title: Optimized refolding and characterization of S-peroxidase (CWPO_C of Populus alba) expressed in E. coli.
abstract: Cationic cell wall peroxidase (CWPO_C) from poplar tree (Populus alba L) was heterologously expressed in Escherichia coli as an inclusion body. The insoluble inclusion body was solubilized and reactivated via a refolding procedure. The condition for this procedure was optimized by varying the refolding pH, and the concentrations of the oxidizing agent (GSSG), denaturing agent (GndCl), and hemin, respectively. The optimal conditions for refolding CWPO_C were 100 mM Tris-HCl at pH 8.5, 0.6mM GSSG, 5 μM hemin, 0.6 M GndCl and 5 mM CaCl₂. The fact that the absorbance spectrum was identical to that of wild CWPO_C from poplar tree suggests that the protein folding, heme insertion and iron coordination were correctly archived. The binding affinity and turnover rate values of refolded CWPO_C were compared with those of HRP_C. k(cat)/K(m) for sinapyl alcohol of CWPO_C was over 170 times higher than that of HRP_C, on the while k(cat)/K(m) for coniferyl alcohol showed similar values for both peroxidase. The kinetic parameters showed that refolded CWPO_C possesses a very unique property of S-peroxidase, preferentially oxidizes sinapyl alcohol rather than coniferyl alcohol. The successful expression of CWPO_C in E. coli provides a valuable tool to elucidate the structure and functional relationship of S-peroxidase, which plays an important role in the lignification of angiosperm woody plant cell walls.
authors.affiliation: Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea.
authors.forename: Le Thanh Mai
authors.initials: le TM
authors.lastname: Pham
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Kim
authors.forename: Bong Keun
authors.initials: BK
authors.lastname: Song
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
mesh_terms: D052160:Benzothiazoles / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D010666:Phenylpropionates / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D058849:Protein Refolding; D012995:Solubility; D013451:Sulfonic Acids / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D052160:Benzothiazoles; D010636:Phenols; D010666:Phenylpropionates; D010940:Plant Proteins; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006427:Hemin; C496130:sinapyl alcohol; C010559:coniferyl alcohol; D006735:Horseradish Peroxidase; D009195:Peroxidase; D019803:Glutathione Disulfide