利用定点突变法研究精氨酸脱亚胺酶活性的影响机制
Mechanism of arginine deiminase activity by site-directed mutagenesis
投稿时间:2011-09-20  
DOI:  
中文关键词:精氨酸脱亚胺酶,定点突变,蛋白质纯化,酶活力,最适pH,抗癌
英文关键词:arginine deiminase, site-directed mutagenesis, protein purification, enzyme activity, optimal pH, anti-tumor
基金项目:国家自然科学基金 (No. 30900030),国家重点基础研究发展计划 (973计划) (No. 2011CB710800),教育部新世纪优秀人才支持计划(No. NCET- 11-0658)资助。
作者单位E-mail
李利锋 江南大学生物工程学院 工业生物技术教育部重点实验室江苏 无锡 214122  
倪晔 江南大学生物工程学院 工业生物技术教育部重点实验室江苏 无锡 214122 yni@jiangnan.edu.cn 
孙志浩 江南大学生物工程学院 工业生物技术教育部重点实验室江苏 无锡 214122  
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中文摘要:
      精氨酸脱亚胺酶 (ADI) 是一种针对精氨酸缺陷型癌症 (如:肝癌、黑素瘤) 的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1 (A128T)、M2 (H404R)、M3 (I410L) 和M4 (A128T/H404R)。将突变株在大肠杆菌BL21 (DE3) 中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性 (pH 7.4) 条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。
英文摘要:
      Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and I410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced Km value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.
李利锋,倪晔,孙志浩.利用定点突变法研究精氨酸脱亚胺酶活性的影响机制[J].生物工程学报,2012,28(4):508~519
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