Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein

Protein Expression and Purification
K R StichaCarston R Wagner

Abstract

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site 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 isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hams...Continue Reading

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Citations

Apr 23, 2002·Pharmacogenomics·Frédérique PompeoEdith Sim
Jun 26, 2012·Protein Expression and Purification·Venuka DuraniThomas J Magliery
May 5, 2011·Protein Expression and Purification·James M Vergis, Michael C Wiener
Feb 26, 2008·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Xindu Geng, Lili Wang
Jul 22, 2006·Journal of Molecular Biology·Fen LiuKylie J Walters
Mar 10, 2001·Trends in Pharmacological Sciences·A UptonE Sim

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