Peptide domains that bind small molecules of industrial significance

Patent metadata

Field	Value
Publication number	US-9447150-B2
Application number	US-201113575834-A
Country	US
Kind code	B2
Filing date	Jan 28, 2011
Priority date	Jan 29, 2010
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

Described herein are small peptide domains and consensus sequences that bind small target molecules of industrial importance, e.g., metals such as nickel, β carotene, and isoflavones such as genistein. Also described are fusion proteins containing such binding domains fused to proteins or to peptide domains like GST or CBD that bind other ligands and can be used to immobilize the target binding domain on a support. One class of fusion proteins that is useful in industrial settings are fusions that contain concatemers of target binding domains, which increases the binding equivalents per molecule.

First claim

What is claimed is:

1. An isolated polypeptide or isolated peptide comprising a fusion of a naturally or non naturally occurring polypeptide or peptide and at least one metal binding peptide, wherein the sequence of the at least one metal binding peptide comprises a core sequence of the formula

H-X-H-(Z′)-H-(Z″)-H
(SEQ ID NO: 116)

wherein
H is histidine,
X is a single amino acid selected from the group consisting of asparagine, arginine, valine, phenylalanine, alanine, glycine, threonine, and isoleucine;
Z′ is amino acid selected from the group consisting of glycine, threonine, and tryptophan,
Z″ is 1 to 4 amino acids,

wherein when Z″ is one amino acid, the amino acid is selected from the group consisting of alanine, arginine, threonine and leucine, and wherein when Z″ is two to four amino acids, at least one amino acid is selected from the group consisting of aspartic acid, alanine, lysine, leucine, threonine, and histidine,
wherein there are no more than 2 residues of lysine and arginine in or within 1 to 5 residues of the core sequence, or no more than 4 residues of aspartate, glutamate, lysine or arginine in or within 1 to 5 residues of the core sequence,
wherein the peptide binds a metal with a dissociation constant of 9.0×10 −11 M or less than 9.0×10 −11 M,
with the proviso that no more than 5 histidine residues occur in 12 contiguous residues that include SEQ ID NO:116.

2. The isolated polypeptide or peptide of claim 1 wherein Z″ does not contain glutamine.

3. The isolated polypeptide or peptide of claim 1 or 2 wherein at least one proline, tyrosine or tryptophan is in, or within 1 to 5 residues of, the core sequence.

4. The isolated polypeptide or peptide of claim 1 wherein X is arginine, valine, phenylalanine, alanine, glycine, threonine, or isoleucine.

5. The isolated polypeptide or peptide of claim 1 wherein the peptide binds a metal selected from the group consisting of nickel, zinc and copper.

6. The isolated polypeptide or peptide of claim 1 wherein a sequence of 12 amino acids that includes the core sequence has a hydropathy measure of between −0.215 and −2.215.

7. The isolated polypeptide or peptide of claim 1 which further contains a ligand binding domain that binds a ligand other than the metal.

8. The isolated polypeptide or peptide of claim 7 wherein the ligand binding domain binds glutathione.

9. The isolated polypeptide or peptide of claim 1 which contains a silk protein.

10. The isolated polypeptide or peptide of claim 1 which is soluble in a first solvent mixture that includes the metal for the metal binding domain, but insolvent in a second solvent mixture.

11. A method of detecting the presence of a target molecule of less than 1600 daltons in a sample, comprising:
immobilizing a peptide having the core sequence of the polypeptide or peptide of claim 1 that binds the target molecule with a dissociation constant of at least 10 −9 M or less on a substrate;
contacting the immobilized peptide with the sample;
washing the substrate to remove unbound material; and
detecting whether the target molecule is bound to the peptide.

12. The method of claim 11 wherein the binding is detected by a surface plasmon resonance detector.

13. The method of claim 11 wherein the binding is detected using a fluorescence detector.

14. The method of claim 11 wherein the binding is detected using a radioisotope detector.

15. The method of claim 11 wherein the binding is detected using a spectrophotometer.

16. The method of claim 11 wherein the binding domain comprises at least one domain within a larger fusion protein.

17. The method of claim 15 wherein the fusion protein is comprised of a plurality of the binding domains.

18. The method of any one of claims 11 to 17 wherein the target molecule is a metal.

19. A sensing device comprising an immobilized polypeptide or peptide of claim 1 .

20. A method of isolating a target molecule from a sample, comprising:
providing a fusion protein having the polypeptide or peptide of claim 1 and an isolation domain comprising amino acid residues that can be used to isolate the fusion protein by at least one of binding to a ligand;
contacting the fusion protein with a sample suspected of containing the target molecule to bind the target molecule to the target domain; and
isolating the fusion protein bound to the target molecule using the isolation domain, thereby isolating the target molecule.

21. The isolated polypeptide or peptide of claim 1 which comprises at least two metal binding peptides, wherein the sequence of each of the at least two metal binding peptides independently comprises the core sequence.

22. The isolated polypeptide or peptide of claim 21 wherein the at least two metal binding peptides form a concatemer.

23. The isolated polypeptide or peptide of claim 22 wherein the concatemer has two different metal binding peptides of the core sequence.

24. The isolated polypeptide or peptide of claim 22 wherein the concatemer has two of the same metal binding peptides of the core sequence.

25. An isolated polypeptide or isolated peptide comprising a fusion of a naturally or non naturally occurring polypeptide or peptide and at least one metal binding peptide, wherein the sequence of the at least one metal binding peptide comprises a core sequence of the formula

(SEQ ID NO: 116)

H-X-H-(Z′)-H-(Z″)-H

wherein
H is histidine,
X is a single amino acid selected from the group consisting of asparagine, arginine, valine, phenylalanine, alanine, glycine, threonine, and isoleucine;
Z′ is 1 or 2 amino acids, wherein when Z′ is two amino acids at least one of the amino acids is selected from the group consisting of arginine, glutamine, methionine, phenylalanine, glycine, leucine, tryptophan, and histidine, and wherein when Z′ is one amino acid, the amino acid is selected from the group consisting of glycine, threonine, and tryptophan,
Z″ is an amino acid selected from the group consisting of alanine, arginine, threonine and leucine,
wherein there are no more than 2 residues of lysine and arginine in or within 1 to 5 residues of the core sequence, or no more than 4 residues of aspartate, glutamate, lysine or arginine in or within 1 to 5 residues of the core sequence,
wherein the peptide binds a metal with a dissociation constant of 9.0×10 −11 M or less than 9.0×10 −11 M,
with the proviso that no more than 5 histidine residues occur in 12 contiguous residues that include SEQ ID NO:116.

26. The isolated polypeptide or peptide of claim 5 wherein Z′ includes arginine, phenylalanine, tryptophan, glycine, or threonine or Z″ is alanine, arginine, or leucine.

27. The isolated polypeptide or peptide of claim 25 wherein Z′

Assignees

• Binder Thomas P
• Rao Aragula Gururaj
• Yamamoto Yasufumi
• Hanke Paul D
• Univ Iowa State Res Found Inc
• Archer-Daniels-Midland Company

Inventors

• Binder Thomas P
• Rao Aragula Gururaj
• Yamamoto Yasufumi
• Hanke Paul D

Classifications

• C07K14/00 Primary

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K17/00

  Carrier-bound or immobilised peptides (carrier-bound or immobilised enzymes C12N11/00); Preparation thereof · CPC title

• C07K7/08 Primary

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• C07K7/06 Primary

  having 5 to 11 amino acids · CPC title