Purification, crystallization and preliminary X-ray diffraction analysis of GumB, an outer membrane lipoprotein essential for secretion of xanthan

JACOBS MELISA, IELPI LUIS

Congreso; 3rd Latin American Protein Society Meeting (LAPSM); 2010

Most bacteria produce different kinds of extracellular polysaccharides which are essential for the cellular viability and for interactions between bacteria and eukaryotic cells. The phytopathogenic bacterium Xanthomonas campestris secretes xanthan, an exopolysaccharide essential for pathogenesis. Besides, xanthan has diverse applications in the industry as thickener and viscosifier. GumB from X. campestris is a 23 kDa outer membrane lipoprotein involved in the secretion of xanthan. GumB belongs to the outer membrane polysaccharide export protein family, which only shares PES motif (Pfam 02563) as primary sequence similarity. Overexpression of GumB results toxic to the cell probably because of its localization in the outer membrane. An efficient overexpression of GumB was obtained from gumB ORF lacking the signal peptide for transport to the membrane. Recombinant cytoplasmic GumB (GumBcyt) was purified to homogeneity by affinity chromatography followed by size exclusion chromatography. Crystals of recombinant GumBcyt were obtained, diffracting to 3.0 Å. Data analysis suggest that GumBcyt crystals belong to the orthorhombic space group P212121 (a=84.27, b=89.31, c=119.21, α=β=γ=90º). Knowledge of the crystal structure of GumB will help to understand the structure and function of the xanthan assembly system.