Abstract:In the N-glycosylation pathway, the bifunctional mannosyltransferase Alg2 protein catalyzes the assembly of two mannose onto dolichol linked oligosaccharide Man1GlcNAc2-PP-Dol by α-1,3/1,6 linkages to form Man3GlcNAc2-PP-Dol. In this study, the function of human Alg2 protein (hAlg2) in S. cerevisiae cells was explored and its activity in vitro after expressed and purified from E. coli was investigated. The growth of constructed yeast strain w303a-GAL1pr-ALG2 was suppressed by glucose, while the overexpression of hALG2 gene could rescue such growth defect, confirming the functional homology of hALG2 and ScALG2. In addition, TrxA-hAlg2 was successfully purified from E.coli and its activity in vitro was further tested using Man1GlcNAc2-PP-Phy (PPGn2M1), a natural substrate analogue of hAlg2. LC-MS analysis showed that the purified TrxA-hAlg2 was capable of producing PPGn2M2 and PPGn2M3 from PPGn2M1, providing the foundation for in vitro reaction system of hAlg2 purified from E. coli.
