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*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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 -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).

Original languageEnglish
Pages (from-to)103-106
Number of pages4
JournalActa crystallographica section F : Structural biology communications
Volume71
DOIs
Publication statusPublished - Jan 2015
MoE publication typeA1 Journal article-refereed

Keywords

  • Lactobacillus rhamnosus GG
  • SpaFED pili
  • fimbria
  • adhesion
  • in-house iodide SAD phasing
  • limited proteolysis
  • ADHESION
  • SPACBA

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