食管癌相关基因1在甲醇酵母中的表达与纯化  

Expression and Purification of Esophageal Cancer Related Gene(ECRG1) in Pichia Pastoris

作　　者：赵念玺 郭黎萍[1] 陆士新[1] 

机构地区：[1]中国医学科学院,中国协和医科大学肿瘤研究所癌变与病因研究室,北京市100021

出　　处：《医学分子生物学杂志》2005年第1期5-9,共5页Journal of Medical Molecular Biology

基　　金：国家高技术研究发展计划(863计划)(No.2002CB5130101);英国科协资助(No.142291)~~

摘　　要：目的为获得食管癌相关基因1(ECRG1)编码的活性蛋白,利用甲醇酵母系统表达ECRG1。方法将本研究室构建的PCDNA6-HA-ECRG1重组到分泌表达载体pPIC9k中,然后转入酵母GS115,通过G418筛选,在甲醇的诱导下,获得表达的蛋白。结果成功构建了分泌表达重组体pPIC9k-ECRG1,并转入了GS115,筛到抗2.5mg/ml G418的重组菌株,在甲醇的诱导下,ECRG1蛋白得到了分泌表达,发酵培养获得的融合蛋白经镍离子亲和层析,得到纯度80%左右的蛋白。结论用酵母表达系统得到了活性蛋白,可用于进一步的生物功能及作用机理的研究,也为今后研究基因-蛋白的功能与结构,提供了分离和纯化蛋白的重要方法。Objective To obtain active protein for the further study of the function of genes, the Pichia pastoris expression system was employed to express the protein encoded by ECRG1 gene. Methods cDNA of ECRG1 was subcloned into _PPIC9k from _pCDNA6-HA-ECRGl. Verified recombinant was then transformed into Pichia pastoris GS115. His^+ yeast was recombined with multicopy inserts, and was screened by resistance to G418. The clone with ECRG1 secreted target protein when it was induced by methanol. Results Insert of recombinant expression vector was exactly sequenced to the sequence encoding ECRG1 protein. The recombinant strain could resist 2. 5 mg/ml G418, showing that it included multi-copy target genes. SDS-PAGE indicated that the molecular weight of the induced protein was about 43 kD, which was consistent with the expected result. Western blot also showed that the induced protein was target protein. Furthermore, we obtained some proteins by fermentation under optimal growth condition Conclusion The active protein encoded by ECRG1 could express in Pichia pastrois, which provides an important means for further study of the function and structure of proteins.

关 键 词：甲醇酵母 食管癌相关基因1 分泌表达 

分 类 号：Q786[生物学—分子生物学]