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  

Expression and Detection the Enzyme Activity of the Wild and Mutation Type of CYP51 Protein of Candida albicans

作　　者：陈双红[1,2] 盛春泉[2] 徐晓辉[2] 姜远英[2] 张万年[2] 何成[2] 

机构地区：[1]海军医学研究所,上海200433 [2]第二军医大学,上海200433

出　　处：《微生物学通报》2009年第10期1564-1570,共7页Microbiology China

基　　金：国家自然科学基金重点项目(No.30430750)

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。The Y118A, Y118F, Y118T, S378A, S378T, H310A, H310R mutants ofCandidaalbicans sterol 14α-demethylase （CACYP51） were constructed and heterologously expressed in D 12667, 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 qualitative and quantitative detect the expressed protein, the GC-MS was used to detect the metabolism activity of the protein. The results showed that, the target protein expressed successfully in the reconstructed strains, with the expression level up to 25% of the total microsome proteins. The results also showed that, the wild type protein had the catalytic activity to its nature substrate. While after alteration the wild gene with Y 118A, Y 118F, Y 118T, S378A, S378T, H310A, H310R by a single base substitution, the catalytic activity of protein markedly decreased respectively. So the wild type and mutation CYP51 were expressed successfully in Saccharomyces cerevisiae and the expression products preserved the activity to metabolism their nature substrate.

关 键 词：白念珠菌 14α-去甲基化酶 突变 活性 

分 类 号：Q78[生物学—分子生物学] Q51