Cloning, Expression And Bioactivity Identification Of BirA In E.coli;cloning, Expression And Bioactivity Identification Of BirA In E.coli

BirA was widely used as a reagent for studying reaction between proteins especially the interaction between cell surface receptors and their ligands.Objective: 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 pET32a to construct the recombinant plasmid pET32a-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in E.coli BL21 (DE3). The expressed product obtained from high-level density fermentation was purified through Ni-Chelating sepharose FF. The enzyme activity of the expressed product was identified by ELISA.Results: The recombinant prokaryotic expression vector pET32a-BirA was constructed and the fusion protein Trx-BirA with Mr being 51779 and bioactivity was expressed successfully. The density of the high-level expression strain reached to 80 g (wet cells)/L in 30L fementation tank. The expression level reached to 2.62×107 U/g (wet cells) After the expressed product was purified through Ni-Chelating sepharose FF, the purity was more than 90%. The results of ELISA showed that the expressed product could make VD111-BSP peptide complex biotinylation. The Michaelis constant Km of BirA was 19.4μM/min and maximum velocity Vmax was 0.68μM/min.Conclusion: BirA enzyme with bioactivity was successfully prepared and its bioactivity was higher than co mMercial BirA while the cost was lower, which provide an effective reagent for studying the interaction between proteins.