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

다국어 초록 (Multilingual Abstract)

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, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDPglycosyltransferase activity and could attach ^(14)C-glucose to β-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with β-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 β-sitosterol and stigmasterol reactants.

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