Method for making acylated polypeptides

摘要

The present invention related to a method of producing polypeptides in transformed host cells by expressing a precursor molecule of the desired polypeptide which are to be acylated in a subsequent in vitro step. The invention is also related to DNA-sequences, vectors and transformed host cells for use in the claimed method. Further, the present invention is related to certain precursors of the desired polypeptides and certain acylation methods. The invention provides a method for making polypeptides being preferentially acylated in certain lysine ε-amino groups.

主权项

1. A method for making an acylated glucagon-like peptide-1 (GLP-1), wherein said GLP-1 comprises at least one lysine residue that is acylated on its ε-amino group, and wherein said GLP-1 comprises GLP-1(7-37) acylated at position Lys26, said method comprising:
(i) expressing in a yeast cell a GLP-1 precursor of said GLP-1, wherein said GLP-1 precursor comprises said GLP-1 and an N-terminal extension, wherein the N-terminal extension is up to 15 amino acids in length, said N-terminal extension being cleavable from the GLP-1 at a lysine cleavage site; (ii) acylating the ε-amino group of said at least one lysine residue in the GLP-1 without acylating the ε-amino group of the lysine cleavage site, to produce an acylated GLP-1 precursor; and (iii) removing the N-terminal extension from the acylated GLP-1 precursor by enzymatic cleavage to produce said acylated GLP-1, wherein said N-terminal extension comprises at least one histidine residue that is capable of establishing a salt bridge with the lysine cleavage site N-terminal to the GLP-1.