Expression and purification of the nucleotide binding domains of P-glycoprotein

P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide binding domains (NBDs). The domain boundaries for the NBDs are poorly defined. To determine the boundaries different N- and C-terminal domain boundaries were engineered based on sequence alignments with bacterial homologues. NBDs were overexpressed in E. coli as thioredoxin fusion proteins and extracted as soluble protein in the presence of ATP. Isolation of NBD protein from the thioredoxin fusion was achieved through digestion with thrombin and gel filtration. All NBDs aggregated and precipitated throughout purification. One C-terminal NBD was analyzed to determine its biochemical characteristics. The NBD was shown to have basal ATPase activity and to exist as a large protein aggregate with some detectable secondary structure as analyzed by CD Spectroscopy. Manipulation of the purification protocol and additives such as SDS and urea had minimal effects on the aggregation state of the C-terminal protein. A new protocol must be developed to yield pure, soluble and correctly folded protein necessary for structural studies.