Functional Expression of Arabidopsis thaliana Sterol Glycosyltransferase from Stably Transformed Drosophila melanogaster S2 Cells

Arabidopsis thaliana sterol glycosyltransferase (SGT), UGT80A2, was expressed from stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Recombinant SGT was detected in both intracellular and extracellular fractions with a molecular mass of approximately 76 kDa. Secreted recombinant SGT accounted for approximately 60% of the total recombinant SGT production. Recombinant SGT in the extracellular fractions was purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. Radiometrical assay using uridine diphospho-D-[U-(14)C] glucose (UDP-(14)C-glucose) as a sugar donor and sterols, beta-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDP-glycosyltransferase activity and could attach (14)C-glucose to beta-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with beta-sitosterol, which was similar to the recombinant SGT produced by a bacterial expression system. The transfer of (14)C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated (14)C-glucosetransferred beta-sitosterol and stigmasterol reactants.