改变毕赤酵母信号肽对人三叶因子3表达的影响

Effect of Different Signal Peptide on the Secretion of hTFF3 in Pichia Pastoris

为了表达具有天然N 末端的人三叶因子 3,构建了含两种信号肽序列的毕赤酵母表达载体 ,其中一种表达载体在分泌信号肽序列α factor后保留STE13信号肽切割序列 ,另一种表达载体去除了该序列。将 2种表达载体分别转化到毕赤酵母GS115中进行表达。经甲醇诱导后目的蛋白分泌到发酵液上清中 ,SDS PAGE和Western印迹证明 2种重组蛋白均以二体的形式分泌表达 ,分子质量约为 13ku ,都能被TFF3抗体所识别。N 端氨基酸测序证明其中一个重组蛋白具有天然人TFF3的N端 ,质谱检测分子质量与天然蛋白一致 ;另一重组蛋白N 端则带有 2个未切割完全的信号肽序列GluAla ,质谱检测该蛋白中还含有部分切割完全的蛋白。

To expression the native N-termius human trefoil factor 3, two kinds of Pichia pasotris expression vectors were constructed , one contained the STE13 signal sequence and another without the sequence. The two vectors were transformed into the Pichia pastoris. Molecular weight of the expressed peptides was about 13ku identified by SDS-PAGE and Western-blotting.N-terminus amino acids analysis proved one yeast expressed TFF3 with the native N-terminus and the right molecular weight. Another recombinant protein has ahybrid leader sequence GluAla at the N-termius and has two protein MS peaks.