Sortase-mediated protein purification and ligation
View Patent ↗The invention relates to a sortase-mediated protein purification and ligation. Specifically, the invention relates to a technique that links protein expression/purification with conjugation to therapeutic agents, imaging agents, or linkers.
1. A conjugation method comprising:
cloning a nucleic acid encoding a polypeptide, the nucleic acid comprises in series from 5′ to 3′ a coding sequence of a ligand with a coding sequence of a sortase recognition sequence, a coding sequence of a flexible linker, a coding sequence of sortase A, and a coding sequence of an affinity tag;
expressing and purifying the polypeptide encoded by the nucleic acid; and
adding calcium and a peptide or protein with an N-terminal glycine,
wherein the addition of said calcium and said peptide or protein with an N-terminal glycine allows the sortase to catalyze ligand release and conjugation of the released ligand to said peptide or protein with an N-terminal glycine.
2. The method of claim 1 , wherein said sortase recognition sequence comprises LPXTG.
3. The method of claim 1 , wherein said affinity tag is a histidine tag.
4. The method of claim 1 , wherein said peptide or protein with an N-terminal glycine comprises a functional group.
5. The method of claim 1 , wherein said peptide or protein with an N-terminal glycine is linked to a drug molecule, an imaging agent, a click chemistry group, an alkyne, an azide, a hapten, a biotin, a protein, a small molecule, or a nanoparticle.
6. The method of claim 1 , said peptide or protein with an N-terminal glycine comprises a plurality of N-terminal glycines.
7. A method for purifying a protein, the method comprising: the conjugation method of claim 1 .
8. A method for purifying a protein, the method comprising:
cloning a nucleic acid encoding a polypeptide, the nucleic acid comprises in series from 5′ to 3′ a coding sequence of a ligand protein with a coding sequence of sortase recognition sequence, a coding sequence of a flexible linker, a coding sequence of sortase A, and a coding sequence of an affinity tag;
expressing and purifying the polypeptide encoded by the nucleic acid; and
adding calcium and a peptide or protein with an N-terminal glycine, wherein the addition of said calcium and said peptide or protein with an N-terminal glycine allows the sortase to catalyze ligand release and conjugation of the released ligand to said peptide or protein with an N-terminal glycine.
9. The method of claim 1 , wherein said flexible linker comprises a glycine-serine (GS)-rich linker.
10. The method of claim 9 , wherein said flexible linker comprises a (GGS) n linker, where n is an integer indicating the number of (GGS) repeats.
11. The method of claim 10 , wherein n is greater than or equal to 3.
12. The method of claim 11 , wherein n is 5.
13. The method of claim 8 , wherein said flexible linker comprises a glycine-serine (GS)-rich linker.
14. The method of claim 13 , wherein said flexible linker comprises a (GGS) n linker, where n is an integer indicating the number of (GGS) repeats.
15. The method of claim 14 , wherein n is greater than or equal to 3.
16. The method of claim 15 , wherein n is 5.