摘要
从水母雪莲Saussurea medusa Maxim.cDNA文库中得到一段查尔酮合酶基因(SmCHS)片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。
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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2011年第9期1363-1370,共8页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.30873452)资助~~
关键词
水母雪莲
查尔酮合酶
原核表达
纯化
酶活检测
Saussurea medusa, chalcone synthase, prokaryotic expression, purification, enzymatic activity assay
