西兰花硫代葡萄糖苷酶基因BoMyr2在毕赤酵母中的表达

Functional Expression of the Myrosinase BoMyr2 from Brassica oleracea var. italica in Yeast Pichia pastoris

以西兰花种子总RNA为模板,通过RT-PCR方法扩增西兰花BoMyr2成熟蛋白基因,将目的片段连接到pMD18-T载体,测序结果显示扩增片段与目的序列一致。通过目的片段的酵母表达载体pPIC9K的构建,获得重组酵母表达载体pPIC9K-BoMyr2,经SacⅠ线性化后电转导入毕赤酵母菌株(Pichia pastoris KM71)。经甲醇诱导表达试验,SDS-PAGE结果显示,表达上清中含有大小约65 kD的蛋白条带,与预期分子质量大小相符。以sinigrin为底物测定表达上清的酶活性,结果显示,通过毕赤酵母KM71表达的培养液具有硫代葡萄糖苷酶活性。硫代葡萄糖苷酶在KM71中的异源表达,为进一步研究与应用提供理论基础。

Myrosinases are thioglncosidases that bydrolyze the natural plant products glueosinolates which are regarded as precursors of anticancer compounds in the diet. Pichia pastoris expression system was used to express recombinant myrosinase BoMyr2 from Brassica oleracea var. italica. The interested BoMyr2 gene was got by digesting the pMD-BoMyr2 vector using restriction cndonuclease, then it was inserted into the secretory pP1C9K Pichia pastoris expression vector and transformed into E.coli. Positive recombinant plasmids were selected,sequenced and named pPIC9K-BoMyr2 and it was linearized by Sac I , then the linear DNA transferred into Pichia pastoris KM71 by electroporation. The recombinant expression vector pPICgK-BoMyr2 integrated into KM71 via homologous recombination between the transforming DNA and regions of homology within the genome. The positive transformants were screened by MD His-plates and colony PCR. The recombinants were induced expression with methanol and the expression products were tested by SDS-PAGE and enzy- matically activity. The results showed that the BoMyr2 gene had been expressed successfully in Pichia pastoris and secreted in the culture medium. The expression product is a 65 kD protein and can degrade sinigrin in suitable condition. This is the first report on successful heterologous expression of a myrosinase in Pichia pastoris KM71 and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.