Purification and properties of recombinant rat catalase produced in Escherichia coli.
Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.
Furuta
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Department of Biochemistry, Shinshu University School of Medicine, Nagano.
Hayashi
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D000818:Animals; D001483:Base Sequence; D002374:Catalase / Q000235:genetics / Q000302:isolation & purification*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004274:DNA, Recombinant / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D004247:DNA; D002374:Catalase