Recombinant Ganglioside GM2 Synthase—Expression in Insect Cells and Enzyme Assay

Abstract

Publisher Summary This chapter investigates the recombinant ganglioside GM2 synthase; and describes the expression of murine β-1,4-N-acetylgalactosaminyltransferase both as a complete membrane-bound enzyme and as a soluble form in the baculovirus insect -cell expression system. A novel assay based on radiochemically labeled GM3 prepared from the tissue ganglioside, which permits the evaluation of potential UDP-GalNAc analogs is presented. Schematic protein structure of GM2 synthase showing the topology of type II transmembrane proteins is presented. For the expression of full-length GM2 synthase, the entire cDNA of GM2 synthase in the vector pCMV Blue is used as a template in the polymerase chain reaction (PCR) reaction. For expression of soluble GM2 synthase, the medium of infected cells is harvested 72 h postinfection by centrifugation (2000 rpm, 10 min) and concentrated 15-fold with a Vivaspin 4-ml concentrator with a PES membrane and 30,000 moleculor weight cut off (MWCO). This concentrated supernatant is prepared fresh for the assays and is always kept on ice. The chapter discusses the immunoblot analysis, standard glycosyltransferase assays, and kinetic analysis of recombinant GM2 synthase.