Stabilized bioactive peptides and methods of identification, synthesis and use
View Patent ↗An intracellular selection system allows concurrent screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at either the N-terminus, the C-terminus, or both. The stabilizing group can take the form of a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, or one or more proline residues.
1. A method for making a protease resistant bioactive peptide comprising:
attaching a first stabilizing group to the N-terminus of a bioactive peptide; and
attaching a second stabilizing group to the C-terminus of the bioactive peptide to yield the protease resistant bioactive peptide;
wherein the first stabilizing group is selected from the group consisting of a small stable protein selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase, Pro-, Pro-pro-, Xaa-Pro- and Xaa-Pro-Pro-, and wherein the second stabilizing group is selected from the group consisting of a small stable protein selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase, -Pro, -Pro-Pro, -Pro-Xaa and -Pro-Pro-Xaa, wherein Xaa is any amino acid residue.
2. The method of claim 1 wherein the protease resistant bioactive peptide is chemically synthesized.
3. The method of claim 1 wherein attaching either or both of the first and second stabilizing groups to the N-terminus or/and the C-terminus of the bioactive peptide comprises the following steps in the order recited:
(a) providing the bioactive peptide; and
(b) attaching either or both of the first and second stabilizing groups to the N-terminus or/and the C-terminus of the bioactive peptide.
4. The method of claim 1 wherein either or both of the first and second stabilizing groups is attached to the N-terminus or/and the C-terminus of the bioactive peptide during synthesis of the bioactive peptide.
5. The method of claim 1 wherein the stabilizing group is isolated from an endogenous source, chemically synthesized, enzymatically synthesized, or produced using recombinant DNA technology.
6. A method for making a protease resistant bioactive peptide, the method comprising:
providing a host cell transformed with a nucleic acid encoding the protease resistant bioactive peptide, which comprises the bioactive peptide, a first stabilizing group attached to the N-terminus of the bioactive peptide, and a second stabilizing group attached to the C-terminus of the bioactive peptide, wherein the first stabilizing group is selected from the group consisting of a small stable protein selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase, Pro-, Pro-pro-, Xaa-Pro- and Xaa-Pro-Pro-, and wherein the second stabilizing group is selected from the group consisting of a small stable protein selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin. maltose binding protein, and glutathione reductase, -Pro, Pro-Pro, -Pro-Xaa and -Pro-Pro-Xaa, wherein Xaa is any amino acid residue;
expressing the said protease resistant bioactive peptide in the host cell; and
recovering the expressed protease resistant bioactive peptide from the host cell.
7. The method of claim 6 wherein the host cell is a bacterial cell.
8. The method of claim 6 wherein the host cell is a eukaryotic cell.
9. The method of claim 1 or 6 wherein at least one of the first and second stabilizing groups comprises a small stable protein.
10. The method of claim 1 or 6 wherein the small stable protein is a four-helix bundle protein.
11. The method of claim 1 or 6 wherein the small stable protein is selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase.
12. The method of claim 1 or 6 wherein the small stable protein is Rop protein.
13. The method of claim 1 or 6 wherein the first stabilizing group is Pro-Pro- and the second stabilizing group is -Pro-Pro.
14. The method of claim 1 or 6 wherein said peptide further comprises a cleavage site immediately preceding the first stabilizing group.
15. The method of claim 1 or 6 wherein protease resistant bioactive peptide further comprises a cleavage site immediately following the second stabilizing group.
16. The method of claim 1 or 6 wherein the bioactive peptide is an antimicrobial peptide.
17. The method of claim 1 or 6 wherein the bioactive peptide is a therapeutic peptide drug.
18. The method of claim 1 or 6 wherein the bioactive peptide is a naturally occurring bioactive peptide.
19. A method for making a protease resistant bioactive peptide comprising:
attaching a stabilizing group to either or both of the N-terminus or/and C-terminus of a naturally occurring bioactive peptide; wherein the stabilizing group attached to the N-terminus comprises Xaa-Pro-Pro-, and the stabilizing group attached to the C-terminus comprises -Pro-Pro-Xaa.
20. The method of claim 19 , wherein protease resistant bioactive peptide is chemically synthesized.
21. The method of claim 19 , wherein attaching the stabilizing group to the bioactive peptide comprises the following steps in the order recited:
(a) providing said protease resistant bioactive peptide, and
(b) attaching the stabilizing group to said protease resistant bioactive peptide.
22. A method for making a protease resistant bioactive peptide comprising:
providing a host cell transformed with a nucleic acid encoding protease resistant bioactive peptide comprising a naturally occurring bioactive peptide and a stabilizing group attached to either or both of the N-terminus or/and the C-terminus of said bioactive peptide, wherein the stabilizing group attached to the N-terminus comprises Xaa-Pro-Pro-, and the stabilizing group attached to the C-terminus comprises -Pro-Pro-Xaa;
expressing the protease resistant bioactive peptide in the host cell; and
recovering protease resistant bioactive peptide from the host cell.