Functional expression of Arabidopsis thaliana sterol glycosyltransferase from stably transformed Drosophila melanogaster S2 cells |
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| Authors: | Ha Young Chung Jeon Hwang-Bo Seong-Ki Kim Nam In Baek Youn Hyung Lee In Sik Chung and Jong-Hwa Park |
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| Institution: | (1) Department of Advanced Technology Fusion and Bio/Molecular Informatics Centers, Konkuk University, Seoul, 143-701, South Korea;(2) Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Suwon, 449-701, South Korea;(3) Department of Dental Hygiene, Yeojoo Institute of Technology, Kyonggi-do, 469-705, South Korea;(4) Department of Chemical Engineering, Kyung Hee University, Suwon, 449-701, South Korea; |
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| 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-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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