微生物學(xué)報Acta Microbiologica Sinica
48(8)：1067~1074; 4 August 2008
基金項目: 國家自然科學(xué)基金(30772586)
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作者簡介: 吳靜(1981. ), 女, 江蘇大豐人, 博士研究生, 主要從事細(xì)胞與分子藥理的研究,。E-mail: [email protected]
吳靜1,2,，雷楗勇1，張蓮芬1,2,，花慧1,，金堅1, 2*
（1 工業(yè)生物技術(shù)教育部重點實驗室，2 江南大學(xué)醫(yī)藥學(xué)院分子藥理研究室,，無錫 214122）
摘要：【目的】為提高重組人ADAM (A Disintegrin And Metalloproteinase) 15 去整合素結(jié)構(gòu)域蛋白(rhADAM15)的表達(dá)水平,?！痉椒ā吭谠敱M分析rhADAM15 的cDNA 和GST(谷胱甘肽-S-轉(zhuǎn)移酶)-ADAM15 結(jié)構(gòu)的基礎(chǔ)上，選擇表達(dá)宿主菌并對表達(dá)質(zhì)粒進(jìn)行改造,?！窘Y(jié)果】(1)選擇能為大腸桿菌稀有密碼子提供額外tRNA 的Escherichia coli. Rosetta(DE3)作為宿主菌，將質(zhì)粒pGEX-ADAM15轉(zhuǎn)化于其中在最佳誘導(dǎo)表達(dá)條件下獲得298 mg/L 融合蛋白GST-ADAM15,；(2) 采用PCR 體外定點突變技術(shù)將目標(biāo)蛋白編碼區(qū)稀有密碼子GGA(Gly425)替換為GGC,，使融合蛋白表達(dá)水平提高9.4%；(3) 通過消除凝血酶識別序列附近的Pro-Glu-Phe 殘基,，提高凝血酶酶切效率，使rhADAM15 產(chǎn)量提高了35.7%,；(4)在GGA 替換為GGC 基礎(chǔ)上切除“Pro-Glu-Phe”殘基,，使rhADAM15 產(chǎn)量提高到68 mg/L，比分別切除“Pro-Glu-Phe”殘基,、GGA 替換為GGC 和野生型提高了19.2%,、51.1%和61.9%?！窘Y(jié)論】這一結(jié)果表明,，在充分認(rèn)識目標(biāo)蛋白特性的基礎(chǔ)上定向選擇表達(dá)宿主并改造表達(dá)質(zhì)粒能實現(xiàn)外源蛋白高水平表達(dá)。
關(guān)鍵詞：ADAM15 去整合素結(jié)構(gòu)域,；大腸桿菌Rosetta (DE3),；稀有密碼子；PCR 定點突變,；抑制血管生成
中圖分類號：Q816 文獻(xiàn)標(biāo)識碼：A 文章編號：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
生物谷推薦全文下載：改善稀有密碼子和氨基酸殘基限制提高重組人ADAM15去整合素結(jié)構(gòu)域蛋白表達(dá)水平
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