Site-specific labelling of proteins

• US 7,449,558 B2
• Filed: 10/29/2004
• Issued: 11/11/2008
• Est. Priority Date: 10/30/2003
• Status: Expired due to Fees

• Current Assignee: National University of Singapore
• Original Assignee: National University of Singapore
• Inventors: Yeo, Su-Yin Dawn, Yao, Shao Qin
• Primary Examiner(s): Lankford; L B
• Assistant Examiner(s): Kim; Taeyoon
• Application Number:
  US10/977,396
• Publication Number:
  US 20050158804A1
• Time in Patent Office: 1,474 Days
• Field of Search: 435/7.5
• US Class Current: 530/402
• 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 that is proteolytically processed to expose an N-terminal cysteine;

and washing the cell;

wherein the probe is acetic acid 4-[(benzylsulfanylcarbonylmethyl-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, and is cell-permeable.

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

3. The method of claim 1 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 3 to about 8 hours.

4. The method of claim 1 wherein the protein having an N-terminal cysteine is generated by intein-mediated protein cleavage.

5. The method of claim 1 wherein the protein having an N-terminal cysteine is generated by site-specific protelysis.

6. The method of claim 1 wherein the protein having an N-terminal cysteine is generated by de-ubiquination of a ubiquitin fusion protein.

7. A method for visualizing a protein in a cell, comprising introducing a detectable probe having a thioester group into a cell expressing a protein that is proteolytically processed to expose an N-terminal cysteine;

washing the cell; and

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

wherein the probe is acetic acid 4-[(benzylsulfanylcarbonylmethyl-carbamoyl)-methyl]-2-oxo-2H-chromen-7-yl ester;

N-enzylsulfanylcarbonylmethyl-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 is cell-permeable and wherein said conditions comprise fluorescence microscopy.

8. The method of claim 7 wherein said introducing the probe comprises adding the probe to the cell in a culture medium for about 1 to about 24 hours.

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

10. The method of claim 7 wherein the protein having an N-terminal cysteine is generated by intein-mediated protein cleavage.

11. The method of claim 7 wherein the protein having an N-terminal cysteine is generated by site-specific proteolysis.

12. The method of claim 7 wherein the protein having an N-terminal cysteine is generated by de-ubiquination of a ubiquitin fusion protein.