Cloning of the nucleotide binding domain from the human cystic fibrosis transmembrane conductance receptor

Abstract

NBD1 is the first nucleotide binding domain found in the CFTR protein. Its function is to bind to ATP in order to open the CFTR channel and allow the passage of chloride ions and bicarbonate ions. The NBD1 is also the site of one of the most prevalent mutations, the delta F508 mutation, which is known to cause the genetic disorder cystic fibrosis. The mutation leads to improper folding and processing of the CFTR protein, resulting in a build-up of mucus in the lining of a number of tracts including the respiratory, digestive and reproductive tract. The aim of this study was to clone and purify the NBD1 in order to study its structure, stability and its binding to ATP and ADP. It was successfully sub-cloned into the pTrc-SUMO plasmid through restriction digest and ligation. It was then expressed in XL-1 Blue E. coli competent cells after being transformed with the recombinant plasmid. Metal chelate affinity chromatography was carried out to purify NBD1 using a hexahistidine tag to bind the protein to the column with nickel ions. However, the results demonstrated that the purification was unsuccessful as the protein was not binding effectively to the column. Possible future work would include mutating the NBD1 to contain the delta F508 mutation and then carrying out circular dichroism spectroscopy and isothermal titration calorimetry to study the structure, stability and thermodynamic properties of NBD1 and the mutated NBD1. A better understanding of this would further help to develop new treatments for cystic fibrosis.