Expression,Purification,and Thermal Stability Analysis of the Rhizobial AcrR Protein
In this study,we successfully accomplished the cloning,expression,and purification of the Rhizobia AcrR protein,accompanied by characterization of its thermal stability properties.The AcrR gene was inserted into the expression vector pHAT2 utilizing the Sticky-End methodology to generate the recombinant plasmid pHAT2-AcrR.The recombinant plasmid was subsequently transformed into Escherichia coli BL21(DE3)competent cells via heat shock transformation,the recombinant protein was expressed induced by isopropyl β-D-1-thiogalactopyrano-side(IPTG).High-purity AcrR protein was obtained through a combination of affinity chromatography,ion ex-change chromatography,and gel filtration chromatography,with SDS-PAGE analysis revealing a molecular mass of approximately 23.56 kDa.Bioinformatic analysis predicted that the AcrR gene fragment consists of 606 base pairs encoding 201 amino acid residues,with a predicted isoelectric point(pI)of 5.74,total atomic mass of 3 168,and molecular formula of C993H1577N285O300S13.The protein lacks transmembrane domains and is classified as a hydrophil-ic unstable protein.Further thermal shift assay(TSA)revealed enhanced thermal stability of the AcrR protein in buffer containing 50 mmol/L Citric acid and 500 mmol/L NaCl at pH 5.0.These findings provide experimental data for understanding the biological characteristics of AcrR protein and subsequent crystal structure analysis and func-tional research.
万方数据
维普
