First steps for structural and functional characterization of GumC, an integral membrane protein

BIANCO MI, SILVA M, SALINAS SR, AND IELPI L.

Puerto Madryn, Chubut

Congreso; Reunión Anual – Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2010

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Xanthan is an exopolysaccharide synthesized by different species of Xanthomonas, which is involved in diverse biological functions and in a wide range of industrial applications. Despite biosynthesis of xanthan repetitive units is widely studied, its polymerization and secretion mechanism is poorly understood. Previous studies from our laboratory in Xanthomonas campestris suggest that polymerization and secretion of xanthan could be associated, and the integral inner membrane protein GumC is involved in these processes. Therefore, we are interested in understand its structure and molecular mechanism of action. Because obtaining sufficient amounts of high-quality pure membrane proteins is still the mayor bottleneck in membrane protein studies, the first challenge of this work was to obtain enough amount of pure GumC. We overexpressed GumC in Escherichia coli BL21(DE3) with a hexahistidine-coding sequence. After several trials, we purified GumC to homogeneity by a two-chromatographic-step protocol and obtained a good yield of pure protein (8 mg per liter of culture). Currently, we are carrying out biophysical assays and crystallization screenings. In addition, based on in silico analysis, we are performing functional studies by site-directed mutagenesis, which showed difference in xanthan production when some conserved aminoacids are mutated.