融合标签谷胱甘肽S-转硫酶高亲和配体的设计与筛选  

Design and screening of high-affinity ligands for glutathione S-transferase as fusion tag

作　　者：杨宪峰[1] 张灵[1] 杨晓兰[1] 龙高波[1] 张奕[1] 廖飞[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2012年第9期791-795,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30472139;81071427);重庆市教委科技资助项目(编号:KJ100313)

摘　　要：目的:设计与筛选融合标签血吸虫谷胱甘肽S-转硫酶(Glutathione S-transferase,GST)的高亲和配体。方法:将原核融合表达载体pGST-MOLUC转入大肠杆菌,异丙基-β-D-硫代半乳糖苷低温诱导GST标签表达,用Glutathione Sepharose 4B亲和纯化此标签GST。初速度法测定GST对底物1-氯-2,4-二硝基苯(1-chloro-2,4-dinitrobenzene,CDNB)和还原型谷胱甘肽(Glutathione,GSH)的Km;测定GST产物S-(2,4-二硝基苯基)-谷胱甘肽(GS-DNB)、3,5-二甲基苯甲酸、对丁基苯甲酸、依他尼酸及3者对应的对称双酰丁二胺对标签GST的抑制作用。结果:纯化标签GST对GSH和CDNB顺序结合且Km都高于0.10mmol/L;GS-DNB相对CDNB的竞争性抑制常数约5μmol/L,相对GSH的非竞争性抑制常数约33μmol/L。3,5-二甲基苯甲酸、对丁基苯甲酸、依他尼酸对此GST抑制常数都大于0.20 mmol/L;N,N’-双依他尼酰-1,4-丁二胺和N,N’-双对丁基苯甲酰-1,4-丁二胺都为GST相对GSH的强竞争性抑制,抑制常数分别为31 nmol/L和(0.61±0.43)μmol/L(n=3)。结论:将标签GST低亲和力芳香羧酸用柔性短链连成对称结构,可获得结合于单个活性中心的高亲和力标签GST抑制剂。Objective：To design and screen high-affinity ligands for schistosoma japonicum glutathione S-transferase（GST） as fusion tag. Methods：pGST-MOLUC was induced to express GST in Escherichia coli BL2I under low temperature and GST was purified by glutathione Sepharose 4B affinity chromatography. By the initial velocity method, K,, of GST aganist glutathione （GSH） and 1-chloro- 2, d-dinitrobenzene （CDNB） was estimated. S- （2,4-dinitrobenzne） -glutathione （GS-DNB）, 3,5-dimethylbenzoic acid, 4-butylben- zoic acid and ethacrynic acid were tested and the inhibition effect of symmetrical biamide of such aromatic carboxylic acid on GST was examed. Results：Homogenous GST was successfully obtained. GSH and CDNB followed sequential bisubstrate kinetic mech anism with both Km over 0.10 mmol/L. GS-DNB had a competitive Ki of about 5.0 ~mol/L against CDNB but a noncompetitive Ki of about 33 μmol/L against GSH. 3,5-dimethylbenzoic acid, 4-butylbenzoic acid, and ethacrynic acid showed Ki over 0.20 mmol/L, but N, N＇ -bis-ethaerynyl- 1,4-butyldiamine and N, N＇ -bis- （4 - （ n-butyl ） -benzoyl） - 1,4-butyldiamine displayed strong competitive inhibition on this GST against GSH with Ki of 31 nmol/L and （0.61 ± 0.43） μmol/L（n=3） ,correspondingly. Concision：Symmetrical biamide of low-affinity aromatic carboxylic acid linked via short linear chain has promise to be high-affinity inhibitors to single active site of GST.

关 键 词：谷胱甘肽S-转硫酶 亲和力 抑制剂 配体 

分 类 号：Q555.8[生物学—生物化学]