Production of cystic fibrosis transmembrane conductance regulator (CFTR) protein in recombinant cells

Abstract

The application of the recombinant DNA technology is essential for the large scale production of therapeutic proteins and for the elucidation of their structures and function. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is an important protein with a possible therapeutical function. In this study, the CFTR gene was cloned into the pYES2 plasmid vector and expressed in Saccharomyces cerevisiae AS102 cells, in which cell lysis was controlled by pMET-regulation cassette.Within the framework of this thesis, the CFTR gene was isolated from pBQ6.2 plasmid vector, and was cloned into the pYES2 plasmid vector under the GAL1 promoter. Two colonies harboring the recombinant plasmid construct containing the CFTR gene insert in correct orientation, namely p57 and p312, were identified by the restriction enzyme analysis. These plasmids were then transformed with Saccharomyces cerevisiae AS102 cells. CFTR protein was found to be expressed when the protein content of the cellular extracts was analyzed by SDS-Polyacrylamide Gel Electrophoresis. Analysis of medium at different time intervals showed an increase in protein concentrations of medium upon methionine addition, by repression of the expression of pMET-regulation cassette, thus confirmed the release of proteins into the culture medium as a result of induced lysis.