限制性核酸内切酶Asc I蛋白质的表达纯化、酶活测定及小角散射结构解析  

Expression,Purification and Biochemical and Structural Characterization of the Type Ⅱ Restriction Endonuclease Asc I

作　　者：方倩倩 陈浩 廖馨源 林忠辉 FANG Qian-Qian;CHEN Hao;LIAO Xin-Yuan;LIN Zhong-Hui(National&Local Joint Biochemical Engineering Research Center on Photodynamic Technologies,College of Chemistry,Fuzhou University,Fuzhou 350108,China)

机构地区：[1]福州大学化学学院生物药光动力国家地方联合工程中心,福州350108

出　　处：《中国生物化学与分子生物学报》2023年第2期259-268,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31971222)资助。

摘　　要：限制-修饰系统(RMS)是细菌为了防御外来DNA入侵而进化产生的一种保护机制。RMS系统可分为Ⅰ型、Ⅱ型、Ⅲ型和Ⅳ型。Asc I是一种Ⅱ型限制性核酸内切酶,能识别8-bp DNA基序。尽管Asc I已经被广泛应用于分子克隆研究中,然而目前尚无关于Asc I蛋白质的表达、纯化以及结构机制等方面的研究报道。本研究基于大肠杆菌重组表达体系建立了Asc I重组蛋白质的高效表达和纯化方法,从每升细菌培养物中可获得约2.5 mg纯度大于95%的Asc I蛋白质。进一步的酶学性质研究表明,Asc I酶切反应的最适温度是37℃,最适pH为7.5~8.5,反应依赖于Mg^(2+)和Mn^(2+)等二价金属离子。基于小角X射线散射(SAXS)分析技术,我们还建立了Asc I蛋白质及其与底物DNA复合物在溶液状态下的三维空间模型,并结合点突变对该模型进行了验证。总之,本研究对Asc I蛋白质的重组表达、纯化、酶活性质以及结构机制进行了比较系统地研究,为了解RMS系统的工作机制提供了结构基础,同时也为Asc I作为分子克隆工具酶的进一步开发和改造提供了理论依据。The bacterial restriction-modification system(RMS) plays a pivotal role in cellular defense against foreign DNA.It can be classified into four major groups:types Ⅰ,Ⅱ,Ⅲ and Ⅳ.Asc I belongs to the type Ⅱ restriction endonucleases and can specifically recognize an 8-bp DNA element.Despite being used extensively in molecular cloning,the information for protein expression,purification and three dimensional structure of Asc I has been lacking.Here,we developed a bacterial expression system and an efficient purification procedure,and obtained recombinant Asc I protein with a yield of ~ 2.5 mg per liter culture for over 95% purity.Further enzymatic characterization showed that the optimum temperature and for enzymatic reaction were 37^(o)C and pH 7.5~8.5,respectively.The enzyme required divalent metal ions for activity,with a strong preference for Mg^(2+) and Mn^(2+).Furthermore,with the Small Angle X-ray Scattering(SAXS) and extensive mutagenesis analyses,we constructed structural models showing how Asc I might interact with its DNA substrate in solution.Together,our work provides biochemical and structural characterizations of Asc I endonuclease,and paves the way for further applications of the enzyme in molecular cloning.

关 键 词：限制性核酸内切酶 Asc I 表达纯化 酶学性质 小角散射 

分 类 号：Q55[生物学—生物化学]