Recombinant zebrafish γ-glutamyl hydrolase exhibits properties and catalytic activities comparable with those of mammalian enzyme

Tseng Ting Kao, Wen Ni Chang, Hua Lin Wu, Guey Yueh Shi, Tzu N. Fu

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

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 folylpoly-glutamate hydrolysis for folate analysis compared with current protocols.

Original languageEnglish
Pages (from-to)302-309
Number of pages8
JournalDrug Metabolism and Disposition
Volume37
Issue number2
DOIs
Publication statusPublished - 2009 Feb

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmaceutical Science

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