Time in Patent Office: 753 Days
• Field of Search: 435/69.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/315 from Streptococcus (G), e.g. Enterococci
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

The invention includes a method for harvesting a polypeptide produced by a host cell, wherein the polypeptide has not undergone intracellular posttranslational modification, such as dehydration of a serine or a threonine, and/or thioether bridge formation. The invention also includes a method for producing thioether-containing peptides and dehydroalanine/dehydrobutyrine-containing.peptides, wherein thioether rings may be formed extracellularly.

Claims

1. A method for producing a thioether bridge containing polypeptide of a non-Gram positive bacterium origin in a host cell, said method comprising:

selecting a host cell having a nucleic acid molecule comprising;

a first nucleic acid fragment encoding a leader peptide;

a second nucleic acid fragment encoding a polypeptide comprising a sequence selected from the group of sequences consisting of Ser-Cys, Ser-Xaa_1-20-Cys (SEQ ID NO;

     310), Thr-Cys, and Thr-Xaa_1-20-Cys (SEQ ID NO;

     311), wherein Xaa is any amino acid and the polypeptide is of the non-Gram positive bacterium origin;

wherein the first and second nucleic acid fragments are within the same open reading frame as the nucleic acid molecule; and

wherein the leader peptide acts as a translocation signal sequence and a recognition signal such that the thioether bridge formation may occur;

selecting the host cell for the presence of the transporter protein LanT or a functional equivalent thereof having LanT activity;

translating the nucleic acid molecule, thus producing the leader peptide and the polypeptide comprising the sequence selected from the group of sequences consisting of Ser-Cys, Ser-Xaa_1-20-Cys (SEQ ID NO;

     310), Thr-Cys, and Thr-Xaa_1-20-Cys (SEQ ID NO;

     311); and

harvesting the polypeptide containing the thioether bridge from a medium of the host cell.

2. The method according to claim 1, further comprising harvesting the polypeptide containing the thioether bridge after the leader peptide is detected in the medium of the host cell.

3. The method according to claim 1, wherein the leader peptide is selected from the group consisting of the leader peptides of Table 2.

4. The method according to claim 1, wherein the host cell is of a Gram-negative or eukaryotic origin.

5. The method according to claim 1, wherein the polypeptide containing the thioether bridge has not undergone intra-cellular post-translational modification.

6. The method according to claim 1, wherein the host cell is not provided with a LanB protein or a functional equivalent thereof having LanB activity.

7. The method according to claim 1, wherein the host cell is not provided with a LanC protein or a functional equivalent thereof having LanC activity.

8. The method according to claim 6, wherein the host cell is of Gram-negative or eukaryotic origin.

9. The method according to claim 7, wherein the host cell is of Gram-negative or eukaryotic origin.

10. The method according to claim 5, wherein the intra-cellular post-translational modification comprises dehydration of a serine, dehydration of a threonine or thioether bridge formation.

11. The method according to claim 1, wherein the polypeptide comprises the sequence Ser-Xaa_2-15-Cys (SEQ ID NO:

312) or Thr- Xaa_2-15-Cys (SEQ ID NO;

     313).

12. A method for producing a thioether bridge containing polypeptide of a non-Gram positive bacterium origin, said method comprising:

providing a host cell having a nucleic acid molecule, the nucleic acid molecule comprising;

a first nucleic acid fragment encoding a leader peptide of a nisin peptide origin;

a second nucleic acid fragment encoding a pre-polypeptide comprising a sequence selected from the group of sequences consisting of Ser-Cys, Ser-Xaa_1-20-Cys (SEQ ID NO;

     310), Thr-Cys, and Thr-Xaa_1-20-Cys (SEQ ID NO;

     311), wherein Xaa is any amino acid and the pre-polypeptide is of the non-Gram positive bacterium origin;

wherein the first and second nucleic acid fragments are within the same open reading frame as the nucleic acid molecule;

translating the nucleic acid molecule, thus expressing the leader peptide and the pre-polypeptide; and

harvesting the thioether bridge containing polypeptide from a medium of the host cell.

13. The method according to claim 12, wherein the pre-polypeptide comprises the sequence Ser-Xaa_2-15-Cys (SEQ ID NO:

312) or Thr- Xaa_2-15-Cys (SEQ ID NO;

     313).