EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF A NOVEL G-PROTEIN, GP, FROM STREPTOCOCCUS-MUTANS

The sgp gene of Streptococcus mutans was recently detected immediately downstream from the dgk gene within the same operon. In this study, t he sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein wi th the maltose-binding protein at a level of 40% of total cellular pro tein. One-step amylose affinity chromatography purification of this fu sion protein yielded a product of approximately 95% purity. SGP was pu rified from this fusion protein following cleavage with protease facto r Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, r ecombinant SGP showed specific binding for GTP and GDP, but not ATP, C TP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Ki netic studies revealed that the SGP K-m value for GTP in this reaction was approximately 5.9 mu M. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labellin g demonstrated that SGP was associated with both membrane and cytoplas mic fractions. SGP not only had structural similarities with other G p roteins but also proved to have high-level intrinsic GTPase activity. Therefore, SGP appears to be a new member of the G protein superfamily and may participate in transmembrane signaling in the responses of S. mutans cells to environmental stimuli.