title: Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG.
abstract: 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 P2(1)2(1)2(1), 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).
authors.affiliation: Regional Centre for Biotechnology, Gurgaon, Haryana 122 016, India.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Chaurasia
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Ingemar
authors.initials: I
authors.lastname: von Ossowski
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Airi
authors.initials: A
authors.lastname: Palva
authors.affiliation: Regional Centre for Biotechnology, Gurgaon, Haryana 122 016, India.
authors.forename: Vengadesan
authors.initials: V
authors.lastname: Krishnan
mesh_terms: D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000235:genetics; D005798:Genes, Bacterial; D052201:Lacticaseibacillus rhamnosus / Q000235:genetics*; D009876:Operon; D059748:Proteolysis
chemical_list: D036781:Fimbriae Proteins; D002918:Chymotrypsin
keywords: Lactobacillus rhamnosus GG; SpaFED pili; adhesion; fimbria; in-house iodide SAD phasing; limited proteolysis