何首乌中Ⅲ型聚酮合酶基因FmPKS2的原核表达及鉴定  

The Prokaryotic Expression and Identification of FmPKS2 Gene of Fallopia Multiflora

作　　者：陆娣[1,2] 赵炜[1] 夏晚霞[1] 赵树进[1] 

机构地区：[1]广州军区广州总医院药学部,广东广州510010 [2]南方医科大学,广东广州510515

出　　处：《现代生物医学进展》2013年第2期216-220,共5页Progress in Modern Biomedicine

基　　金：广东省自然科学基金项目(10151001002000012)

摘　　要：目的:对传统中药何首乌中Ⅲ型聚酮合酶基因FmPKS2进行原核表达并鉴定重组蛋白酶活性,为研究该酶基因在何首乌有效成分代谢合成及其调控中的功能奠定基础。方法:根据何首乌FmPKS2(GenBank登录号:GQ984139)基因序列,通过PCR扩增其全长的编码区,克隆至原核表达载体PET-28a,构建重组质粒PET-FmPKS2,将其转化原核表达菌株E.coli BL21(DE3),并用IPTG诱导表达,SDS-PAGE鉴定融合蛋白可溶性,通过Ni-NTA亲和树脂纯化可溶性蛋白后以丙二酰-COA和香豆酰-COA为底物进行催化反应,TLC鉴定反应产物。结果:经过诱导,重组菌表达分子量为42KD左右的融合蛋白,其中可溶性重组蛋白最优表达条件为IPTG浓度0.5 mmol/L,诱导时间6h,温度25℃。纯化后的可溶性重组蛋白催化丙二酰-COA和香豆酰-COA得到的产物经TLC鉴定为二苯乙烯类化合物白藜芦醇。结论:成功实现FmPKS2的原核表达且融合蛋白以可溶形式存在,催化反应证明FmPKS2融合蛋白具有二苯乙烯合酶的活性。Objective： To provide information for further research on the functions of FmPKS2 in the biosynthesis pathway and metabolic regulation of major bioactive principles of Fdlopia multi.flora. Methods： According to the sequences of FmPKS2 （GenBank accession number： GQ984139） gene available in GenBank, the full length of FmPKS2 coding region was amplified by PCR. The amplified products was ligated to prokaryotic vector PET-28a and the recombinant expression plasmid named pET-FmPKS2 was constructed. The recombinant plasmid was transformed into prokaryotic expression cells E.coli BL21（DE3） and the fusion expression was induced by IPTG. The solubility of the fusion protein was identified by SDS-PAGE and soluble protein was purified with Ni-NTA chelating sepharose affinity chromatography. The soluble protein was used to catalyze malonyl-CoA and coumaroyl-CoA, of which catalysate was identified by TLC. Results： The molecular weight of the fusion protein was about 42 kD and FmPKS2 was expressed in the form of soluble proteins. The best expression conditions of soluble protein： inductive time 6h, IPTG concentration 0.5 mmol/L, temperature 25℃. The TLC showed the soluble FmPKS2 proteins after purified catalyzed malonyl-CoA and coumaroyl-CoA to form resveratrol. Conclusion： The FmPKS2 Gene of Fallopia multiflora successfully expressed in the form of soluble proteins in the prokaryotic system. The purified soluble FmPKS2 protein showed the same bioactivity with natural Stilbene synthase.

关 键 词：FmPKS2 原核表达 融合蛋白 活性鉴定 

分 类 号：Q516[生物学—生物化学] Q78