Expression, purification, and characterization of a novel G protein, SGP, 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, the sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein at a level of 40% of total cellular protein. One-step amylose affinity chromatography purification of this fusion protein yielded a product of approximately 95% purity. SGP was purified from this fusion protein following cleavage with protease factor Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, recombinant SGP showed specific binding for GTP and GDP, but not ATP, CTP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Kinetic studies revealed that the SGP Km value for GTP in this reaction was approximately 5.9 microM. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labelling demonstrated that SGP was associated with both membrane and cytoplasmic fractions. SGP not only had structural similarities with other G proteins 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.
Wu
J
J
Department of Oral Biology, State University of New York at Buffalo 14214, USA.
Cho
M I
MI
Kuramitsu
H K
HK
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins; D013295:Streptococcus mutans / Q000737:chemistry*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C094290:SGP protein protein, Streptococcus mutans; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins