Construction, Expression, And Purification Of Colicin Ia Channel Domain-CSP Fusion Protein And Its Antibacterial Activity Evaluation

Dental plaque biofilm, a well-organized microbial community located on tooth or dental prosthetic restoration surface, has not been able to be rinsed out by water. The biofilm formation may increase the bacterium resistance to host defense and decrease the effectiveness of the antibiotics . It can also act as an appropriate microenvironment in which the bacteria can play their pathogenesis roles. S.mutans, one of the most important pathogens in the development of dental caries, can also reside in the biofilm and, according to the change in the surrounding environment, regulate the function of itself through Quorum Sensing.In S.mutans, the induction of genetic competence is regulated by a competence stimulating peptide(CSP)-mediated quorum-sensing system. This system may be involved in the regulation of S.mutans biofilm formation, glucose metabolism, antiacid capacity, and the production of virulence factors, thus, contributing significantly to the survival of S.mutans in the complex environment of oral cavity. Colicin Ia is a bacteriocin produced by E. coli that can act on the lipid bilayer of cell membranes. It kills target bacterium by forming a voltage-activated channel in the cell membrane, mediated by its C-terminal channel-forming domain.The aim of this study was to obtain colicin Ia channel domain-CSP fusion protein by gene engineering and purification, and evaluate its antibiotic activity, so as to develop a new way for the control of tooth decay.Our research is composed of three parts:1. Construction of the expression factor containing fused Colicin Ia channel domain-CSPBased on nucleotide sequence data of colicin Ia channel domain registered in GenBank, a pair of primers were designed and used to clone the fragment encoding colicin Ia channel domain by PCR, the size of the product was the same as expected, and it was inserted into vector pMAL-c2x which contains maltose-binding protein (MBP). Fragments of CSP obtained by biosynthesis was also inserted into this vector. The resulted plasmid was sequenced, no mutation or absence was found.2. Induced expression of the recombinant plasmid in E. coli and purification of the fusion proteinThe constructed expression vector was called pMAL-c2x-Colicin Ia channel domain-CSP, which was finally transformed into E. coli BL21 (DE3) following the standard procedure. The expression of fusion protein was induced by usinging IPTG. SDS-PAGE analysis showed that the molecular weight of the fused protein was about 62KD, which was the same as expected. Detailed studies did not show any association between the amount of the protein and IPTG concentration, and the maximum amount of protein was obtained at the 4th hour of induction. Bacterium wre lysed by supersonics, and centrifugated to collect supernatant fluid which were purified by affinity chromatograph. The aim protein were obtained from eluants.The targeted antibiotic peptides were obtained from fusion protein after digested by Factor Xa. Then, it was further purified by SP cationic exchange chromatography. The SDS-PAGE assay showed a molecular weight of about 19.3 KD, similar with the speculated MW.3.Antimicrobial bioactivity assay of the targeted antibiotic peptides.The antimicrobial bioactivity assay of antibiotic peptides showed that the peptides exhibit antimicrobial activity. The antimicrobial activity is intensive to S.mutans, but is relatively weak to S.sanguis and S.sobrinus.