Probe incorporation mediated by enzymes
The invention provides compositions and methods of use thereof for labeling peptide and proteins in vitro or in vivo. The methods described herein employ lipoic acid ligase or mutants thereof, and lipoic acid analogs recognized by lipoic acid ligase and lipoic acid ligase mutants.
1. A method for preparing a protein conjugate, the method comprising: contacting a fusion protein with a lipoic acid analog in the presence of a lipoic acid ligase polypeptide to produce a protein conjugate in which the lipoic acid analog is linked to the fusion protein, wherein:
the lipoic acid analog is a substrate of the lipoic acid ligase polypeptide and comprises either a functional group handle or a directly detectable moiety,
the fusion protein comprises the target protein and an acceptor polypeptide,
the acceptor polypeptide consists of the amino acid sequence Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Xaa 6 Xaa 7 Xaa 8 Xaa 9 Lys Xaa 10 Xaa 11 Xaa 12 Xaa 13 Xaa 14 or a fragment thereof having at least 12 amino acid residues, each of Xaa 3 , Xaa 6 and Xaa 14 , independently, being a hydrophobic amino acid residue, each of Xaa 1 , Xaa 2 , Xaa 4 , Xaa 5 , Xaa 8 , Xaa 10 , Xaa 11 , and Xaa 12 , independently, being any amino acid residue, and each of Xaa 7 , Xaa 9 , and Xaa 13 , independently, being either E or D; and
the lipoic acid ligase polypeptide comprises the amino acid sequence of a wild-type lipoic acid ligase, or is a functional mutant of a wild-type lipoic acid ligase, which has an amino acid sequence at least 85% identical to SEQ ID NO:11, has lipoic acid ligase activity, and comprises an amino acid residue substitution at the position corresponding to W37 in SEQ ID NO:11.
2. The method of claim 1 , wherein the lipoic acid ligase polypeptide comprises an amino acid sequence of a wild-type lipoic acid ligase.
3. The method of claim 1 , wherein the lipoic acid analog comprises a functional group handle.
4. The method of claim 3 , wherein the functional group handle is an azide moiety, an alkyne moiety, or an alkyl halide moiety.
5. The method of claim 4 , further comprising contacting the protein conjugate with a detectable label to allow linkage of the detectable label to the lipoic acid analog in the protein conjugate via reaction with the functional group handle.
6. The method of claim 5 , wherein the detectable label comprises a cyclooctyne moiety and a moiety selected from the group consisting of a fluorescent moiety, a chemiluminescent moiety, a phosphorescent moiety, or a radioisotope.
7. The method of claim 1 , wherein the lipoic acid analog comprises a directly detectable moiety.
8. The method of claim 7 , wherein the directly detectable moiety is an aryl azide moiety, a diazirine moiety, a benzophenone moiety, a chloroalkane moiety, or a fluorophore moiety.
9. The method of claim 8 , wherein the fluorophore moiety is coumarin, resofurin, or an xanthene-type fluorophore.
10. The method of claim 1 , wherein Xaa 7 and Xaa 9 are E and D, respectively.
11. The method of claim 10 , wherein the lipoic acid analog comprises a functional group handle.
12. The method of claim 11 , wherein the functional group handle is an azide moiety, an alkyne moiety, or an alkyl halide moiety.
13. The method of claim 12 , further comprising contacting the protein conjugate with a detectable label to allow linkage of the detectable label to the lipoic acid analog in the protein conjugate via reaction with the functional group handle.
14. The method of claim 13 , wherein the detectable label comprises a cyclooctyne moiety and a moiety selected from the group consisting of a fluorescent moiety, a chemiluminescent moiety, a phosphorescent moiety, or a radioisotope.
15. The method of claim 10 , wherein the lipoic acid analog comprises a directly detectable moiety.
16. The method of claim 15 , wherein the directly detectable moiety is an aryl azide moiety, a diazirine moiety, a benzophenone moiety, a chloroalkane moiety, or a fluorophore moiety.
17. The method of claim 16 , wherein the fluorophore moiety is coumarin, resofurin, or an xanthene-type fluorophore.
18. The method of claim 1 , wherein the mutant comprises an amino acid sequence at least 85% identical to SEQ ID NO:11, has lipoic acid ligase activity, and comprises an amino acid residue substitution at the position corresponding to W37 in SEQ ID NO:11.
19. The method of claim 18 , wherein the mutant comprises an amino acid sequence at least 90% identical to SEQ ID NO:11.