Preliminary Crystallization Of The Protein Coding By LuxS From Streptococcus Mutans

Dental caries is a tape of bacterial infections on tooth hard tissue,which is recognized asone of the most common diseases in humans,especially in children.Evidence frombacteriological researches and animal models confirm that S.mutans is the most principalcariogenic bacteria. The virulence properities of S. mutans is closely related to itsacidogenicity. The S-ribosylhomocysteinase encoded by luxS gene from Streptococcusmutans synthetize antoinducer-2(AI-2) though a series of enzymatic reaction.AI-2is anuniversal language for interspecies communication.It plays a crucial role in monitoring celldentity,regulating biological function of bacteria,effecting biofilm formation,strengtheningthe acid resistance and drug resistance.To date,the mechanisms of Streptococcusmutans’aciduricity has not been reported.In this study,we determined the structure ofS-ribosylhomocysteinase encoded by luxS gene from Streptococcus mutans.The3D-structure of S-ribosylhomocysteinase will help us identify its functional role in acidresistance mechanisms and provide a structural basis for prevention and cure dental caries.Methods：1)Cloning of S-ribosylhomocysteinase The luxS gene encoding S-ribosylhomo-cysteinase was PCR amplified using genomic DNA of Streptococcus mutans UA159astemplate and cloned into the expression vector pGEX-6p-1.2)The expression and purification of S-ribosylhomocysteinase The construct wastransformed into E.coli BL21(DE3),and the target protein was expressed by inducing withIPTG.The target protein was identified by SDS-PAGE and mass spectrography.Then thetarget protein was purified by GST tag affinity chromatography,anion exchangechromatography and gel filtration.3)The crystallization and structure analysis of S-ribosylhomocysteinase The growthconditions of the crystal of S-ribosylhomocysteinase were screened by using the Screen kitsfrom Hampton Research.The initial screening conditions of crystal growth wereoptimized.The diffraction data sets were collected in Beijing Sychrotron Radiation Facility(BSRF).The structure of S-ribosylhomocysteinase was analyzed by using the CCP4softwarepackage.Results：1) S-ribosylhomocysteinase was successfully expressed in E. coli in a soluble form.The product was identified and confirmed by SDS-PAGE and mass spectrography. 2) After optimizing the conditions of expression and purification, we got the targetprotein fused with GST tag with a purity more than95%.3) The crystal of S-ribosylhomocysteinase was obtained.4) The crystal of S-ribosyihomocysteinase which diffracted to2.4A and belongs to thespace group C2221.We collected several sets of diffraction data of the crystal andpreliminary analyzed of the data.Conclusion：We have successfully expressed S-ribosylhomocysteinase in E. coli, established aperfect purification protocol of the recombinant protein and resolved its structure by usingmolecular replacement method...