A biotin capture assay for oligosaccharyltransferase.
Oligosaccharyltransferase (OST) catalyzes the en bloc transfer of dolichylpyrophosphate oligosaccharides to an asparagine residue found in the sequon Asn-Xaa-Thr/Ser of newly synthesized proteins. Currently the method most commonly used to monitor this reaction, involving multiple solvent extractions and HPLC, is extremely time consuming and tedious. Herein, we present the use of a biotinylated peptide as the acceptor substrate and dolichylpyrophosphate [3H]chitobiose as the donor substrate for the OST-catalyzed reaction. This allows for separation (avidin-agarose beads) and quantitative analysis (scintillation counting) of only the biotinylated glycopeptide product of the OST-catalyzed reaction. This new assay yields highly reproducible results in a rapid manner.
Srinivasan
Anjali
A
Department of Medicinal Chemistry, University of Michigan, Ann Arobr 48109-1055, USA.
Coward
James K
JK
D001681:Biological Assay / Q000379:methods; D001710:Biotin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006602:Hexosyltransferases; D007700:Kinetics; D008565:Membrane Proteins; D010455:Peptides / Q000138:chemical synthesis; D012680:Sensitivity and Specificity; D014166:Transferases / Q000032:analysis*
D008565:Membrane Proteins; D010455:Peptides; D001710:Biotin; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase