research communications
SpaD is the predicted backbone-pilin subunit of the SpaFED pilus, whose loci are encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG, a Gram-positive gut-adapted commensal strain with perceived probiotic benefits. In this study, soluble recombinant SpaD protein was overproduced in Escherichia coli and 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 group P212121, with unit-cell parameters a = 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).
Keywords: Lactobacillus rhamnosus GG; SpaFED pili; fimbria; adhesion; in-house iodide SAD phasing; limited proteolysis.
Supporting information
Portable Document Format (PDF) file https://doi.org/10.1107/S2053230X14027216/hc5185sup1.pdf |