靶向性DNA甲基化酶在毕赤酵母中的表达和鉴定

Expression and Identification of Targeting DNA Methyltransferase in Pichia pastoris

目的:在毕赤酵母中表达融合Myc-His标签的靶向性甲基化酶B1-3a并进行鉴定。方法:以含有B1-3a基因的pcDNA4.0-myc/his质粒为模板,通过PCR扩增获得融合有myc/his标签序列的目的区段B1-3a基因,然后克隆入表达载体pPIC3.5k;电穿孔转化毕赤酵母菌株GS115,经G418筛选后进行甲醇诱导表达,并以SDS-PAGE和Western印迹对表达产物进行鉴定。结果:表达产物中可见与目的蛋白相对分子质量(50000)相符的条带,该条带可被Myc标签单克隆抗体特异识别。结论:正确构建了靶向性甲基化酶B1-3a的酵母表达载体,靶向性甲基化酶能够在毕赤酵母中成功表达。

Objective： To clone and express the targeting methyltranferase B1-3a fused with Myc-His tag in P/ch/a pastor/s expression system and to identify the protein of fused expression. Methods： The B1-3a gene was amplified from pcDNA4.0-his/myc plasmid with PCR method and cloned into the expression vector pPIC3.5k. The linearized DNA was transferred into the yeast host cell GSll5 with electroporation method. After screening with G418, the positive yeast clone was induced to express with methanol, and the expression product was analyzed with SDS-PAGE and Western blot. Restilts： A positive protein band with molecular weight about 50 kD was found in the expression product, which is accordant with interest protein, and this band could be specifically recognized by anti-Myc monoclonal antibody. Conclusion： The gene of targeting DNA methyltransferase B1-3a was cloned into the expression vector correctly and was expressed successfully in P.pastor/s expression system.