大肠杆菌二氢叶酸还原酶基因folA的克隆、表达纯化及分子间交联

Cloning,expression,purification and protein cross-linking of Escherichia coli dihydro folate reductase

利用PCR技术从大肠杆菌DH5α中获取二氢叶酸还原酶(DHFR)基因folA。用限制性内切酶BamHI与PstI将该片段插入到克隆载体pUC18上,DNA测序鉴定目的基因。而后再将该基因亚克隆到表达载体pTrcHisC上,IPTG诱导表达重组蛋白。在非变性条件下,用TALON金属亲和层析树脂纯化含组氨酸标记的重组DHFR。纯化产物在热诱导条件下行SDS-PAGE分析,除23000大小的单体外,还出现了交联的二聚体和多聚体;而当反应体系中含有还原剂β-巯基乙醇时,二聚体和多聚体都被减弱。推断蛋白质在热诱导条件下二级结构发生改变而产生交联,并且有二硫键的参与。

By using the PCR technique,the0.49kb DNA fragment of dihydrofolate reductase(DHFR)gene(folA)was ampli-fied from the Escherichia coli DH5αcell lysate.The fragment was inserted into plasmid pUC18by the sites of two re-striction enzyme Bam HI and PstI,and the target gene was confirmed by DNA sequencing.Then the target gene was sub-cloned into the expression plasmid pTrcHisC.The recombinant protein expression was induced by IPTG.The recombinant DHFR protein containing his-tag was purified by using TALON metal affinity resin under nondenaturing conditions.Under the thermal unfolding conditions,dimer /polymer bands in addition to the major23kD band were observed on the SDS-PAGE.In contrast,the dimer /polymer was attenuated when reaction solution contained reducerβ-mercaptoethanol.It sug-gested that protein cross-linking involved in a change in protein conformation and the formation of interchain disulfide bonds.