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High-throughput screening of cell lysates for ganglioside synthesis

TitleHigh-throughput screening of cell lysates for ganglioside synthesis
Publication TypeJournal Article
Year of Publication2008
AuthorsHancock, SM, Tarling, CA, Withers, SG
JournalANALYTICAL BIOCHEMISTRY
Volume382
Pagination48-54
Date PublishedNOV 1
ISSN0003-2697
Abstract

A high-throughput screen to detect the synthesis of natural and non-natural gangliosides by cell lysates has been developed and automated. Utilizing the binding specificity of cholera toxin B-subunit for the oligosaccharide moiety of the ganglioside G(M1), the synthesis of sugar-sphingolipid glycosidic linkages was detected using a modified enzyme-linked immunosorbent assay (ELISA)/enzyme-linked lectin assay (ELLA). The screen was optimized and validated for high-throughput screening of cell lysates by evaluating different vectors, promoters, substrates and detection strategies. The extent of ganglioside synthesis was found to be proportional to enzyme concentration and length of incubation time. As a test of the finalized screen efficacy, individual colonies from a saturation mutagenesis library of nucleophile mutants of an endoglycoceramidase were screened to identify the most active enzyme for ganglioside synthesis. This screen should find general application in assaying both glycolipid biosynthesis and glycolipid hydrolysis, as it is highly sensitive and can be used with crude cell extracts. (C) 2008 Elsevier Inc. All rights reserved.

DOI10.1016/j.ab.2008.07.023