p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响，应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变，并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体，在大肠杆菌中经ＩＰＴＧ诱导表达，Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体，并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白

Expression and Purification of Wild and P48L Mutant p16 Tumor Suppressor protein in E.cloi

To study the effect of P48L mutation on the function of p16 protein in vitro ,P48L mutant was constructed by site directed mutagenesis in vitro using two step PCR.Then wild type and mutant p16 cDNA were cloned into expression vector pGEX 5T and transformed E.coli .After 3 h induced by IPTG,the expression of recombinant 42 kD GST p16 and GST p16P48L protein were confirmed by SDS PAGE and Western blotting.Then wild type and mutant p16 GST fusion proteins were purified with single step affinity chromatography using Glutathione Sepharose 4B.The wild type and P48L mutant p16 proteins were expressed in E.coli as GST fusion protein and purified to homogeneity.