Site-specific labelling of proteins

• US 20050158804A1
• Filed: 10/29/2004
• Published: 07/21/2005
• Est. Priority Date: 10/30/2003
• Status: Active Grant

• Current Assignee: National University of Singapore
• Original Assignee: National University of Singapore
• Inventors: Yeo, Su-Yin Dawn, Yao, Shao Qin
• Granted Patent: US 7,449,558 B2
• Time in Patent Office: Days
• US Class Current: 435/7.500
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

An in vivo method for labelling a protein having an N-terminal cysteine is disclosed. A probe comprising a thioester group and a detectable label is introduced into a cell expressing the N-terminal cysteine protein, allowing for the N-terminal cysteine to cleave the thioester bond and resulting in the label being covalently attached to the N-terminus of the protein by an amide bond.

Claims

1. A method for labelling a protein in a cell, comprising introducing a detectable probe having a thioester group into a cell expressing a protein having an N-terminal cysteine.

2. The method of claim 1 wherein the probe is cell-permeable.

3. The method of claim 2, further comprising washing the cell.

4. The method of claim 3 wherein the probe comprises a fluorescent group, a chemiluminescent group, a radioactive group, a ligand, a photolabile fluorescent group, a reactive group, an antigen, an epitope, a paramagnetic group, or a heavy metal complex.

5. The method of claim 3 wherein the probe is acetic acid 4-[9-benzyl sulfanylcarbonylmethyl-carbamoyl)-methyl]-2-oxo-2H-chromen-7-yl ester;

N-benzylsulfanylcarbonylmethyl-6-(3,6-dihydroxy-3H-xanthen-9-yl)-isophthalamic acid;

amino thioacetic acid S-benzyl ester of 5-carboxy-tetramethylrhodamine;

amino thioacetic acid S-benzyl ester of acetoxynapthofluorescein;

[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-thioacetic acid S-benzyl ester;

(4-benzoyl-benzoylamino)-thioacetic acid S-benzyl ester;

or amino thioacetic acid S-benzyl ester of caged fluorescein.

6. The method of claim 5 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 1 to about 24 hours.

7. The method of claim 5 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 3 to about 8 hours.

8. The method of claim 5 wherein the protein having an N-terminal cysteine is generated by intein-mediated protein cleavage.

9. The method of claim 5 wherein the protein having an N-terminal cysteine is generated by site-specific proteolysis.

10. The method of claim 5 wherein the protein having an N-terminal cysteine is generated by de-ubiquination of a ubiquitin fusion protein.

11. A method for visualizing a protein in a cell, comprising introducing a detectable probe having a thioester group into a cell expressing a protein having an N-terminal cysteine;

and observing the cell under conditions in which the detectable probe may be visualized.

12. The method of claim 11 wherein the probe is cell-permeable.

13. The method of claim 12, further comprising washing the cell.

14. The method of claim 13 wherein the probe comprises a fluorescent group, a chemiluminescent group, a paramagnetic group, or a radioactive group.

15. The method of claim 13 wherein the probe is acetic acid 4-[9-benzyl sulfanylcarbonylmethyl-carbamoyl)-methyl]-2-oxo-2H-chromen-7-yl ester;

N-benzylsulfanylcarbonylmethyl-6-(3,6-dihydroxy-3H-xanthen-9-yl)-isophthalamic acid;

amino thioacetic acid S-benzyl ester of 5-carboxy-tetramethylrhodamine;

amino thioacetic acid S-benzyl ester of acetoxynapthofluorescein;

or amino thioacetic acid S-benzyl ester of caged fluorescein, and wherein said conditions comprise fluorescence microscopy.

16. The method of claim 15 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 1 to about 24 hours.

17. The method of claim 15 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 3 to about 8 hours.

18. The method of claim 15 wherein the protein having an N-terminal cysteine is generated by intein-mediated protein cleavage.

19. The method of claim 15 wherein the protein having an N-terminal cysteine is generated by site-specific proteolysis.

20. The method of claim 15 wherein the protein having an N-terminal cysteine is generated by de-ubiquination of a ubiquitin fusion protein.

21. A method for isolating a protein from a cell, comprising introducing a detectable probe having a thioester group into a cell expressing a protein having an N-terminal cysteine;

lysing the cell; and

capturing the detectable probe.

22. The method of claim 21 wherein the probe is cell-permeable.

23. The method of claim 22, further comprising washing the cell.

24. The method of claim 23 wherein the probe comprises a ligand, an antigen, an epitope, or a heavy metal complex.

25. The method of claim 23 wherein the probe is [5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-thioacetic acid S-benzyl ester and wherein said capturing comprises contacting the lysed cell with avidin or streptavidin.

26. The method of claim 25 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 1 to about 24 hours.

27. The method of claim 25 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 3 to about 8 hours.

28. The method of claim 25 wherein the protein having an N-terminal cysteine is generated by intein-mediated protein cleavage.

29. The method of claim 25 wherein the protein having an N-terminal cysteine is generated by site-specific proteolysis.

30. The method of claim 25 wherein the protein having an N-terminal cysteine is generated by de-ubiquination of a ubiquitin fusion protein.