BirA酶基因表达载体的构建、原核表达及表达产物的活性鉴定

Prokaryotic expression of BirA enzyme and identification of the bioactivity of expressed product

目的:构建生物素-蛋白连接酶(BirA酶)基因的表达载体,并在大肠杆菌BL-21(DE3)中表达具有生物学活性的BirA酶。方法:用PCR法扩增BirA酶基因。将PCR产物克隆入pGEX-4T-2中构建BirA酶-GST融合蛋白基因的重组表达载体pGEX-BirA。经测序验证后,在大肠杆菌BL-21(DE3)中诱导表达,表达产物采用谷胱苷肽-琼脂糖层析柱进行纯化。以带有生物素酶底物肽(BirAsubstratepeptide,BSP)的HLA-A2-肽复合物为底物,用ELISA和Westernblot鉴定表达产物的生物素化活性。结果:构建了pGEX-BirA原核表达质粒,并在大肠杆菌BL-21(DE3)中诱导表达Mr为61300的BirA酶-GST融合蛋白,表达产物经谷胱苷肽-琼脂糖层析柱纯化后,得到N端带有GST标签的BirA酶蛋白。ELISA和Westernblot的结果显示,表达产物能使HLA-A2-肽复合物生物素化。结论:成功地制备了具有生物学活性的Bi-rA酶,为研究蛋白质分子的相互作用提供了有效的制剂。

AIM： To construct the expression vector of biotin-protein ligase （BirA enzyme） gene and express the BirA enzyme with bioactivity in E. coli BL-21 （ DE3 ）. METHODS. The BirA gene was amplified from E. coil genome by PCR and cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E. coil BL-21（DE3）. The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot. RESULTS. The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA was expressed successfully. The results of ELISA and Western blot showed that the purified BirA enzyme biotinylated HLA-A2 peptide complex. CONCLUSION： BirA enzyme with bioactivity is prepared successfully, which can be used for studying the interaction between protein and protein,