定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究

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。

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

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.