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

• US 20040009550A1
• Filed: 02/07/2003
• Published: 01/15/2004
• 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
• Granted Patent: US 6,861,236 B2
• Time in Patent Office: Days
• 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 intra-cellular post-translational 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 extracellularly thioether rings may be formed.

Claims

1. A method for harvesting a desired polypeptide produced by a host cell, said method comprising:

selecting a host cell having first nucleic acid comprising;

a first nucleic acid fragment encoding a leader peptide;

a second nucleic acid fragment encoding the polypeptide;

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

wherein the leader peptide is at least functionally equivalent to a N-terminal leader peptide found within a prepeptide of a lantibiotic;

selecting the host cell for the presence of the transporter protein LanT or a functional equivalent thereof,

translating the first nucleic acid; and

harvesting the polypeptide from the culture medium of the host cell.

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

3. The method according to claim 1, wherein the polypeptide originates from a eukaryote or a virus.

4. The method according to claim 1, wherein the polypeptide is selected from the group consisting of the polypeptides of Table I.

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

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

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

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

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

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

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

12. The method according to claim 10, wherein the host cell is of Gram-negative or eukaryotic origin.

13. The method according to claim 7, wherein the intra-cellular post-translational modification comprises dehydration of a serine, dehydration of a threonine or thioester bridge formation.