Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrialspaFEDoperon inLactobacillus rhamnosusGG

Abstract

SpaD is the predicted backbone-pilin subunit of the SpaFED pilus, whose loci are encoded by the fimbrialspaFEDoperon inLactobacillus rhamnosusGG, a Gram-positive gut-adapted commensal strain with perceived probiotic benefits. In this study, soluble recombinant SpaD protein was overproduced inEscherichia coliand then purified by Ni2+-chelating affinity and gel-filtration chromatography. After limited proteolysis with α-chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 Å resolution. These crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 50.11,b= 83.27,c= 149.65 Å. For phasing, sodium iodide-derivatized crystals were prepared using the halide quick-soaking method and diffraction data were collected in-house to a resolution of 2.2 Å. An interpretable electron-density map was successfully obtained using single-wavelength anomalous diffraction (SAD).