Design of new glycoproteins via in vivo incorporation of non-natural amino acid

Abstract

Incorporation of non-canonical amino acids is fascinating subject for substantial change in the physical properties of proteins and chemical modification at multiple sites. In this study, target proteins, extracellular matrix protein (aECM) {LD-GEEIQIGHIPREDVDYHLYPG [(VPGVG)2VPGFG(VPGVG) 2]5VP}3 and mouse dihydrofolate reductase (mDHFR) in which Phe can be replaced by p-acetylphenylalanine (pAcPhe). Introduction of pAcPhe was accomplished via use of an E. Coli strain equipped with a mutant phenylalanyl-tRNA synthetase (PheRS) with altered substrate specificity (Ala294Gly, Thr251Gly). Glycosylation of the mDHFR-Ac (54% incorporated) was carried out in 8M urea solution (pH 4.5, protein 0.5mg/mL) at 37°C for 20 h, using 430 eq. of N-acetyl-D-glucosamine hydroxylamine derivative as the glycosyl donor. MALDI TOF-mass spectra revealed that mDHFR-Ac was 63% glycosylated.