白色念珠菌CYP51蛋白功能性氨基酸残基定.PDFVIP

微生物学通报 OCT 20, 2009, 36(10): 1564~1570 Microbiology © 2009 by Institute of Microbiology, CAS tongbao@ 研究报告 白色念珠菌CYP51 蛋白功能性氨基酸残基定 点突变、蛋白表达及活性测定 1,2 2 2 2* 2 2 陈双红 盛春泉 徐晓辉 姜远英 张万年 何 成 (1. 200433) (2. 200433) 摘 要: 实验设计了白色念珠菌CYP51 蛋白功能性氨基酸残基突变体Y118A 、Y118F 、Y118T 、 S378A、S378T、H310A 、H310R, 并转入基因工程菌 D12667 中表达。用Western 及紫外分光光 度法定性、定量检测蛋白, 用GC-MS 法测定蛋白代谢活性。结果表明, 成功表达目标蛋白, 蛋白 诱导表达量接近微粒体蛋白总量的 25% 。活性测定表明, 表达的野生型蛋白保持其对天然底物的 代谢能力; 相较于野生型蛋白, 突变体蛋白代谢活性不同程度改变, 最多可下降 1/2 左右。因此, 本研究中成功表达了野生型和突变型CYP51 蛋白, 表达的蛋白保留了对天然底物的代谢活性。 关键词: 白念珠菌, 14α-去甲基化酶, 突变, 活性 Expression and Detection the Enzyme Activity of the Wild and Mutation Type of CYP51 Protein of Candida albicans CHEN Shuang-Hong1,2 SHENG Chun-Quan2 XU Xiao-Hui2 JIANG Yuan-Ying2* ZHANG Wan-Nian2 HE Cheng2 (1. The Institution of Naval Medicine, Shanghai 200433, China) (2. Second Military Medical University, Shanghai 200433, China) Abstract: The Y118AY118FY118TS378AS378TH310AH310R mutants of Candida albicans sterol 14α-demethylase (CACYP51) were constructed and heterologously expressed in D12667, the recon- structed strain with the deletion of CYP51 gene of the Y12667. With the strains obtained and microsome enzymes separated, the western blot and the ultraviolet absorption spectrophotometry were used to qualita- tive and quantitative d