OVEREXPRESSION AND LARGE-SCALE PURIFICATION OF RECOMBINANT HAMSTER POLYMORPHIC ARYLAMINE N-ACETYLTRANSFERASE AS A DIHYDROFOLATE-REDUCTASE FUSION PROTEIN

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxificati on and/or bioactivation of a variety of xenobiotics, Rapid kinetic, bi ophysical, structural, and bioactivation studies on NATs require quant ities of purified enzyme capable of being obtained only through recomb inant DNA technology. This laboratory has previously developed a prote in expression and purification system in which NATs are expressed as p roteins fused to a FLAG octapeptide followed by a thrombin-cleavage si te to allow liberation of the rNAT. Typically, however, only 0.5 - 1.5 mg of the recombinant NAT's could be readily purified in a single iso lation sequence by immunoaffinity chromatography. Therefore, the expre ssion system was modified by inserting the L54F dihydrofolate reductas e (DHFR) mutant gene sequence between the FLAG octapeptide and the thr ombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH d ependence of binding to a methotrexate (MTX)-affinity column by the L5 4F DHFR mutant. Unfortunately, the affinity chromatography strategy di d not work satisfactorily. Although the specific activity of the purif ied rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered, The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Io n-exchange chromatography was investigated as an alternative purificat ion method, An initial DEAF anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved wit h thrombin and reapplied to a DEAE anion-exchange column. The second L EAF column resulted in not only the separation of rNAT2-70D from FLAG- L54F DHFR, but also the purification of rNAT2-70D to near homogeneity, Application of the nearly homogeneous rNAT2-70D to a gel-filtration c olumn resulted in recovery of homogeneous protein, The ion-exchange me thod of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture. (C) 1997 Academ ic Press.