Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose binding protein

Many membrane proteins that belong to the ATP-binding cassette (ABC) superf amily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (mult idrug resistance gene product; MDR1). These proteins contain two multispann ing transmembrane domains, each followed by one nucleotide-binding domain ( NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBD s is critical for ABC protein function; the linker region seems to have a r egulatory role. Previous attempts to express soluble NBDs and/or linker reg ions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system f or the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD), a comparison of the expressions of NBD1MLD fus ed to glutathione S-transferase, thioredoxin and maltose-binding protein (M BP) shows that a high level of expression in the soluble fraction (approx. 8 % of total E. coli protein) can be achieved only for MBP-NBD1MLD. The add ition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purif ied with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteol ytic site between MBP and NBD1MLD. The approach described here could be gen erally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.