PEGylation of cytochrome c at the level of lysine residues mediated by a microbial transglutaminase

Objectives To establish a method for microbial trans glutaminase (mTG)-mediated PEGylation of proteins at the level of lysine (Lys) residues. Results Carboxybenzyl-glutaminyl–glycinyl-methoxypolyethylene glycol (CBZ-QG-mPEG) was prepared by introducing carboxybenzyl-glutaminyl-glycine (CBZ-QG) to mPEG amine. The analysis by Fourier transform infrared spectroscopy and SDS-PAGE showed that CBZ-QG-mPEG was successfully synthesized and can be recognized by mTG as an acyl donor to modify therapeutic protein, cytochrome c (cyt c ). Finally, under an optimized condition (cyt c 0.5 mg/ml, CBZ-QG-mPEG 11.25 mg/ml, mTG 0.5 mg/ml, 37 °C, 2 h), the PEGylation yield reached 76.5 %. Conclusions This is the first study regarding the PEGylation of protein at the level of Lys residues catalyzed by mTG. The novel method could be employed to immobilize active proteins and modify therapeutic proteins.