Epitope-tagging of Cystic Fibrosis Transmembrane Conductance Regulator, CFTR, for the detection of interacting proteins during its biosynthesis
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Date
2000-05-01T00:00:00Z
Authors
Chen, Kathy
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Abstract
Cystic Fibrosis (CF) represents the most common life-threatening recessive genetic trait among the caucasian population, especially those of northern European descent. The disease is caused by the functional absence of a plasma membrane chloride channel, designated as the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). We are interested in investigating which proteins associate with CFTR during its biosynthesis in a manner that may determine whether or not the CFTR protein would mature into a functional plasma membrane channel. Co-immunoprecipitation procedures have been previously applied in detecting such protein-protein interactions. In these experiments, antibodies to known chaperone proteins were assessed for their ability to co-immunoprecipitate newly synthesized CFTR. While such trials successfully identified interactions co-immunoprecipitate newly synthesized CFTR. While such trials successfully identified interactions with Hsp70, calnexin and Hsp90, they do not assess the issue of whether any other, perhaps unknown, proteins associate with CFTR. We wish to do the converse of the previous experiments and immunoprecipitate CFTR a d characterize the proteins found in these co-precipitates. A lack of suitable, high affinity antibodies to CFTR led us to explore the use of short polypeptide tags for affinity purification. The tags were well characterized epitopes or ligands for affinity matrices and were added to the carboxyl-terminus of the CFTR coding sequence. (Abstract shortened by UMI.)