吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (5): 1109-1115.doi: 10.13481/j.1671-587X.20220502

• 基础研究 • 上一篇    

重叠延伸PCR技术定点突变TFEB原核表达载体的构建及体外诱导表达和纯化

焦凤娟(),刘俊杰,卢思维,姜东君   

  1. 济宁医学院精神卫生学院 山东省行为医学重点实验室,山东 济宁 272067
  • 收稿日期:2022-01-12 出版日期:2022-09-28 发布日期:2022-11-15
  • 通讯作者: 焦凤娟 E-mail:jiaofengjuan8516@163.com
  • 基金资助:
    国家自然科学基金青年基金项目(82101340)

Construction of prokaryotic expression vector of site directed mutant TFEB by SOE PCR technique and its induced expression and purification in vitro

Fengjuan JIAO(),Junjie LIU,Siwei LU,Dongjun JIANG   

  1. Shandong Key Laboratory of Behavioral Medicine,School of Mental Health,Jining Medical University,Jining 272067,China
  • Received:2022-01-12 Online:2022-09-28 Published:2022-11-15
  • Contact: Fengjuan JIAO E-mail:jiaofengjuan8516@163.com

摘要:

目的 基于重叠延伸PCR(SOE PCR)技术构建转录因子EB(TFEB)丝氨酸114位点突变体原核表达质粒,并进行体外诱导表达和纯化。 方法 根据SOE PCR技术原理设计突变引物,以pGEX-6p-1-TFEB质粒为模板,分别采用外侧引物F和R及突变引物Fn和Rn进行第1步PCR扩增,获得含突变位点的产物1和产物2。经第2步PCR退火延伸对产物1和产物2进行重叠拼接,以拼接后DNA片段为模板,采用外侧引物F和R进行第3步PCR扩增获得含突变位点的目的DNA;将其克隆至pGEX-6p-1载体,采用BamH Ⅰ和Sal Ⅰ进行酶切鉴定,DNA测序验证突变结果。于大肠杆菌(E.coli)中分别采用不同浓度异丙基-β-D-硫代半乳糖苷(IPTG)在不同条件下诱导TFEB及其突变体重组蛋白表达。Glutathione-Sepharose 4B琼脂糖凝珠分离纯化蛋白,SDS-PAGE凝胶电泳检测纯化产物蛋白浓度和相对分子质量。 结果 第1步PCR扩增获得均含有突变位点的2条DNA片段,经第2步PCR退火延伸和第3步PCR扩增后获得大量含有突变位点的完整DNA片段。双酶切鉴定,连接的DNA片段与目的片段大小一致。DNA测序显示TFEB的114位丝氨酸(TCT)成功突变为丙氨酸(GCT)。在0.5?mmol·L-1 IPTG、16?℃诱导过夜条件下获得TFEB及其突变体的可溶性蛋白表达。SDS-PAGE凝胶电泳,纯化后蛋白浓度较高,分子大小正确。 结论 利用SOE PCR技术成功实现了TFEB丝氨酸114位点的定点突变,并且TFEB及其突变体基因在E.coli中成功表达。

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 coliE. 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

中图分类号: 

  • Q78