PUBLICATION
Recombinant Zebrafish γ-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme
- Authors
- Kao, T.T., Chang, W.N., Wu, H.L., Shi, G.Y., and Fu, T.F.
- ID
- ZDB-PUB-081114-22
- Date
- 2009
- Source
- Drug metabolism and disposition: the biological fate of chemicals 37(2): 302-309 (Journal)
- Registered Authors
- Chang, Wen-Ni, Fu, Tzu-Fun, Kao, Tseng-Ting
- Keywords
- analytical chemistry, anticancer agents, drug disposition, enzyme mechanism, HPLC, kinetics
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Catalytic Domain
- Genetic Engineering
- Mammals/metabolism*
- Protein Conformation
- Recombinant Proteins/chemistry
- Recombinant Proteins/metabolism*
- Zebrafish
- Zebrafish Proteins/chemistry
- Zebrafish Proteins/metabolism
- gamma-Glutamyl Hydrolase/chemistry
- gamma-Glutamyl Hydrolase/metabolism*
- PubMed
- 19005029 Full text @ Drug Metab. Dispos.
Citation
Kao, T.T., Chang, W.N., Wu, H.L., Shi, G.Y., and Fu, T.F. (2009) Recombinant Zebrafish γ-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme. Drug metabolism and disposition: the biological fate of chemicals. 37(2):302-309.
Abstract
A cDNA encoding for zebrafish gamma-glutamyl hydrolase (gammaGH) 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-zgammaGH). 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 zgammaGH was similar to mammalian gammaGH. Thrombin digestion of this NH-zgammaGH fusion protein resulted in zgammaGH with approximately 2-fold higher catalytic activity as compared with the NH-zgammaGH fusion enzyme. This recombinant zgammaGH is active and exhibits comparable endopeptidase activity with folate substrate and antifolate drug methotrexate. Use of this recombinant zgammaGH significantly increased efficiency in folylpolyglutamate hydrolysis for folate analysis compared to current protocols.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping