Monellin-EGFP融合蛋白在毕赤酵母中的表达  

Expression of Monellin-EGFP Fusion Protein in Pichia pastoris

作　　者：金筱耘[1] 赵爱春[1] 李军[1] 李镇刚[1] 王茜龄[1] 吴存容[1] 余茂德[1] 

机构地区：[1]西南大学生物技术学院,重庆400716

出　　处：《食品科学》2012年第13期171-175,共5页Food Science

基　　金：国家现代农业产业技术体系建设专项(CARS-22-ZJ0103);重庆市蚕桑重大科技专项(CSTC;2009AA1024)

摘　　要：根据已报道的Monellin甜蛋白氨基酸序列,结合毕赤酵母与桑树密码子的偏好性,人工设计合成单链monellin基因,并与增强型绿色荧光蛋白EGFP基因连接,构建融合表达载体。采用电击转化法成功将表达载体导入毕赤酵母GS115中,并获得高效表达的重组子pPIC9K-M-E。经PCR检测,SDS-PAGE和Western Blot杂交证实已表达出融合蛋白,该蛋白经纯化与盲测实验,结果显示其甜度约为标准蔗糖的500倍。According to the reported amino acid sequence of single-chain monellin, the codon bias of Pichia pastoris and Morus alba L., a synthetic monellin gene was achieved.Then, the enhanced green fluorescent protein （EGFP） gene was added into the downstream of monellin gene to construct an expression vector named as pPIC9K-M-E. Based on transformation of yeast by electroporation, the expression vector pPIC9K-M-E was successfully imported into Pichia pastoris. PCR amplification, fluorescence detection, Western Blot analysis and SDS-PAGE results demonstrated that the fusion protein was expressed and the fusion protein revealed high intensity of sweetness, which was almost 500 times sweeter than the sucrose with same amount.

关 键 词：Monellin甜蛋白 绿色荧光蛋白 真核表达 桑树 

分 类 号：Q786[生物学—分子生物学]