人金属硫蛋白基因(MT1E)在毕赤酵母中的表达及其铬抗性

Transgenic Pichia pastoris over-expressing human metallothionein gene (MT1E) on heavy metal chromium stress

金属硫蛋白(Metallothionein,MT)是一类富含半胱氨酸的蛋白质,巯基含量丰富,其半胱氨酸上的巯基对重金属离子有极强的结合力,能够结合重金属形成无毒或低毒的络合物,具有重金属解毒的功能.本文根据已公布的人金属硫蛋白基因MT1E序列,用Vector NTI 7.0软件设计合成12条寡聚核苷酸片段,并拼接得到MT1E完整基因.将测序正确的MT1E基因克隆至酵母表达载体pPIC9K,酶切验证后的阳性克隆命名为pPIC9K-MT1E,含有该重组基因的毕赤酵母工程菌命名为GS115/pPIC9K-MT1E.通过酶联免疫(Enzyme-Linked Immunosorbent Assay,ELISA)方法检测到GS115/pPIC9K-MT1E工程菌发酵液有金属硫蛋白酶活性,表明人金属硫蛋白MT1E基因能在毕赤酵母GS115菌株中正确分泌表达.铜(Cu)、锌(Zn)、锰(Mn)、铬(Cr)、钴(Co)重金属抗性分析表明,工程菌GS115/pPIC9K-MT1E对Cr(Ⅵ)的抗性较强,有解毒重金属Cr(Ⅵ)的能力.

Metallothionein(MT) is a kind of heavy metal detoxification protein which is rich in cysteine and sulfydryl.The sulfydryl group of cysteine can bind heavy metal to form low toxic or even nontoxic complex owing to its high affinity for heavy metal ions.According to the published human metallothionein gene(MT1E) sequences,12 oligomeric nucleotide fragments were synthesized and ligated in vitro,yielding a complete MT1 E gene.After verified by sequencing,the MT1 E gene was cloned into yeast expression vector pPIC9 K and the recombinant vector pPIC9K-MT1 E was obtained.The positive recombinant Pichia pastoris strain carrying the exogenous pPIC9K-MT1 E was renamed GS115 / pPIC9K-MT1 E.Metallothionein activity could be detected by employing ELISA(Enzyme Linked Immunosorbent Assay) in the fermentation supernatants of the engineering yeast cells.These results indicated that the engineering yeast cells could express and secrete the MT-1E protein.Finally,the results of heavy metal(including Cu,Zn,Mn,Cr,and Co) tolerance analysis showed that,as compared to the control strains,GS115 / pPIC9K-MT1 E strain could grow and multiply in the medium with certain Cr6+,demonstrating that MT-1E expression in engineering yeast strain increases the tolerance to environmental heavy metal Cr.