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HIGH EXPRESSION AND PURIFICATION OF RANA CATESBEIANA RIBONUCLEASE IN E.COLI
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- Author:
- No author available
- Journal Title:
- CANCER RESEARCH AND CLINIC
- Issue:
- 4
- DOI:
- 10.3760/cma.j.issn.1006-9801.2004.04.001
- Key Word:
- 核糖核酸酶;融合蛋白;表达;纯化
Abstract： 目的:利用大肠杆菌系统表达重组RC-RNase融合蛋白,并对其表达产物进行初步纯化.方法:以PCR方法扩增出RC-RNase基因,插入到融合蛋白原核表达载体PET32a中,构建重组表达载体PET-RC-RNase,转化大肠杆菌E.coli BL21(DE3)plyS,利用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达.工程菌经超声碎菌,离心后,采用Ni亲合层析法在变性条件下进行初步纯化.结果:经IPTG诱导后,SDS-PAGE和免疫印迹分析显示,工程菌在相对分子质量为3.1×104处出现一条新生蛋白带,目的蛋白占菌体总蛋白的34%,主要以包涵体形式存在.所得包涵体经纯化,纯度可达90%以上.结论:重组融合蛋白RC-RNase在大肠杆菌中获得成功表达,纯化效果令人满意,为进一步研究其生物学特性和功能奠定了良好的基础.
- [1]付勇,刘彦仿,苏勤,等.编码牛蛙核糖核酸酶成熟蛋白基因的cDNA克隆及序列分析[J].2003,(2).
- [2]M. Irie,K. Nitta,T. Nonaka.Biochemistry of frog ribonucleases[J].1998,54(8).
- [3]P, Laccetti,D, Spalletti-Cernia,G, Portella,etc.Seminal ribonuclease inhibits tumor growth and reduces the metastatic potential of Lewis lung carcinoma.[J].1994,54(16).
- [4]Matousek J,Fulka J,Slavik T,etc.Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation.[J].2000,287(5).
- [5]W, Ardelt,S M, Mikulski,K, Shogen.Amino acid sequence of an anti-tumor protein from Rana pipiens oocytes and early embryos. Homology to pancreatic ribonucleases.[J].1991,266(1).
- [6]Wu YN.,Boix E.,Ardelt W.,etc.Role of the N Terminus in Rnase a Homologues - Differences in Catalytic Activity, Ribonuclease Inhibitor Interaction and Cytotoxicity[J].1996,257(5).
- [7]Ardelt W,Saxena SK,Mikulski SM,etc.Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family.[J].2002,277(17).
- [8]Kotchetkov R,Vogel JU,Kornhuber B,etc.Bovine seminal ribonuclease exerts selective cytotoxicity toward neuroblastoma cells both sensitive and resistant to chemotherapeutic drugs.[J].2000,20(2A).
- [9]Martin Michaelis,Josef Matousek,Jens-Uwe Vogel,etc.Bovine seminal ribonuclease attached to nanoparticles made of polylactic acid kills leukemia and lymphoma cell linesin vitro[J].2000,11(5).
- [10]Kleineidam, R.G.,Jekel, P.A.,Beintema, J.J.,etc.Seminal-type ribonuclease genes in ruminants, sequence conservation without protein expression?[J].1999,231(1).