序列特异的三锌指多肽的构建及其在大肠杆菌中的表达

Constructing and Expression of Three Zinc-fingers Peptide with Specific DNA Recognition Property in Escherichia coli

在获得单一锌指突变体的基础上 ,以小鼠转录因子Zif2 6 8的三锌指DNA结合区为模板 ,利用重叠 (Over lap)PCR技术 ,获得了关键氨基酸位点同时突变的三锌指突变体ZF12 3、2ZF12 3。ZF12 3、2ZF12 3分别克隆进pUC 18质粒 ,序列测定正确后 ,以pGEX 2T为表达质粒 ,在大肠杆菌JM10 9中实现了功能性的表达。经SDS PAGE分析 ,表达出了分子量 34 0kD的融合蛋白 ,扫描分析其含量在 2 0 %左右。菌体经超声波破碎后 ,对可溶性融合蛋白进行了纯化得到了游离的目的蛋白 ,为进一步的DNA结合特性分析、杂交转录因子的构建等奠定了基础。

For investigating the DNA binding property of classical zinc finger protein Zif268,an \%in vivo\% transcription interference experiment was once utilized to develop a genetic selection assay.By screening a library in which the key amino acids of the third zinc finger from Zif268 were randomized,some single fingers with new binding specificity were obtained.In this sutdy,by combining the single fingers,two three-finger peptides cDNA ZF123 and 2ZF123 were constructed by an over-lap PCR technique using the DNA binding domain of Zif268 as the template.After three times PCR,the products were inserted into pUC18 for cloning.The ZF123 and 2ZF123 cDNA were also inserted into pGEX-2T for expression in \%Escherichia coli\% after sequencing confirmation.The result showed that the three-finger peptides were expressed at a high level in \%E.coli\% JM109.The fusion protein GST-ZF123/2ZF123 have the relative molecular weight of 34 0kD and consisted about 20% of the total soluble cell protein as detected by SDS-PAGE.After supersonic treatment,the soluble part of the bacterial extract was purified.After two additional thrombin cleavage and Sepharose 4B affinity purification steps,the free three-fingers peptide proteins were also obtained.The construction and obtaining of the three-fingers peptide cDNA and its products will facilitate the \%in vivo\% and \%in vitro\% DNA binding specificity study and the design of the hybrid transcription factors.