Monellin—EGFP融合蛋白在毕赤酵母中的表达

摘要

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.%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。