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Cloning, expression and charaterization of chalcone synthase from Saussurea medusa
水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

Keywords: Saussurea medusa,chalcone synthase,prokaryotic expression,purification,enzymatic activity assay
水母雪莲,查尔酮合酶,原核表达,纯化,酶活检测

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Abstract:

A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1 313 bp, containing an open reading frame (1 170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

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