Cloning And Expression Of A Peroxiredoxin Gene From Phanerochaete Chrysosporium In Escherichia Coli

Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases wildly distributed among prokaryotes and eukaryotes. Antioxidant enzymes in fungi play an important role in protection against the oxygen radicals generated during aerobic metabolism. The results of cloning and functional characterization of a cDNA encoding peroxiredoxin from a 3-day culture of the white-rot fungus Phanerochaete chrysosporium designed as PcPrx are described in this thesis.The full-length PcPrx cDNA (932 bp) contains an open reading frame of 200 amino acid residues with a molecular mass of 22.1 kDa and a pI of 4.8. The deduced primary structure of PcPrx polypeptide in P.chrysosporium shows a high level of sequence homology to recently identified 2-cys peroxiredoxins. PcPrx also exhibits a significant level of sequence similarity to non-fungi 2-cys peroxiredoxins in fungi, plant and bacteria with 60%,40%,41%, respectively.RT-PCR analysis was carried out to determine the relative level variability of expression of PcPrx gene in P.chrysosporium and the expression of PcPrx slightly increased in 3-day culture. The recombinant PcPrx protein was expressed as a histidine fusion protein in Escherichia coli and purified with Ni-column. The purified protein was shown to have a protective effect against plasmid DNA cleavage by reactive oxygen species. Meanwhile, PcPrx protein displayed ability to remove H₂O₂ in ferrithiocyanate system. The PcPrx existed as dimmers under non-reducing conditions and dissociated into monomer in the presence of DTT. All above results are likely to provide clue on the physiological importance of the 2-cys Prx for adaption to diverse stresses.