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 folylpolyglutamate hydrolysis for folate analysis compared with current protocols.
Footnotes
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This work was supported by the National Science Council, Taiwan [Grant NSC 96-2320B-006-023-MY3]; and the Program for Promoting Academic Excellence of Universities, National Cheng Kung University [Grants D96-3500, D96-2200].
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The amino acid numbering used for zγGH in this study is numbered starting from the first methionine in the full-length peptide with the signal peptide.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.024042.
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ABBREVIATIONS: γGH, γ-glutamyl hydrolase; zγGH, zebrafish γGH; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; NH-zγGH, Nus-His-tag fusion γ-glutamyl hydrolase; IPTG, isopropyl-β-d-thiogalactopyranoside; THF, tetrahydrofolate; 5-CHO-THF-Glu3, 5-formyltetrahydrofolate triglutamate; 5-CHO-THF-Glu1, 5-formyltetrahydrofolate monoglutamate; PAGE, polyacrylamide gel electrophoresis; MTX, methotrexate.
- Received August 19, 2008.
- Accepted November 7, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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