Cloning, expression and characterization of theta-class glutathione S-transferase from the silkworm, Bombyx mori.
This study focused on glutathione S-transferase (GST), one of the detoxification enzymes, from the silkworm, Bombyx mori (GSTT1). A cDNA encoding a putative GST was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 59%, 57% and 56% identities to theta-class GSTs of Musca domestica, Anopheles gambiae and Drosophila melanogaster, respectively. GSTT1 was also estimated to be close to those GSTs in a phylogenetic tree. Recombinant GST (rGSTT1) was functionally overexpressed in Escherichia coli in a soluble form, purified to homogeneity, and characterized. The pH-optimum of rGSTT1 was broad from pH 4 to 9 and rGSTT1 retained more than 75% of its original activity after incubation at pH 5-11. Incubation for 30 min at temperatures below 50 degrees C also affected the activity insignificantly. The Michaelis constant for 1-chloro-2,4-dinitrobenzene was 0.48 mM.
Yamamoto
Kohji
K
Laboratory of Insect Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan. yamamok@agr.kyushu-u.ac.jp
Zhang
Pingbo
P
Miake
Fumio
F
Kashige
Nobuhiro
N
Aso
Yoichi
Y
Banno
Yutaka
Y
Fujii
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D018076:DNA, Complementary; D011994:Recombinant Proteins; C413545:glutathione S-transferase T1; D005982:Glutathione Transferase