公开(公告)号：KR1020040087358A

公开(公告)日：2004-10-14

申请号：KR1020030021542

申请日：2003-04-07

Applicant: 재단법인서울대학교산학협력재단

Inventor： 오규희 ,  방옥선 ,  정진하

IPC: C07K19/00

CPC classification number: C07K14/00 ,  C07K2319/00 ,  C12N15/52 ,  C12N15/62 ,  C12N15/70

Abstract: PURPOSE: A screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage is provided, thereby easily screening ubiquitin and ubiquitin-like protein specific proteases and simultaneously measuring activity of enzymes, so that the enzymes are useful for development of drugs for treating brain neuronal diseases. CONSTITUTION: The screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage comprises the steps of: (1) cloning Ub, UbI and cI genes, preparing a mutant of cI gene, preparing an expression plasmid of Ub-cI* or UbI-cI* fusion protein, and preparing transformed E. coli using the expression plasmid; (2) introducing cDNA library into the transformed E. coli using recombinant lambda-phage; (3) selecting E. coli expressing desired protein by heat shock at 42 deg. C for 2 hours; and analyzing enzyme activity of the expressed protein, wherein the Ub-cI* or UbI-cI* fusion protein is produced by fusing ubiquitin-like protein Nedd8 or SUMO-1 with cI protein mutant(cI*); and the fusion protein expression plasmid is pACYC184.

Abstract translation: 目的：提供使用λ噬菌体cI基因的泛素和泛素样蛋白特异性蛋白酶的筛选方法，从而轻松筛选泛素和泛素样蛋白特异性蛋白酶，同时测定酶的活性，使酶可用于开发 用于治疗脑神经元疾病的药物。 构成：使用λ噬菌体cI基因的泛素和泛素样蛋白特异性蛋白酶的筛选方法包括以下步骤：（1）克隆Ub，UbI和cI基因，制备cI基因突变体，制备Ub- cI *或UbI-cI *融合蛋白，并使用表达质粒制备转化的大肠杆菌; （2）使用重组λ噬菌体将cDNA文库导入转化的大肠杆菌; （3）通过42度热休克选择表达所需蛋白质的大肠杆菌。 C 2小时; 并分析表达蛋白的酶活性，其中通过将泛素样蛋白Nedd8或SUMO-1与cI蛋白突变体（cI *）融合产生Ub-cI *或UbI-cI *融合蛋白; 融合蛋白表达质粒为pACYC184。