PT  - JOURNAL ARTICLE
AU  - Tseng-Ting Kao
AU  - Wen-Ni Chang
AU  - Hua-Lin Wu
AU  - Guey-Yueh Shi
AU  - Tzu-Fun Fu
TI  - Recombinant Zebrafish γ-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme
AID  - 10.1124/dmd.108.024042
DP  - 2009 Feb 01
TA  - Drug Metabolism and Disposition
PG  - 302--309
VI  - 37
IP  - 2
4099  - http://dmd.aspetjournals.org/content/37/2/302.short
4100  - http://dmd.aspetjournals.org/content/37/2/302.full
SO  - Drug Metab Dispos2009 Feb 01; 37
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