Methods for chemically synthesizing immunoglobulin chimeric proteins
View Patent ↗The invention provides methods of chemically synthesizing chimeric proteins comprising at least a portion of an immunoglobulin constant region and a biologically active molecule.
1. A method of synthesizing a chimeric monomer-dimer hybrid comprising
a) recombinantly expressing a fusion protein comprising a portion of an immunoglobulin containing an Fc neonatal receptor (FcRn) binding site and a splicing protein capable of forming a thioester intermediate on the C terminus of the portion containing the FcRn binding site;
b) adding a thiol cofactor to the fusion protein of a);
c) adding at least one biologically active molecule having an N terminal cysteine, thereby linking the biologically active molecule with the C terminus of the portion containing the FcRn binding site; and
d) adding a second polypeptide containing an FcRn binding site, thereby forming a chimeric monomer-dimer hybrid;
wherein the biologically active molecular is a polypeptide or a nucleic acid molecule.
2. The method of claim 1 , wherein the splicing protein is intein.
3. The method of claim 1 , wherein the thiol cofactor is 2-mercaptoethanesulfonic acid.
4. The method of claim 1 , wherein the monomer-dimer hybrid is a dimerically linked monomer-dimer hybrid.
5. The method of claim 1 , wherein the biologically active molecule is a polypeptide.
6. The method of claim 1 , wherein the biologically active molecule is a nucleic acid molecule.
7. The method of claim 1 , wherein both the biologically active molecule and the fusion protein are produced recombinantly.
8. The method of claim 1 , wherein at least one of the biologically active molecule and the fusion protein is produced by chemical synthesis.
9. The method of claim 1 , wherein the chimeric protein further comprises a linker.
10. The method of claim 1 , wherein the reaction is performed in solution.
11. The method of claim 1 , wherein the reaction is performed on a solid support.
12. The method of claim 11 , wherein the solid support is a chitin bead.
13. The method of claim 1 , wherein the biologically active molecule is selected from the group consisting of erythropoietin (EPO), interferon, a viral fusion inhibitor, and a clotting factor.
14. The method of claim 1 , wherein the biologically active molecule is EPO.
15. The method of claim 1 , wherein the biologically active molecule is interferon α.
16. The method of claim 1 , wherein the biologically active molecule is interferon β.