Expression and purification of transmembrane segments 3 and 4 of the cystic fibrosis transmembrane conductance regulator
Year: 1997
Type: dissertation
Abstract: Structural characterization of the membrane-associated chloride channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene is expected to contribute substantively to our understanding and treatment of cystic fibrosis. Accordingly, we are examining the structure of CFTR, focusing on its transmembrane (TM) segments, in an effort to generate a molecular description of CFTR channel activity. To this end, we developed a novel His-tag/thioredoxin fusion expression system to allow for efficient production and purification of the hydrophobic TM segments of CFTR in soluble form in bacteria. Using this system, we have purified TM segments 3 and 4 of CFTR to near homogeneity, with a yield of 5-10 mg/L of culture. The system allowed cleavage of the TM segments from the leader peptide (fusion carrier protein), in vitro, while placement of an HA-epitope tag permitted sensitive detection of the TM peptides by immuno-blotting. Although designed specifically for the production of the TM segments of CFTR, this expression system should also permit routine expression and purification of other polytopic eukaryotic membrane proteins.
Author: Alia Qureshi Emili
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2771128238