The Expression,Purification And Characterization Of CpxR In Pectobacterium Carotovorum Subsp.Carotovorum

The Cpx signal transduction system is one prevalent two-component systems in gram-negative bacteria consisted of the membrane integrated sensor histidine kinase CpxA and the cytoplasmic response regulator CpxR..The Cpx is involved in the expression of virulence genes in E.Coli.However,the functions of Cpx in Pectobacterium carotovorum are still unknown.This study aims to construct a expression vector of CpxR protein in vitro,obtain high purity protein,and explore the conditions of crystal growth.In this work,cpxR gene was amplified from Pectobacterium carotovorum subsp.carotovorum(PC1)and cloned into the pET-15 b vector.The results showed that cpxR contained 699 bp of a complete ORF encoding a protein(232 amino acids,26.4 kDa,pI5.23).The cpxR gene and its protein structure were analyzed and predicted by bioinformatics technology.The results indicated that the CpxR protein was a kind of non-secretory cytoplasmic protein,included two domains(REC and HTH)and no signal peptide.The secondary structure was mainly composed of α-helix,β-sheet,β-turn,and randon coil.In addition,the CpxR protein was a functional part in signal transduction and drug resistance for KEGG analysis.The expression of CpxR in vitro was essential for research the relationship between structure and biological function,so the CpxR was expressed in E.coli BL21(DE3)cells and induced by IPTG.The results showed the optimal concentration of IPTG was 0.5mmol/L.Then,the CpxR protein was purified by Nickel affinity chromatography and FPLC.The purified protein was digested by thrombin and analysed by Circular dichroism,which could be crystallized.The results of Western blot demonstrated that 0.14 μg fusion protein could be totally digested by 0.3 U thrombin at 20°C for 16 h.The results of Circular dichroism suggested that the secondary structure of CpxR was α-helix 52.6%,β-sheet 8.6%,β-turn 15.6%,and unordered 22.4%.The protein stability indicated the CpxR had the favourable thermal stability and was suitable for crystallization.Micro-crystals were obtained in the crystallizing tank with Magnesium sulfate hydrate and Tris.This study established a complete protocal of expression and purification of CpxR in vitro,predicted the protein secondary structure and screened out a micro-crystal growth condition,which provide a basis for further study.