Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5′ to 3′ direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with an amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.
