Expression of nucleotide binding domain 1 (NBD1) from the cystic fibrosis transmembrane conductance receptor (CFTR)

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease which arises due to mutations in the cystic fibrosis transmembrane conductance receptor (CFTR) gene. The majority of disease-causing mutations are located on the first nucleotide binding domain (NBD1) of the protein. Purification of wild type NBD1 was shown to be very difficult due to poor understanding of its folding and unfolding mechanism. This project aimed to establish a purification protocol for wild type NBD1, in an attempt to characterise the protein and assess the affinity of its interaction with Thymosin-α1 (T-α1). NBD1 was expressed in BL-21 cells from a pTrcSUMO-NBD1 plasmid. The expression of SUMO-NBD1 was optimised, each condition followed by purification. The level of expression and level of purity were assessed through SDS-PAGE. Purified protein samples were treated with ULP-1 in order to cleave off the SUMO moiety from the protein. Site directed mutagenesis was also carried out via PCR as means of creating SUMO-3S-NBD1, having F429S, F494N, and Q637R. Purification of wild type NBD1 proved to be very challenging, most likely as a result of lack of comprehension of the mechanisms governing its folding and unfolding mechanisms. The high protein concentration produced by the expression of pTrcSUMO3S-NBD1 was inferred to be highly stable by circular dichroism and a temperature ramp assay. The findings of this project indicate little to no affinity between 3S-NBD1 and Thymosin-α1, however, this should not discourage further research in this area, due to the difference in structural arrangements of different NBD1 disease-causing mutants.