PubMed:16143056 JSONTXT 5 Projects

[Prokaryotic expression of BirA enzyme and identification of the bioactivity of expressed product]. 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.coli 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.coli 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.