Pham Le Thanh Mai, Kim Su Jin, Song Bong Keun, Kim Yong Hwan
Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea.
Protein Expr Purif. 2011 Dec;80(2):268-73. doi: 10.1016/j.pep.2011.08.003. Epub 2011 Aug 12.
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.
来自杨树（银白杨）的阳离子细胞壁过氧化物酶（CWPO_C）在大肠杆菌中作为包涵体进行异源表达。通过重折叠程序使不溶性包涵体溶解并重新激活。通过分别改变重折叠pH值、氧化剂（GSSG）、变性剂（GndCl）和血红素的浓度来优化此程序的条件。重折叠CWPO_C的最佳条件是pH 8.5的100 mM Tris-HCl、0.6 mM GSSG、5 μM血红素、0.6 M GndCl和5 mM CaCl₂。吸收光谱与杨树野生CWPO_C的吸收光谱相同这一事实表明蛋白质折叠、血红素插入和铁配位均正确完成。将重折叠的CWPO_C的结合亲和力和周转速率值与HRP_C的进行比较。CWPO_C对芥子醇的k(cat)/K(m)比HRP_C的高170倍以上，而对松柏醇的k(cat)/K(m)在两种过氧化物酶中显示出相似的值。动力学参数表明重折叠的CWPO_C具有S-过氧化物酶非常独特的性质，优先氧化芥子醇而非松柏醇。CWPO_C在大肠杆菌中的成功表达为阐明S-过氧化物酶的结构和功能关系提供了有价值的工具，S-过氧化物酶在被子植物木质植物细胞壁的木质化过程中起重要作用。
