Cloning, expression, purification and properties of a putative multidrug resistance efflux protein from Helicobacter
Scott Morrison1, Alison Ward, Christopher J Hoyle
1Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Abstract:
Helicobacter pylori is a common pathogen of humans, which predisposes individuals to gastric inflammation and a variety of diseases including gastric cancer. Sequencing of the organism's genome reveals an extensive portfolio of predicted membrane transport proteins, although few of the proteins encoded by these genes have yet been isolated. We describe here the cloning and expression in Escherichia coli of the H. pylori gene hp1181 encoding a putative multidrug resistance membrane transport protein. Substantial overexpression was accomplished, and the protein was tagged with RGS(His)(6) at the C-terminus, which enabled its purification in mg quantities. Identification of the full-length protein was achieved by N-terminal amino acid sequencing and Western blotting using an antibody to the RGS(His)(6) epitope. The retention of structural integrity and occurrence of predicted alpha-helix in the protein were verified by circular dichroism spectroscopy.
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