Structure-activity relationships

Patent metadata

Field	Value
Publication number	US-9228223-B2
Application number	US-201313757554-A
Country	US
Kind code	B2
Filing date	Feb 1, 2013
Priority date	Feb 12, 2007
Publication date	Jan 5, 2016
Grant date	Jan 5, 2016

Abstract

The present disclosure relates to compositions and methods for screening a plurality of polypeptide variants.

First claim

We claim:

1. A method useful for identifying at least one polypeptide having a desired activity, comprising:
(i) screening a plurality of polypeptide variants against a plurality of ligands of interest, wherein each variant reacts with at least one ligand to produce a detectable signal, and wherein at least two members of the plurality of polypeptide variants are related to a parent polypeptide and react with different ligands; and
(ii) identifying at least one variant that produces a signal upon reaction with at least one ligand of interest, thereby identifying at least one polypeptide having a desired activity.

2. The method of claim 1 , wherein the at least two members of the plurality of polypeptide variants are related to a parent polypeptide and react with differing levels of activity upon a given ligand.

3. The method of claim 1 , further comprising: (iii) identifying at least one second variant that produces a signal upon reaction with the ligand of interest; (iv) postulating a sequence-activity relationship between the amino acid residues within the identified variants and the ability of each variant to produce a detectable signal; (v) generating a second plurality of polypeptide variants based on the sequence-activity relationship; (vi) screening the second plurality against the ligand of interest; and (vii) identifying at least one variant from the second plurality that produces an enhanced signal of interest as compared to the identified variants.

4. The method of claim 3 , in which the identity of the ligand of interest is unknown.

5. The method of claim 3 , in which each variant is an enzyme and the ligand of interest is a candidate substrate for the enzyme.

6. The method of claim 3 , in which each variant is a receptor and the ligand of interest is a candidate ligand for the receptor.

7. The method of claim 3 , in which two-or more members of the plurality of polypeptide variants differ in the signal produced.

8. The method of claim 7 , in which the signal produced is selected from a signal indicating substrate specificity, chemoselectivity, regioselectivity, stereoselectivity, stereospecificity, ligand specificity, receptor agonism, receptor antagonism, conversion of a cofactor, or any combination thereof.

9. The method of claim 3 , in which two or more-members of the plurality of polypeptide variants differ in the level of signal produced.

10. The method of claim 9 , in which the difference in level is a difference in rate of product formation, percent conversion of a substrate to a product, or percent conversion of a cofactor.

11. The method of claim 1 , in which the identity of the ligand of interest is unknown.

12. The method of claim 1 , in which each variant is an enzyme and the ligand of interest is a candidate substrate for the enzyme.

13. The method of claim 1 , in which each variant is a receptor and the ligand of interest is a candidate ligand for the receptor.

14. The method of claim 1 , in which at least two or more members of the plurality of polypeptide variants differ in the signal produced.

15. The method of claim 14 , in which the signal produced is selected from a signal indicating substrate specificity, chemoselectivity, regioselectivity, stereoselectivity, stereospecificity, ligand specificity, receptor agonism, receptor antagonism, conversion of a cofactor, or any combination thereof.

16. The method of claim 1 , in which two or more members of the plurality of polypeptide variants differ in the level of signal produced.

17. The method of claim 16 , in which the difference in level is a difference in rate of product formation, percent conversion of a substrate to a product, or percent conversion of a cofactor.

Assignees

• Codexis Inc

Inventors

• Mundorff Emily
• Davis Simon Christopher
• Huisman Gjalt W
• Krebber Anke
• Grate John H
• Fox Richard

Classifications

• C12Q1/25 Primary

  involving enzymes not classifiable in groups C12Q1/26 {- C12Q1/66} · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C40B10/00

  Directed molecular evolution of macromolecules, e.g. RNA, DNA or proteins · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title