人小肠三叶因子缺失半胱氨酸57突变体的构建及其生物活性

用 PCR方法构建了一个不能形成二聚体的 C端缺失半胱氨酸 57的 h ITF突变体 .将其克隆到大肠杆菌表达载体 p GEX- 4T- 1中 ,ITPG诱导表达 ,融合蛋白经 Glutathione- Sepharose 4B亲和层析 ,凝血酶酶切和 Sephacryl S 1 0 0纯化 ,得到突变体蛋白 .SDS- PAGE,氨基酸组成 ,飞行质谱 ,N端氨基酸序列测定结果与期望值一致 .研究表明突变体的生物学活性有所降低 .对胃蛋白酶作用的稳定性降低 .

Construction and Bioactivity of ACys57 Mutant of Human Intestinal Trefoil Factor

A mutant that deleted Cys57 of human intestinal trefoil factor was construcuted through PCR amplification and expressed as fusion protein with GST in E.coli. The fusion protein was purified through three steps,Glutathione Sepharose 4B affinity chromatography,thrombin digestion and gel filtration on Sephacryl S 100.The results of SDS PAGE,mass spectrometry,amino acid composition and sequence of seven amino acids at the N terminus of the expressed protein were as expected.The biological activity of the mutant was much lower than that of hITF due to its sensitivity to pepsin digestion.