Covalent tethering of functional groups to proteins

Patent metadata

Field	Value
Publication number	US-11028424-B2
Application number	US-201916359581-A
Country	US
Kind code	B2
Filing date	Mar 20, 2019
Priority date	Jan 31, 2003
Publication date	Jun 8, 2021
Grant date	Jun 8, 2021

Abstract

A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.

First claim

What is claimed is:

1. A compound of formula (I): R-linker-A-X,
wherein R is selected from the group consisting of a nucleic acid molecule, an amino acid, a peptide, a receptor protein, a glycoprotein, an antibody, a lipid, a hapten, a receptor ligand, a fluorophore, a drug, a toxin, and an affinity molecule,
wherein R is not biotin,
wherein the linker is a multiatom straight or branched chain including C, N, S and/or O and comprising no more than 30 carbon atoms,
wherein A-X is a substrate for a dehalogenase,
wherein A is (CH2) n and n=2-10, and
wherein X is a halogen.

2. The compound of claim 1 , which is a substrate for a Rhodococcus dehalogenase.

3. The compound of claim 1 , wherein X is Cl or Br.

4. The compound of claim 1 , wherein the linker separates R and A by at least 11 atoms.

5. The compound of claim 1 , wherein R is separated from A-X by up to 500 angstroms.

6. The compound of claim 1 , wherein the linker comprises —C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.

7. The compound of claim 1 , wherein R is a small organic affinity molecule.

8. The compound of claim 7 , wherein the small organic affinity molecule selectively interacts with an acceptor protein.

9. A method of forming a complex comprising (a) a fusion protein; (b) a mutant dehalogenase; and (c) the compound of claim 8 , the method comprising: contacting a fusion of a protein of interest and the mutant dehalogenase with the compound of claim 8 , wherein the mutant dehalogenase comprises at least one amino acid substitution relative to the corresponding wild-type dehalogenase, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with its substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the substrate, wherein the at least one amino acid substitution in the mutant dehalogenase is a substitution (i) at an amino acid residue in the corresponding wild-type dehalogenase that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type dehalogenase and the substrate or (ii) at an amino acid residue in the corresponding wild-type dehalogenase that forms an ester intermediate with the substrate.

10. The method of claim 9 , further comprising contacting the complex with the acceptor protein for the small organic affinity molecule.

11. The method of claim 9 , further comprising allowing the complex to be contacted by with the acceptor protein for the small organic affinity molecule.

12. The method of claim 9 , wherein the fusion protein is expressed within a cell.

13. The method of claim 12 , wherein the compound is administered to the cell and enters into the cell.

14. The method of claim 1 , wherein R is a drug.

15. The compound of claim 1 , wherein R is selected from the group consisting of a nucleic acid molecule, an amino acid, a peptide, a receptor protein, a glycoprotein, an antibody, a lipid, a hapten, a receptor ligand, a fluorophore, and a toxin.

16. A compound of formula (I): R-linker-A-X, wherein R is selected from the group consisting of a nucleic acid molecule, an amino acid, a peptide, a receptor protein, a glycoprotein, an antibody, a lipid, a hapten, a receptor ligand, a fluorophore, and a toxin, wherein the linker is a multiatom straight or branched chain including C, N, S and/or O and comprising no more than 30 carbon atoms, wherein A-X is a substrate for a dehalogenase, wherein A is (CH 2 ) n and n=2-10, and wherein X is a halogen.

17. The compound of claim 16 , which is a substrate for a Rhodococcus dehalogenase.

18. The compound of claim 16 , wherein X is Cl or Br.

19. The compound of claim 16 , wherein the linker separates R and A by at least 11 atoms.

20. The compound of claim 16 , wherein the linker comprises —C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.

Assignees

• Promega Corp

Inventors

• Wood Keith V
• Los Georgyi V
• Bulleit Robert F
• Klaubert Dieter
• Mcdougall Mark
• Zimprich Chad

Classifications

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title

• C07C217/08

  the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom · CPC title

• C07F5/022

  without C-boron linkages · CPC title