The Recombinant Expression And Activities Assay Of Catalytic Region Of Glucosyltransferase

Dental caries which is very common in human beings is associated with the acidogenic and aciduric properties of the Streptococci mutans. The colonization and accumulation of these cariogenic oral streptococci on the tooth surface have been associated with the ability of mutans streptococci to synthesize extracellular glucan from sucrose.GTF have been implicated as important constituents in the active accumulation of mutans streptococci on teeth⁹. The glucans synthesized by GTFs not only promote the accumulation of cariogenic streptococci on the tooth surface, but also contribute significantly to the bulk of dental plaque^17,18 .GTF in oral streptococci all have three major domains, including an N-terminal highly variable region, a conserved core catalytic region, and a C-terminal glucan-binding domain¹⁵ The catalytic domain, which appears to function to stabilize glucosyl intermediates formed during the hydrolysis of sucrose^2,13 This accretion is facilitated by extracellular glucan,which is synthesized from sucrose by a group of GTF¹¹ This enzyme contains an N-terminal catalytic site (CAT) and a C-terminal repetitive glucan-binding region (GLU) which is presumably involved in chain extension of the growing glucan polymersIn this study, the catalytic (CAT) region of gtfC was amplified by PCR from the genome of S. mutans GS5, and then cloned into pET-28a-SWG. It was found that recombinant expressed CAT-C was insoluble. The antibodies against to the rCAT-C were prepared. In order to obtain the soluble fusion protein, the fragment of CAT coding region was recloned into the vector of pET-32-NusA. The SDS-PAGE results showed that the rNusA-CAT-C is soluble.The activity of recombinant protein was tested using the mothod of anthrone- sulphuric acid. Our results showed that the soluble rNusA-CAT-C could catalyze the sucrose effectively.