Methods For The Production And Secretion Of Modified Peptides

• US 20090042246A1
• Filed: 12/07/2005
• Published: 02/12/2009
• Est. Priority Date: 12/07/2004
• Status: Active Grant

• Current Assignee: LanthioPep BV (Morphosys Ag)
• Original Assignee: Morphosys Ag
• Inventors: Driessen, Arnold Jacob Mathieu, Kuipers, Anneke, Moll, Gert Nikolaas, Rink, Rick, Kuipers, Oscar Paul
• Granted Patent: US 8,835,616 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence

Abstract

The invention relates to the production of heterologous polypeptides in a recombinant host cell. More specifically, it relates to the production and secretion of peptides, such as biologically active peptides, that are modified by one or more lantibiotic-synthesizing enzymes. Provided is a nucleic acid construct encoding a polypeptide comprising 1) a non-lantibiotic export signal that is recognized by a non-lantibiotic export system; 2) a lantibiotic leader peptide that is recognized by at least a lantibiotic dehydratase such as LanB and, C-terminally of said export signal and said leader peptide, 3) a peptide of interest containing one or more serine or threonine residue(s) which can be posttranslationally dehydrated by said dehydratase. Also provided is a method for producing a polypeptide in a host cell, comprising providing a host cell with a nucleic acid construct according to the invention and allowing expression and secretion of the encoded polypeptide by said host cell, wherein said host cell comprises at least a dehydratase capable of modifying said encoded polypeptide and a non-lantibiotic export system capable of secreting said modified polypeptide.

Claims

1. A nucleic acid construct encoding a polypeptide comprising:

a non-lantibiotic export signal that is recognized by a non-lantibiotic export system;

a lantibiotic leader peptide that is recognized by at least a lantibiotic dehydratase and, C-terminally of said export signal and said leader peptide,

a peptide of interest comprising one or more serine or threonine residue(s) that can be posttranslationally dehydrated by said dehydratase.

2. The nucleic acid construct of claim 1, wherein said lantibiotic leader peptide enables recognition by a LanB or LanM dehydratase.

3. The nucleic acid construct of claim 1, wherein said peptide of interest further comprises at least one cysteine residue which can be coupled enzymatically or chemically to a dehydrated serine or threonine residue.

4. The nucleic acid construct of claim 3, wherein said lantibiotic leader peptide furthermore enables recognition by a lantibiotic cyclase.

5. The nucleic acid construct of claim 3, wherein said at least one cysteine residue is separated by 0 to 30 amino acid residues from said serine or threonine residue.

6. The nucleic acid construct of claim 5, comprising the consensus motif sequence X1-D/E-E-V/L-S/T-D/E-X2-E-L-D/E, wherein X1 is any hydrophobic amino acid and wherein X2 is any amino acid;

or the consensus motif sequence F-D/E/N-L-D-X3, wherein X3 is L, I or V.

7. The nucleic acid construct of claim 1, wherein the lantibiotic leader peptide comprises a consensus motif derivable from the sequence alignment as set out in Table 1.

8. The nucleic acid construct of claim 1, wherein the lantibiotic leader peptide is the leader peptide of a lantibiotic selected from the group consisting of nisin A, nisin Z, subtilin, ericin S, ericin A, streptin, epidermin, val1-leu6-epidermin, gallidermin, mutacin 1140, mutacin B-Ny266, mutacin III, mutacin I, pep5, epilancin K7, epicidin 280, lacticin 481, variacin, mutacin II, streptococcin A-FF22, salivaricin A, lactocin S, cypemycin, plantaricin C, actagardine, Ala(0)-actagardine, lacticin 3147A1, lacticin 3147A2, staphylococcin C55α

, staphylococcin C55b, plantaricin Wa, plantaricin Wb, cytolysin_L1, cytolysin Ls, ruminococcin A, carnocin U149, macedocin, bovicin HJ50, nukacin ISK-1, sublancin 168, butyrivibriocin OR79A, cinnamycin, duramycin, ancovenin, mersacidin, sapB (see Table

     2), and a functional derivative of any thereof that is recognized by at least a lantibiotic dehydratase.

9. The nucleic acid construct of claim 1, wherein said non-lantibiotic export signal is a signal for a protein transport system with broad substrate specificity.

10. The nucleic acid construct of claim 1, wherein said non-lantibiotic export signal is a signal for a transport system selected from the group consisting of the Sec-dependent secretion system, the Twin-Arginine Translocation (TAT) system and a non-lantibiotic ABC transporter.

11. The nucleic acid construct of claim 1, wherein said non-lantibiotic export signal is located N- or C-terminally from the lantibiotic leader peptide.

12. The nucleic acid construct of claim 1, wherein said non-lantibiotic export signal and said lantibiotic leader peptide are separated by a spacer sequence.

13. The nucleic acid construct of claim 12, wherein said spacer sequence allows for isolation, immobilization or purification of said polypeptide.

14. The nucleic acid construct of claim 1, wherein said peptide of interest is selected from the group consisting of a bioactive peptide, a hormone, an enzyme, an inhibitory protein, a therapeutic protein, a lantibiotic protein, a viral protein and a mutant of any thereof.

15. A host cell provided with the nucleic acid construct of claim 1.

16. The host cell of claim 15, wherein said host cell is selected from the group consisting of a prokaryote, a eukaryote, a Gram-positive prokaryote, a Lactococcus spp. and a Bacillus spp.

17. A method for producing a polypeptide in a host cell, said method comprising:

providing the host cell of claim 15

expressing the polypeptide in the host cell, and

secreting the polypeptide from said host cell, wherein said host cell comprises a lantibiotic dehydratase able to modify said polypeptide and a non-lantibiotic export system able to secrete a modified polypeptide.

18. The method according to claim 17, wherein said host cell further comprises a lantibiotic cyclase so as to allow production of a thioether-ring comprising the peptide of interest in said host cell.

19. The nucleic acid construct of claim 12, wherein said spacer sequence encodes 2-250 amino acid residues lengthwise.

20. The nucleic acid construct of claim 4, wherein said lantibiotic leader peptide furthermore enables recognition by LanC or LanM.