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A systematic approach to the amplified expression, functional characterisation and purification of inositol transporters from Bacillus subtilis

DOI: 10.3109/09687688.2012.729093 DOI Help
PMID: 23078035 PMID Help

Authors: Kim Bettaney (University of Leeds) , Preethi Sukumar (University of Leeds) , Rohanah Hussain (Diamond Light Source) , Giuliano Siligardi (Diamond Light Source) , Peter Henderson (University of Leeds) , Simon Patching (University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Molecular Membrane Biology , VOL 30(1) , PAGES 3-14

State: Published (Approved)
Published: February 2013

Abstract: A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ? 10–15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of 3H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for 3H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography

Journal Keywords: Inositol Transport Protein; Amplified Expression; Purification; Ligand Recognition; Bacillus Subtilis

Subject Areas: Biology and Bio-materials, Chemistry, Medicine

Instruments: B23-Circular Dichroism

Other Facilities: No