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目的构建葡萄糖调节蛋白75(GRP75)不同结构域、不同形式突变体与线粒体信号肽重组蛋白并鉴定其亚细胞定位。方法通过重叠延伸PCR,将线粒体靶向信号肽序列分别与GRP75不同结构域基因拼接。利用定点突变PCR分别扩增实现62和65位氨基酸突变的磷酸化失活型、磷酸化激活型突变体、482位氨基酸突变的底物结合缺陷型突变体及三位点同时突变重组体。将上述重组基因克隆入p EGFPC1真核表达质粒,酶切和测序验证后分别用脂质体转染He La细胞,Western blot法检测重组蛋白表达水平,激光共聚焦显微镜观察比较重组蛋白亚细胞定位情况。结果成功构建了线粒体信号肽融合或缺失的GRP75结构域、磷酸化失活和激活突变体、底物结合缺陷突变体真核表达质粒。各重组质粒的片段插入、位点突变、信号肽融合情况均符合设计目的,在He La细胞表达后产物的相对分子质量大小均符合预期。EGFP蛋白C端插入信号肽能引导下游融合的GRP75全长蛋白、结构域片段、突变体蛋白表达定位于He La细胞线粒体中,而缺失信号肽的对应重组蛋白则主要分布于胞质和局部核周。结论构建了一系列GRP75重组真核表达质粒,EGFP融合信号肽能引导重组蛋白在线粒体中表达定位。
Objective To construct different domains of glucose regulatory protein 75 (GRP75), different forms of mutants and mitochondrial signal peptide recombinant proteins and identify their subcellular localization. Methods By overlap extension PCR, the mitochondrial targeting signal peptide sequences were spliced with different GRP75 domain genes, respectively. The site-directed mutagenesis PCR was used to amplify the phosphorylation-inactivating and phosphorylation-activating mutants with amino acid mutations at positions 62 and 65, the substrate binding defect mutant with amino acid mutation at position 482, and the simultaneous mutagenesis of the three loci. The recombinant plasmids were cloned into the eukaryotic expression vector p EGFPC1. The recombinant plasmids were transfected into HeLa cells by lipofectamine. The expression of recombinant protein was detected by Western blot. The expression of recombinant protein was detected by confocal laser scanning microscopy Happening. Results Mitochondrial signal peptide fusion or deletion of GRP75 domain, phosphorylation inactivation and activation mutant, substrate binding defect mutant eukaryotic expression plasmid were successfully constructed. The insertion of each recombinant plasmid, site mutation, signal peptide fusion are in line with the design purpose, the relative molecular mass of the product after He La cells expressed are in line with expectations. The signal peptide inserted into the C terminus of EGFP protein can guide the downstream fusion GRP75 full-length protein, domain fragment, mutant protein expression located in He La cell mitochondria, and the corresponding recombinant protein lacking signal peptide is mainly distributed in the cytoplasm and the local nucleus week. Conclusion A series of recombinant eukaryotic expression plasmids for GRP75 were constructed. EGFP fusion signal peptide can direct the expression of recombinant protein in mitochondria.