Patent ID: 8476232

Claim:
The method of preparing lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant comprising (A) cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding a fusion protein comprising insulin-like growth factor I (IGF-I) (SEQ ID NO: 1) or an IGF-I variant N-terminally linked to the C-terminus of a propeptide, said C-terminus of said propeptide comprising the amino acid sequence Y-Pro, wherein Y is selected from the group consisting of Pro; Pro-Ala; Pro-Gly; Pro-Thr; Ala-Pro; Gly-Pro; Thr-Pro; Arg-Pro; Pro-Arg-Pro; Ala-Pro-Arg-Pro (SEQ ID NO: 12); and Pro-Ala-Pro-Arg-Pro (SEQ ID NO: 13) and said variant being a polypeptide which differs from SEQ ID NO: 1 in that one or two of the lysine residues at positions 27, 65, or 68 thereof is independently substituted by a polar amino acid selected from the group consisting of cysteine; aspartic acid; glutamic acid; histidine; asparagine; glutamine; arginine; serine; and threonine; and the remainder of the amino acid sequence of said variant is the same as that of SEQ ID NO: 1 and causing said cell to express said fusion protein; (B) recovering said fusion protein; (C) lysine-PEGylating said fusion protein; (D) cleaving said lysine-PEGylated fusion protein with IgA protease, and (E) recovering said lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant produced in step (D).