Title
Improving activity of an N-glycosyltransferase using a medium throughput HPLC assay
Conference Dates
September 24-28, 2017
Abstract
The targeted synthesis of glycoproteins with defined structure remains a major hurdle to our understanding (and exploitation) of their biological activities. The recently discovered class of enzymes, cytoplasmic N-glycosyltransferases (NGTs), represent a powerful new tool in our glycoengineering toolbox. These enzymes transfer a single glucose monosaccharide onto the asparagine residue of proteins in the sequon Asn-X-Ser/Thr (where X is any amino acid except proline). The ability of NGTs to catalyze site-specific glycosylation of recombinant proteins in E. coli has been exploited for the targeted biosynthesis of novel glycoprotein structures. To improve NGT activity, we have developed a medium throughput HPLC assay using fluorescently labelled peptides as a proxy for the protein substrate. We have screened partial-saturation mutagenesis libraries at over 50 sites in the protein, focusing on flexible regions distal to the active site. Subsequent combination of the beneficial mutations yielded mostly additive effects on enzymatic activity. We present a heavily mutated protein with several-fold increased activity compared to the wild type.
Recommended Citation
Timothy G. Keys, "Improving activity of an N-glycosyltransferase using a medium throughput HPLC assay" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/enzyme_xxiv/12