Chaurasia, Priyanka and von Ossowski, Ingemar and Palva, Airi and Krishnan, Vengadesan (2015) Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrialspaFEDoperon inLactobacillus rhamnosusGG. Acta Crystallographica Section F Structural Biology Communications, 71 (1). pp. 103-106. ISSN 2053-230X

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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).

Item Type:	Article
Subjects:	Biochemical and Biophysical Sciences
Depositing User:	RCB Library
Date Deposited:	13 Nov 2017 10:47
Last Modified:	13 Nov 2017 10:47
URI:	http://rcb.sciencecentral.in/id/eprint/133

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