定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究

中国生物工程杂志

2012, Vol. 32

Issue (05): 79-84

技术与方法

定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究

荀启静, 林瑛, 邱沛然, 孟清

东华大学生物科学与技术研究所上海 201620

High Splicing Activity of Ter DnaE-3 Mini-intein through Directed Evolution

XUN Qi-jing, LIN Ying, QIU Pei-ran, MENG Qing

Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai 201620, China

全文: PDF(594 KB) HTML

摘要：

目前,蛋白质内含子在蛋白质工程领域中得到越来越广泛的应用。为提高微小蛋白质内含子Ter DnaE-3 (Trichodesmium erythraeum) 在异源宿主中的剪接活性,采用易错PCR技术,通过改变反应体系中dNTP、Mg²⁺、Mn²⁺的浓度等手段,借助依赖卡那霉素的蛋白质内含子筛选系统进行筛选。Western印迹结果表明:通过定向进化,其中5号突变体的剪接活性从原始的约20%提高至约85%;9号突变体能够避免发生剪接副反应,即N端断裂反应。氨基酸突变位点与剪接活性变化的相关性分析表明:参与α-helix形成的氨基酸的突变极有可能影响蛋白质内含子的断裂反应,参与β-sheet形成的氨基酸的突变则有可能影响蛋白质内含子结构的紧凑性。通过定向进化提高微小蛋白质内含子Ter DnaE-3在异源宿主中的剪接活性,进一步验证依赖卡那霉素抗性的筛选系统的可行性,为扩大蛋白质内含子的应用范围奠定基础。

关键词： 蛋白质内含子; 卡那筛选系统; 定向进化; 蛋白质剪接

Abstract:

Natural and artificially engineered inteins have been more widely used in the field of protein engineering. However, inteins are often inactive when cloned into a heterologous host protein, and also need the presence of the native exteins, which would be left in the target protein. Inteins should overcome these limitations. In order to improve splicing activity of Ter DnaE-3 (Trichodesmium erythraeum) mini-intein in heterologous host, random mutagenesis was based on error PCR by change the concentration of dNTP、Mg²⁺、Mn²⁺, and then mutants were screened in kanamycin resistance-dependent system. After directed evolution, mutant 5th increased splicing activity from ~20% to ~85%, and mutant 9th can avoid occurring cleavage compared with other mutants by western blot analysis. The relationship of amino acid mutations and splicing activity showed that amino acids involved in the formation of α-helix structure may influence intein cleavage reaction, and amino acids involved in the formation of β-sheet structure may provide some help for intein structure. The resulting improved inteins showed high activity in heterologous host, which verify the kanamycin resistance-dependent system feasibility and expand intein application.

Key words: Intein Kanamycin resistance-dependent system Directed evolution Protein splicing

收稿日期: 2011-12-30 出版日期: 2012-05-25

ZTFLH:

Q816

基金资助:

国家"863"计划专题项目(2006AA03Z451)、国家自然科学基金面上项目(31070698)、国家自然科学基金委青年基金(30800186)、教育部博士点新教师基金(200802551026)资助项目

通讯作者: 孟清 E-mail: mengqing@dhu.edu.cn

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引用本文:

荀启静, 林瑛, 邱沛然, 孟清. 定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究[J]. 中国生物工程杂志, 2012, 32(05): 79-84.

XUN Qi-jing, LIN Ying, QIU Pei-ran, MENG Qing. High Splicing Activity of Ter DnaE-3 Mini-intein through Directed Evolution. China Biotechnology, 2012, 32(05): 79-84.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I05/79

[1] Perler F B, Davis E O, Dean G E, et al. Protein splicing elements: inteins and exteins-a definition of terms and recommended nomenclature. Nucleic Acids Res, 1994, 22(7): 1125-1127.

[2] Perler F B. InBase: the Intein Database. Nucleic Acids Res, 2002, 30(1): 383-384.

[3] Perler F B. Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution. Cell, 1998, 92(1): 1-4.

[4] Liu X Q. Protein-splicing intein: Genetic mobility, origin, and evolution. Annu Rev Genet, 2000, 34: 61-76.

[5] Saleh L, Perler F B. Protein splicing in cis and in trans. Chem Rec, 2006, 6(4): 183-193.

[6] Mills K V, Perler F B. The mechanism of intein-mediated protein splicing: variations on a theme. Protein Pept Lett, 2005, 12(8): 751-755.

[7] Muir T W, Sondhi D, Cole P A. Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci USA, 1998, 95(12): 6705-6710.

[8] Ludwig C, Pfeiff M, Linne U, et al. Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angew Chem Int Ed Engl, 2006, 45(31): 5218-5221.

[9] Vila-Perello M, Muir T W. Biological Applications of Protein Splicing. Cell, 2010, 143: 191-200.

[10] Mills K V, Lew B M, Jiang S, et al. Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein. Proc Natl Acad Sci USA, 1998, 95(7): 3543-3548.

[11] Ludwig C, Schwarzer D, Mootz H D. Interaction studies and alanine scanning analysis of a semi-synthetic split intein reveal thiazoline ring formation from an intermediate of the protein splicing reaction. J Biol Chem, 2008, 283(37): 25264-25272.

[12] 林瑛,周倩,荀启静,等. 高剪接活性断裂蛋白质内含子的体内切割. 中国生物工程杂志, 2011, 31(8): 97-101. Lin Y, Zhou Q, Qun Q J, et al. Cleavage of Split-Inteins with High Splicing Activity. China Biotechnology, 2011, 31(8): 97-101.

[13] 周倩,孟清. 异源生物中筛选高剪接活性Intein系统的建立. 中国生物化学与分子生物学报, 2009, 25(3): 282-286. Zhou Q, Meng Q. An Efficient Screening System of Active Intein in Heterologous Hosts. Chinese Journal of Biochemistry and Molecular Biology, 2009, 25(3): 282-286.

[14] 李曼,孟清. 易错PCR/卡那霉素筛选系统定向进化蛋白质内含子:中国,C12N15/65. 2011-10-05. Li M, Meng Q. Error prone PCR/Kanamycin directed evolution system for improving inteins: China, C12N15/65. 2011-10-05.

[15] Oeemig J S, Aranko A S, Djupsjobacka J, et al. Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett, 2009, 583(9): 1451-1456.

[16] Appleby-Tagoe J H, Thiel I V, Wang Y, et al. Highly efficient and more general cis- and trans-splicing intein through sequential directed evolution. J Biol Chem, 2011, 286(39):34440–34447.

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