Site-specific labelling of proteins
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.
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.