Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA

Abstract

Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA(25-460)) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA(25-460) were collected to 2.25 Å resolution from a crystal that belonged to space group P21, with unit-cell parameters a = 50.96, b = 131.41, c = 123.40 Å, α = 90.0, β = 94.8, γ = 90.0°, and which was predicted to contain two BabA(25-460)-nanobody complexes per asymmetric unit.

Keywords: BabA; Helicobacter pylori; adhesin; nanobody.

Figure 1

(a) MS spectra of BabA^1–460 reveal spontaneous proteolysis leading to the loss of up to 23 N-terminal amino acids. (b) Immunoblot detection (using α-His antibody) of BabA fragments in the respective periplasmic extracts. Lane M contains molecular-mass marker (labelled in kDa).

Figure 2

(a) Size-exclusion chromatogram and SDS–PAGE analysis (inset) of BabA^25–460 run on Superdex 75 10/300 (GE Healthcare). (b) CD spectrum of BabA^25–460 in phosphate buffer (50 mM KH₂PO₄/K₂HPO₄ pH 8.0) reveals a predominantly α-helical secondary-structure content.

Figure 3

(a) Image of BabA^25–460–Nb-ER19 crystals and SDS–PAGE of dissolved crystals alongside purified BabA^25–460 (inset, labelled C and P, respectively; *, BabA^25–460; **, Nb-ER19). (b) Representative diffraction image (0.2° rotation, 0.2 s exposure at 20% transmission) from a crystal of BabA^25–460 in complex with Nb-ER19 obtained by the sitting-drop vapour-diffusion method.