Engineering human seleno-glutaredoxin containing consecutive rarecodons as an artificial glutathione peroxidase  

作　　者：ZHANG Wei LUO Quan WANG XiaoPing ZHANG DongMei MIAO Lu XU JiaYun LUO GuiMin SHEN JiaCong LIU JunQiu 

机构地区：[1]State Key Laboratory of Supramolecular Structure and Materials,Jilin University,Changchun 130012,China [2]Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education,Jilin University,Changchun 130012,China

出　　处：《Chinese Science Bulletin》2012年第1期25-32,共8页

基　　金：supported by the National Natural Science Foundation of China(91027023,20874036,20921003,and 21004028);the Natural Science Foundation for the Youth(21004028);the Natural Science Foundation of China for Outstanding Younger Scientist(20725415);the 111 pro-ject(B06009);the National Basic Research Program(2007CB808006)

摘　　要：The active center of human glutaredoxin(hGrx1)shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH)with natural glutathione peroxidase(GPx).hGrx1 was redesigned to introduce the catalytic selenocysteine residue to imi- tate the function of antioxidant selenoenzyme GPx in vivo.The human hGrx1 scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds.Two consecutive rare codons(AGG-AGG)in the open reading frame of hGrx1 mRNA encoding Arg26-Arg27 residues can reduce seleno-hGrx1 expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51.Therefore,we optimized the rare codons,which resulted in a remarkable in- crease of the expression level in the Cys auxotrophic cells,which may be sufficient for future medical production.The engineered artificial selenoenzyme displays high GPx catalytic activity,rivaling that of some natural GPx proteins.Kinetic analysis of the engineered seleno-hGrx1 showed a typical ping-pong kinetic mechanism;its catalytic properties are similar to those of some nat- urally occurring GPx proteins.The active center of human glutaredoxin （hGrxl） shares a common thioredoxin fold and specific affinity for substrate glutathione （GSH） with natural glutathione peroxidase （GPx）. hGrxl was redesigned to introduce the catalytic selenocysteine residue to imitate the function of antioxidant selenoenzyme GPx in vivo. The human hGrxl scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds. Two consecutive rare codons （AGG-AGG） in the open reading frame of hGrxl mRNA encoding Arg26-Arg27 residues can reduce seleno-hGrxl expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51. Therefore, we optimized the rare codons, which resulted in a remarkable increase of the expression level in the Cys auxotrophic cells, which may be sufficient for future medical production. The engineered artificial selenoenzyme displays high GPx catalytic activity, rivaling that of some natural GPx proteins. Kinetic analysis of the engineered seleno-hGrxl showed a typical ping-pong kinetic mechanism; its catalytic properties are similar to those of some naturally occurring GPx proteins.

关 键 词：谷胱甘肽过氧化酶 稀有密码子 氧工程 硒 硫氧还蛋白 营养缺陷型 动力学机制 人造 

分 类 号：Q78[生物学—分子生物学] X835[环境科学与工程—环境工程]