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N端缺失突变对核糖核酸酶抑制因子活性的影响

Activity Effect of Ribonuclease Inhibitor by Deletion-Mutagenesis and Expressing in Pichia pastoris

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【作者】 吴毓赵宝昌王继红任风吴妍宁赵鹏崔秀云

【Author】 WU Yu 1),ZHAO Bao-chang 2)*, WANG Ji-hong 2),REN Feng 2),WU Yan-ning 2),ZHAO Peng 2), CUI Xiu-yun 2) ( 1)College of Life Sciences,Liaoning Normal University,Dalian 116029,China; 2)Department of Biochemistry and Molecular Biology,Dalian Medical University,Dalian 116027, China)

【机构】 辽宁师范大学生命科学学院大连医科大学生物化学与分子生物学教研室大连医科大学生物化学与分子生物学教研室 大连116029大连116027大连116027

【摘要】 人胎盘核糖核酸酶抑制因子(HRI)是一种存在于细胞浆中的50 kD的酸性蛋白质,富含亮氨酸和半肤氨酸.作为胞浆蛋白可保护细胞不受外来胰RN白s。侵袭.HRI主要结构是由7个富含亮氨酸的重复序列组成,7个亮氨酸重复单位有规律环状排列使N端、c端在空间上较为接近.用PcR方法在HRI cl〕NAS,端去除30个核普酸,并将此缺失突变的HRI的cl〕NA片段构建于质粒pPIcgK,电击转化入毕赤酵母(Pi峨i。 Pasto汀S)Gslls中,进行分泌型表达.对表达产物进行亲和层析纯化.实验结果表明,N端缺失突变的HRI与RN白s。A的亲合力较野生型HRI降低1倍,但依然具有竞争性抑制RNas。A的活性,表明HRIN端10个氨基酸残基缺失后并未丧失其抑制活性.

【Abstract】 Human placental ribonuclease inhibitor (HRI) is an acidic protein of M r~50 kD with an amino acid composition characterized by unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI is consisted of 7 leucine-rich repeats. The repeat units must form either a circle, a helix, or a line. Of these, a circular arrangement may bring the N- and C-termini into close proximity. 5′-termini of 30 bp deletion-mutated HRI cDNA was constructed and inserted into plasmid pPIC9K, and then transformed into Pichia pastoris GS115 by electroporation. After screening the colony, the transformant was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with deletion-mutation for RNase A and its capacity of inhibition to RNase A activity were examined. The results indicated that there was two-times decreases in the affinity for RNase A compared with the wild type of RI. Residues 1~10 of HRI could be deleted without destroying its inhibitory activity.

【基金】 辽宁省教育厅专项基金 (No .2 0 2 72 2 80 )~~
  • 【文献出处】 中国生物化学与分子生物学报 ,Chinese Journal of Biochemistry and Molecular Biology , 编辑部邮箱 ,2004年06期
  • 【分类号】R341
  • 【被引频次】4
  • 【下载频次】84
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