Biochemical characterization of 1-Cys peroxiredoxin from Antrodia camphorata.
Antrodia camphorata is a unique medicinal mushroom found only in Taiwan. It has been used as a remedy for various diseases in folk medicine. Antrodia camphorata has been shown to exhibit antioxidative effects. Peroxiredoxins play important roles in antioxidation and cell signaling. A gene encoding an antioxidant enzyme, 1-cysteine peroxiredoxin (1-Cys Prx), was identified in an expressed sequence tag database of the A. camphorata and cloned by polymerase chain reaction. The 1-Cys Prx cDNA (837 bp, accession no. AY870325) contains an open reading frame encoding a protein of 223 amino acid residues with calculated molecular mass of 25,081 Da. The deduced protein shared 44-58% identity with 1-Cys Prx from Homo sapiens, Bos taurus, and Saccharomyces cerevisia. The sequence surrounding the conserved cysteine DFTPVCTTE is conserved. The coding sequence was subcloned into a vector, pET-20b (+), and transformed into Escherichia coli. The recombinant 1-Cys Prx was purified by Ni(2+)-nitrilotriacetic acid (Sepharose). The purified enzyme was characterized under various conditions. The enzyme is thermostable because its half-life of inactivation was 15.5 min at 60 degrees C. It was stable under alkaline pH range from 7.8 to 10.2. The enzyme showed decreased activity with increasing concentration of imidazole. The enzyme is sensitive to trypsin and chymotrypsin treatment.
Wen
Lisa
L
Department of Chemistry, Western Illinois University, 1 University Circle, Macomb, IL, 61455-1390, USA.
Huang
Hui-Ming
HM
Juang
Rong-Huay
RH
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism*; D054464:Peroxiredoxins; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D011994:Recombinant Proteins; D010544:Peroxidases; D054464:Peroxiredoxins