modifications that will be recognized by the skilled person in the present invention the lack of cognate trna to amber stop codon results in early termination at the amber codon in the mrna of amber codon containing dna however when amber codon containing dna was transcribed and then translated in a cell free protein synthesis system supplemented with cognate suppressor trna full length polypeptide or protein could be synthesized noren c j et al science 244 182 188 1989 0029 fig1 shows the schematic process of preparing the mutein of epo with unnatural amino acid site specifically incorporated according to cell free protein synthesis the codon of residue of interest in wild type epo asparagine was mutated by site directed mutagenesis to amber stop codon this mutein coding plasmid was translated in a cell free protein synthesis system supplemented with amber suppressor trna charged with unnatural amino acid containing protected functional group t7 rna polymerase mediated transcription of this plasmid followed by translation and suppression by suppressor trna resulted in the mutein of full length epo with unnatural amino acid at desired position immuno purified mutein of epo was subsequently deprotected to expose free sulfhydryl group at the desired position of epo and pegylated as described as follow specifically peg modified mutein of epo at 38 th residue was produced as briefly described below cdna of wild type epo was cloned into a prokaryotic expression vector pk7 kim d m et al.