title: [hHO-1 structure prediction and its mutant construct, expression, purification and activity analysis].
abstract: Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme. On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E. coli DH5alpha strain. The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography. The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively. The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1. The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function.
authors.affiliation: Department of Pediatrics, Rui Jin Hospital, Shanghai Second Medical University, Shanghai. xzw63@hotmail.com
authors.forename: Zhen Wei
authors.initials: ZW
authors.lastname: Xia
authors.forename: Wen Jun
authors.initials: WJ
authors.lastname: Cui
authors.forename: Wen Pu
authors.initials: WP
authors.lastname: Zhou
authors.forename: Xue Hong
authors.initials: XH
authors.lastname: Zhang
authors.forename: Qing Xiang
authors.initials: QX
authors.lastname: Shen
authors.forename: Yun Zhu
authors.initials: YZ
authors.lastname: Li
authors.forename: Shan Chang
authors.initials: SC
authors.lastname: Yu
mesh_terms: D015153:Blotting, Western; D002845:Chromatography; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary
chemical_list: D051547:Heme Oxygenase-1