Recombinant zebrafish {gamma}-glutamyl hydrolase exhibits properties and catalytic activities comparable with those of mammalian enzyme.
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 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 with current protocols.
Kao
Tseng-Ting
TT
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan.
Chang
Wen-Ni
WN
Wu
Hua-Lin
HL
Shi
Guey-Yueh
GY
Fu
Tzu-Fun
TF
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D005818:Genetic Engineering; D008322:Mammals / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry / Q000378:metabolism; D011623:gamma-Glutamyl Hydrolase / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D011623:gamma-Glutamyl Hydrolase