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KMID : 1094720110160040801
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Biotechnology and Bioprocess Engineering
2011 Volume.16 No. 4 p.801 ~ p.801
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Functional expression of Arabidopsis thaliana sterol glycosyltransferase from stably transformed Drosophila melanogaster S2 cells
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Chung Ha-Young
Hwangbo Jeon
Kim Seong-Ki
Baek Nam-In
Lee Youn-Hyung
Chung In-Sik
Park Jong-Hwa
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Abstract
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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-14C]glucose (UDP-14C-glucose) as a sugar donor and sterols, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDP-glycosyltransferase activity and could attach 14C-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 14C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated 14C-glucosetransferred β-sitosterol and stigmasterol reactants.
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KEYWORD
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Drosophila melanogaster S2 cells, sterol glycosyltransferase, UGT80A2, β-sitosterol, stigmasterol
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