Assembly and functional expression of murine glutathione reductase cDNA: a sequence missing in expressed sequence tag libraries.
Glutathione reductase (GR) is a chemotherapeutic target. Murine GRcDNA, which contains 85% GC in the 38 codons following the start codon, was assembled from the PCR-amplified exon 1 and a downstream cDNA prior to expression in Escherichia coli as a His(6)-tagged protein. Recombinant GR, an FAD-containing homodimer, corresponds in its enzymic and spectral properties to GR isolated from murine Ehrlich ascites tumor cells. Another cDNA, representing GR with a mitochondrial targeting sequence, yielded two distinct enzymically active expression products.
Iozef
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R
Center of Biochemistry, Heidelberg University, Im Neuenheimer Feld 328, D-69120, Heidelberg, Germany.
Becker
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K
Boehme
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CC
Schirmer
R H
RH
Werner
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D
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D020862:GC Rich Sequence; D015723:Gene Library; D005980:Glutathione Reductase / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005980:Glutathione Reductase