RT Journal Article SR Electronic T1 Recombinant Zebrafish γ-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP 302 OP 309 DO 10.1124/dmd.108.024042 VO 37 IS 2 A1 Tseng-Ting Kao A1 Wen-Ni Chang A1 Hua-Lin Wu A1 Guey-Yueh Shi A1 Tzu-Fun Fu YR 2009 UL http://dmd.aspetjournals.org/content/37/2/302.abstract AB A cDNA encoding for zebrafish γ-glutamyl hydrolase (γGH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-zγGH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant zγGH was similar to mammalian γGH. Thrombin digestion of this NH-zγGH fusion protein resulted in zγGH with approximately 2-fold higher catalytic activity compared with the NH-zγGH fusion enzyme. This recombinant zγGH is active and exhibits comparable endopeptidase activity with folate substrate and antifolate drug methotrexate. Use of this recombinant zγGH significantly increased efficiency in folylpolyglutamate hydrolysis for folate analysis compared with current protocols. The American Society for Pharmacology and Experimental Therapeutics