A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries

Analytical Biochemistry
Mattias Persson, Monica M Palcic

Abstract

Protein engineering using directed evolution or saturation mutagenesis at hot spots is often used to improve enzyme properties such as their substrate selectivity or stability. This requires access to robust high-throughput assays to facilitate the analysis of enzyme libraries. However, relatively few studies on directed evolution or saturation mutagenesis of glycosyltransferases have been reported in part due to a lack of suitable screening methods. In the present study we report a general screening assay for glycosyltransferases that has been developed using the blood group alpha-(1-->3)-galactosyltransferase (GTB) as a model. GTB utilizes UDP-Gal as a donor substrate and alpha-L-Fucp-(1-->2)-beta-D-Galp-O-R (H antigen) as an acceptor substrate and synthesizes the blood group B antigen alpha-D-Galp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-R. A closely related alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) uses UDP-GalNAc as a donor with the same H acceptor, yielding the A antigen alpha-D-Galp-NAc-(1-->3)-[alpha-L-Fuc(1-->2)]-beta-D-Gal-O-R. GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. The screening assay is based on the col...Continue Reading

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Citations

Jul 22, 2009·Nature Chemical Biology·Shelley D Copley
Nov 18, 2010·Glycobiology·Zhengliang L WuWeiping Jiang
Aug 3, 2010·Biochemistry. Biokhimii︠a︡·Rui ShenPeng Wang
Sep 18, 2013·Applied Biochemistry and Biotechnology·Wei LiZhiqun Liang
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Jan 7, 2016·Applied Biochemistry and Biotechnology·Yan LiPingkai Ouyang
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Aug 25, 2015·Analytical Biochemistry·Patricia BubnerBernd Nidetzky
Oct 17, 2008·Angewandte Chemie·Richard W GanttJon S Thorson
Dec 6, 2008·Angewandte Chemie·Christopher J ThibodeauxHung-wen Liu
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Jan 31, 2021·Biotechnology Advances·Pei XuWen-Yong Lou
Jul 22, 2021·Chembiochem : a European Journal of Chemical Biology·Nazim OcalLaurence Hecquet

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