title: Expression, refolding, purification and crystallization of the sensory domain of the TlpC chemoreceptor from Helicobacter pylori for structural studies.
abstract: Helicobacter pylori infections are associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinoma. Bacterial chemotaxis, mediated by four different chemoreceptors (also termed transducer-like proteins (Tlp)), plays an important role in initial colonization and development of disease. Chemoreceptor sensory domains of H. pylori share no significant sequence similarity with those of Escherichia coli or any other non-Epsilonproteobacteria. The structural basis of how chemical signals are recognized by chemoreceptors of H. pylori is poorly understood mainly due to the lack of a robust procedure to purify their sensory domains in a soluble form. This study reports a method for extraction of the periplasmic sensory domain of transducer-like protein C (TlpC) from inclusion bodies and refolding to yield 5mg pure crystallizable protein per 1l of bacterial culture. Purified protein was monomeric in solution by size-exclusion chromatography and folded according to the circular dichroism spectrum. Crystals have been grown by the hanging-drop vapor-diffusion method using PEG 4000 as a precipitating agent. The crystals belonged to space group C2, with unit-cell parameters a=189.3, b=103.2, c=61.8Å, β=98.3. A complete X-ray diffraction data set has been collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains three monomers.
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Yu Chih
authors.initials: YC
authors.lastname: Liu
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. Electronic address: Anna.Roujeinikova@monash.edu.
authors.forename: Anna
authors.initials: A
authors.lastname: Roujeinikova
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D016480:Helicobacter pylori / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins
keywords: Chemoreceptor; Chemotaxis; Helicobacter pylori; Transducer-like protein; X-ray crystallography