Molecular cloning, expression and characterization of a recombinant glutathione S-transferase from Echinococcus multilocularis.
We report the identification and characterization of the first cestode glutathione S-transferase (GST) cDNA sequence. A fragment of an Echinococcus multilocularis glutathione S-transferase cDNA was isolated by the polymerase chain reaction. Subsequently, a Lambda zap cDNA library prepared from mRNA from protoscolices of E. multilocularis was screened with this PCR fragment. A complete cDNA clone was isolated and the nucleotide sequence determined. Analysis of the E. multilocularis GST-deduced amino acid sequence indicates that it is clearly related to the mammalian mu-class GSTs. The E. multilocularis GST cDNA was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The 25.5-kDa enzyme subunit was purified to homogeneity using glutathione-sepharose chromatography. Gel filtration demonstrated that this GST is enzymatically active as a homodimer. The recombinant enzyme had conjugating activity with organic hydroperoxides and with members of the trans,trans-2,4 alkadienal and trans-2-alkenal series, which are secondary products of lipid peroxidation.
Liebau
E
E
Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Müller
V
V
Lucius
R
R
Walter
R D
RD
Henkle-Dührsen
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004446:Echinococcus / Q000201:enzymology*; D005210:Fasciola hepatica / Q000201:enzymology; D015723:Gene Library; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012550:Schistosoma mansoni / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D005982:Glutathione Transferase