关键词: 转录因子EB, 重叠延伸PCR, 定点突变, 融合蛋白, 丝氨酸, 丙氨酸

Abstract:

Objective： To construct the prokaryotic expression plasmid of transcription factor EB（TFEB） serine 114 site mutant based on gene splicing by overlap extension PCR （SOE PCR）technique， and to induce the expression and purification in vitro. Methods The mutant primers were designed according to the principle of SOE PCR technique. First，the pGEX-6p-1-TFEB plasmid was used as the template， outer primers F and R and mutation primers Fn and Rn were used for the first step PCR amplification to obtain product 1 and product 2 containing mutation sites. Secondly， overlap splicing of product 1 and product 2 was performed by annealing and extension of the second step PCR. Lastly， using the spliced DNA fragment as a template， the third step PCR amplification was performed to obtain the target DNA containing the mutation sites by using outer primers F and R. Then the target DNA fragments were cloned into the pGEX-6p-1 vector， which were identified by enzyme digestion with BamH Ⅰ and Sal Ⅰ. The mutation results were verified by DNA sequencing. The expressions of TFEB and its mutant recombinant proteins were induced in Escherichia coli（E. coli） with different concentrations of isopropyl-beta-D-thiogalactopyranoside（IPTG） and different conditions. The purified proteins were purified by Glutathione-Sepharose 4B agarose beads， and the protein concentrations and molecular weights of the purified products were detected by SDS-PAGE gel electrophoresis. Results Two DNA fragments containing mutation sites were obtained by the first step of PCR amplification， and a large number of complete DNA fragments containing mutation sites were obtained after the annealing and extension of the second step of PCR and the amplification of the third step of PCR. The double-enzyme digestion identification results showed that the size of the ligated DNA fragment was the same as that of the target fragment. The DNA sequencing results showed that the 114-position serine （TCT） of TFEB was successfully mutated to alanine （GCT）. The soluble protein expression of TFEB and its mutants were successfully obtained under the condition of 0.5 mmol·L^－1 IPTG and overnight at 16 ℃.The results of SDS-PAGE gel electrophoresis showed that the purified protein concentration was higher and the molecular size was correct. Conclusion The site-directed mutagenesis of serine 114 of TFEB is successfully achieved by SOE PCR technique， and TFEB and its mutant genes are successfully expressed in E. coli.

Key words: Transcription factor EB, Gene splicing by overlap extension PCR, Site-directed mutagenesis, Fusion protein, Serine, Alanine