Expression and purification of the cystic fibrosis transmembrane conductance regulator and development of a high throughput screen for drug discovery
The cystic fibrosis transmembrane conductance regulator protein (CFTR) is a chloride channel than plays an important role in epithelial cells. In the lungs it is involved in regulation of the airway surface layer (ASL) and loss of CFTR function leads to clogging of airways with sticky mucus and consequent persistent bacterial infections. Humans carrying mutations in CFTR have several problems as a result of loss of CFTR function and limited life span and quality.
Drugs are needed to treat the most common CFTR mutation (deletion of Phe508) which causes misfolding of the protein. In order to discover new drugs, we are expressing the human CFTR protein in yeast (S. cerevisiae) so that large amounts of the protein can be obtained for drug screens via functional assays.
This project will involve the expression and purification of stable CFTR protein using the yeast system, and the student will be trained in advanced molecular biology, microbiology and membrane protein purification techniques. Purification will be enhanced using protein with both a C-terminal His tag as well as an internal FLAG tag, allowing a simple 2-stage chromatographic purification scheme. Once significant (mg) quantities of purified wild-type protein have been obtained, the aim will be to develop a modified procedure for the purification of the mutated (F508del) version of the protein.
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For this self-funded project, applicants are encouraged to contact the Principal Supervisor directly to discuss their application and the project. Please select any of the subject areas displayed on this project listing when making your online application.
Rosenberg MF, O'Ryan LP, Hughes G, Zhao Z, Aleksandrov LA, Riordan JR, Ford RC. The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate. J Biol Chem. 2011 Dec 9;286(49):42647-54.
O'Ryan L, Rimington T, Cant N, Ford RC Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae. J Vis Exp. 2012 Mar 10;(61). pii: 3860. doi: 10.3791/3860.