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Purification, crystallization and preliminary X‐ray diffraction analysis of SpaD, a backbone‐pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG
Author(s) -
Chaurasia Priyanka,
von Ossowski Ingemar,
Palva Airi,
Krishnan Vengadesan
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14027216
Subject(s) - pilin , operon , lactobacillus rhamnosus , crystallography , pilus , orthorhombic crystal system , chemistry , protein subunit , resolution (logic) , escherichia coli , materials science , biochemistry , lactobacillus , crystal structure , gene , artificial intelligence , fermentation , computer science
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 Ni 2+ ‐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 P 2 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).