The cystic fibrosis transmembrane conductance regulator (CFTR) is an ABC transporter family protein that acts as an ion channel. Mutations in CFTR cause the most common genetic disease in Caucasian populations, cystic fibrosis (CF). The high-resolution X-ray crystal structure of CFTR is now needed to aid the design of CFTR-targeted drugs for CF treatment and also to elucidate the molecular mechanisms behind its unique function as an ATP-ligand gated ion channel. However, until now, such structural studies have been severely limited by the lack of sufficient quantities of purified full-length CFTR protein. This thesis reports the novel over-expression and purification of milligram quantities of the chicken orthologue of CFTR protein from a Saccharomyces cerevisiae (yeast) expression system. A green fluorescent protein (GFP) tag fused to the CFTR C-terminus allowed rapid detection of the protein throughout the purification procedure. CFTR was expressed under an inducible promoter and appeared localised at, or near to, the plasma membrane, where it represented around 1 % of total protein after isolation in yeast microsomes. CFTR was solubilised from microsomes and purified using the detergents dodecylmaltoside (DDM) and lyso-phosphatidyl glycerol (LPG), by nickel affinity and size exclusion chromatography (SEC) to yield 1-2 mg of CFTR protein per 18 L fermentation culture. CFTR thermal stability was probed using fluorescent measurements to reveal a two-state cooperative unfolding transition around 40 °C for the DDM-purified protein, but no such transition was observed for the LPG-purified material. Light scattering and electron microscopy techniques revealed that, in LPG, CFTR was a homogenous population of monomeric particles around 60-Å in length that were soluble up to 8 mg/ml protein concentration. In DDM, CFTR was only soluble below 0.4 mg/ml protein concentration where is existed as a very heterogenous population of different sized amorphous particles, including dimeric particles around 180-Å in length. The DDM-purified CFTR protein could be crystallised as monomers in two-dimensional (2D) crystals with similar lattice parameters to 2D crystals of CFTR purified from mammalian cells. The ATPase activity of DDM-purified and reconstituted CFTR was similar to already published rates, at around 13 nmol Pi/min/mg integrated over a reaction time of 60 min, with an apparent affinity Km for ATP of 0.14 mM. Such a low ATPase rate compared to other ABC transporters may be due to the observed rapid run-down of activity with time and correlation with published CFTR channel gating kinetics. CFTR showed reduced ATPase activity after purification in LPG, suggesting a structural destabilisation in this detergent. The protocols presented here can now be used to provide sufficient quantities of purified CFTR protein for novel biochemical and biophysical studies. The tendency of CFTR to aggregate in a mild detergent remains a major obstacle towards 3D crystallisation trials and a high-resolution structure.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603138 |
| Date | January 2014 |
| Creators | Cant, Natasha |
| Contributors | Ford, Robert; Prince, Stephen |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/expression-and-purification-of-the-cystic-fibrosis-transmembrane-conductance-regulator-from-saccharomyces-cerevisiae-for-highresolution-structural-studies(826ead72-d291-40f6-8e06-a61730eb2d5f).html |
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