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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
引用本文:口如琴,王蔚,李令媛,茹炳根. 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[J]. 中国生物化学与分子生物学报, 2000, 16(5): 606-611
作者姓名:口如琴  王蔚  李令媛  茹炳根
作者单位:北京大学生命科学学院生物化学与分子生物学系,蛋白质工程国家重点实验室,北京,100871
摘    要: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 .

关 键 词:小肠三叶因子  突变体  生物活性  
收稿时间:2000-10-20
修稿时间:1999-11-05

Construction and Bioactivity of ACys57 Mutant of Human Intestinal Trefoil Factor
KOU Ru-qin,WANG Wei,LI Ling-yuan,RU Bing-gen. Construction and Bioactivity of ACys57 Mutant of Human Intestinal Trefoil Factor[J]. Chinese Journal of Biochemistry and Molecular Biology, 2000, 16(5): 606-611
Authors:KOU Ru-qin  WANG Wei  LI Ling-yuan  RU Bing-gen
Affiliation:KOU Ru-qin,WANG Wei,LI Ling-yuan,RU Bing-gen(Department of Biochemistry and Molecular Biology,College of Life Sciences,Peking University,National Laboratory of Protein Engineering;Beijing 100871,China)
Abstract: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.
Keywords:Intestinal trefoil factor  Mutant  Bioactivity
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