title: Overexpression and activities of 1-Cys peroxiredoxin from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode.
abstract: Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame (ORF) of 639 bp, which encoded a protein of 213 amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared 99, 97, and 97 % identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17 and 1-Cys peroxiredoxin of P. fluorescens Pf 0-1, respectively. The ORF was cloned into expressing vector pET-15b and introduced into Escherichia coli BL21 (DE3). Overexpression of a 27-kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni(2+) matrix column. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. Furthermore, E. coli expressing the ORF showed tolerance to hydrogen peroxide stress, which indicated that the gene might help P. fluorescens GcM5-1A resist hydrogen peroxide generated by host pines after pine wood nematode associated with this bacterium infected pine trees.
authors.affiliation: Department of Biology, Qingdao University, Qingdao, 266071, People's Republic of China.
authors.forename: Guohua
authors.initials: G
authors.lastname: Liu
authors.forename: Kai
authors.initials: K
authors.lastname: Feng
authors.forename: Daosen
authors.initials: D
authors.lastname: Guo
authors.forename: Ronggui
authors.initials: R
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009348:Nematoda / Q000382:microbiology*; D016366:Open Reading Frames; D054464:Peroxiredoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D054464:Peroxiredoxins