Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603138
Title: Expression and purification of the cystic fibrosis transmembrane conductance regulator from Saccharomyces cerevisiae for high-resolution structural studies
Author: Cant, Natasha
ISNI:       0000 0004 5354 7165
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Abstract:
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.
Supervisor: Ford, Robert; Prince, Stephen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603138  DOI: Not available
Keywords: CFTR, membrane protein, purification, yeast
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