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

Improving production and bioactivity of recombinant human disintegrin domain of ADAM15 (rhADAM15) in Escherichia Coli

【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298mg/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68mg/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。

[Objective] To enhance the production and bioactivity of recombinant human disintegrin domain of ADAM15 （A Disintegrin and Metalloproteinase 15） （rhADAM15） in Escherichia coll. [Methods] The expression host was chose and the recombinant expression plasmid pGEX-ADAM15 was constructed, based on analysis of the cDNA sequence of rhA- DAM15. [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.