微生物学报Acta Microbiologica Sinica
48(8)：1067~1074; 4 August 2008
基金项目: 国家自然科学基金(30772586)
*通讯作者。Tel/Fax: +86-510-85918219; E-mail: jinjian31@hotmail.com
作者简介: 吴静(1981. ), 女, 江苏大丰人, 博士研究生, 主要从事细胞与分子药理的研究。E-mail: wujing@jiangnan.edu.cn
吴静1,2，雷楗勇1，张莲芬1,2，花慧1，金坚1, 2*
（1 工业生物技术教育部重点实验室，2 江南大学医药学院分子药理研究室，无锡 214122）
摘要：【目的】为提高重组人ADAM (A Disintegrin And Metalloproteinase) 15 去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15 的cDNA 和GST(谷胱甘肽-S-转移酶)-ADAM15 结构的基础上，选择表达宿主菌并对表达质粒进行改造。【结果】(1)选择能为大肠杆菌稀有密码子提供额外tRNA 的Escherichia coli. Rosetta(DE3)作为宿主菌，将质粒pGEX-ADAM15转化于其中在最佳诱导表达条件下获得298 mg/L 融合蛋白GST-ADAM15；(2) 采用PCR 体外定点突变技术将目标蛋白编码区稀有密码子GGA(Gly425)替换为GGC，使融合蛋白表达水平提高9.4%；(3) 通过消除凝血酶识别序列附近的Pro-Glu-Phe 残基，提高凝血酶酶切效率，使rhADAM15 产量提高了35.7%；(4)在GGA 替换为GGC 基础上切除“Pro-Glu-Phe”残基，使rhADAM15 产量提高到68 mg/L，比分别切除“Pro-Glu-Phe”残基、GGA 替换为GGC 和野生型提高了19.2%、51.1%和61.9%。【结论】这一结果表明，在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达。
关键词：ADAM15 去整合素结构域；大肠杆菌Rosetta (DE3)；稀有密码子；PCR 定点突变；抑制血管生成
中图分类号：Q816 文献标识码：A 文章编号：0001-6209 (2008) 08-1067-08
Improving production and bioactivity of recombinant human disintegrin domain of ADAM15 (rhADAM15) in Escherichia Coli
Jing Wu1,2, Jianyong Lei1, Lianfen Zhang1,2, Hui Hua1, Jian Jin1,2*
(1Key Laboratory of Industrial Biotechnology, Ministry of Education, 2Department of Pharmaceutical and Molecular Biotechnology, School of Pharmaceutical and medical, Jiangnan University, Wuxi 214122, China)
Abstract: [Objective] To enhance the production and bioactivity of recombinant human disintegrin domain of ADAM15 (A Disintegrin and Metalloproteinase 15) (rhADAM15) in Escherichia coli. [Methods] The expression host was chose and the recombinant expression plasmid pGEX-ADAM15 was constructed, based on analysis of the cDNA sequence of rhADAM15. [Results] (1) 298 mg/L GST (Glutathione-S-transferase)-ADAM15 and 42 mg/L rhADAM15 were achieved when choosing E. coli Rosetta (DE3) as the expression host that could supply additional tRNA for rare codons. (2) The GST-ADAM15 expression level increased to 326 mg/L after changing the rare codon GGA (Gly425) to GGC by PCR (Polymerase Chain Reaction)-based site-directed mutagenesis. (3) The rhADAM15 concentration increased to 57 mg/L by deleting the “Pro-Glu-Phe” at the GST-ADAM15 junction to improve the thrombin cleavage efficiency. (4) Finally, by combinational introduction of the favorable codons and suitably eliminating of certain amino acid sequence, rhADAM15 concentration reached the highest level (68 mg/L). [Conclusion] The high expression of heterologous protein could be achieved by releasing rare codon usage and amino acids residues restriction.
Keywords: recombinant ADAM15 disintegrin domain; Escherichia coli Rosetta (DE3); rare codons; PCR site-directed mutagenesis; anti-angiogenesis
生物谷推荐全文下载：改善稀有密码子和氨基酸残基限制提高重组人ADAM15去整合素结构域蛋白表达水平
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