Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG

Priyanka Chaurasia, Ingemar von Ossowski, Airi Palva, Vengadesan Krishnan*

*Tämän työn vastaava kirjoittaja

Tutkimustuotos: LehtiartikkeliArticleScientificvertaisarvioitu

Abstrakti

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 -chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 angstrom 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 angstrom. 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 angstrom. An interpretable electron-density map was successfully obtained using single-wavelength anomalous diffraction (SAD).

AlkuperäiskieliEnglanti
Sivut103-106
Sivumäärä4
JulkaisuActa crystallographica section F : Structural biology communications
Vuosikerta71
DOI - pysyväislinkit
TilaJulkaistu - tammikuuta 2015
OKM-julkaisutyyppiA1 Julkaistu artikkeli, soviteltu

Sormenjälki

Sukella tutkimusaiheisiin 'Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial <i>spaFED</i> operon in <i>Lactobacillus rhamnosus</i> GG'. Ne muodostavat yhdessä ainutlaatuisen sormenjäljen.

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