单活性结构域T7核酸酶Ⅰ的制备及性质研究

Preparation and Characterization of T7 EndonucleaseⅠ With Single Active Domain

T7核酸酶Ⅰ(T7EⅠ)是一种能特异识别并拆分重组中间体Holliday Junction(HJ)的多功能核酸酶,该酶不仅能特异识别和拆分HJ,而且能随机切刻双链DNA,特异识别并切割双链DNA中的切刻和单碱基错配位点.构建了两种原核表达载体用于表达MBP和His-tag融合的两种T7EⅠ突变体,MBP-T7EⅠ(E20K)和His6-T7EⅠ(E65K).每一种融合蛋白形成的同源二聚体不具有核酸酶活性,通过共表达或两种蛋白质共变性复性、双标签亲和纯化,最终获得具有单个活性结构域的T7EⅠ异源二聚体(T7EⅠ-SAD).以pUC(AT)和pUC19为底物测试T7EⅠ-SAD的HJ拆分活性、随机切刻活性和切刻位点切割活性,结果显示,T7EⅠ-SAD可特异识别并切刻HJ结构,但丧失了同时引入两个切刻位点的能力,其对pUC(AT)的特异切刻活力和对pUC19的随机切刻活力与野生酶相当.逐级变性梯度洗脱实验显示,T7EⅠ-SAD的稳定性与野生型酶接近,二聚体解离需要5.0～6.0mol/L尿素或1.75～2.0mol/L盐酸胍;凝胶阻滞实验表明T7EⅠ-SAD具备与HJ结构特异结合的能力.为具有细胞毒性蛋白的表达提供了一种新策略,同时提供了一种简便直观的蛋白质二聚体稳定性测试方法.

T7 endonucleaseⅠ （T7EⅠ） is a multiple functional endonuclease from bacteriophage T7, which can act as a reslovase to bind and resolve recombination intermediate Holliday Junction （HJ） specifically, act as a nicking enzyme to nick double-stranded DNA randomly, act as a specific endonuclease to recognize and cleave nicked sites and single-base mismatched sites on duplex DNA. Vector pET21a-MBP-T7E Ⅰ （E20K） and pET28a-T7EⅠ （E65K） were constructed, and MBP and His6 tagged T7EⅠ mutants MBP-T7EⅠ （E20K） and His6-T7EⅠ （E65K） were expressed and purified respectively. Two T7E Ⅰ mutants exist as homodimers, two domains of each mutant are inactive. One MBP-T7EⅠ （E20K） subunit and one His6-T7EⅠ （E65K） subunit can form a heterodimer, which have one active domain and one inactive domain （so named T7EⅠ-SAD）. T7EⅠ-SAD was obtained by two approaches： The first concerns co-expression of two plasmids in E. coli strains ER2566 and purification of T7EⅠ- SAD by amylose and Ni-NTA affinity columns. The second concerns co-denaturing and co-refolding of MBP-T7EⅠ （E20K） and His6-T7EⅠ （E65K） in equal molar and isolation of heterodimer T7EⅠ -SAD by double affinity tagging. Using the cruciform structure-containing plasmid pUC（AT） and general closed circular plasmid pUC19 as substrates, the resolving, random nicking and nicked sites cleavage activity of T7EⅠ -SAD were analyzed. T7EⅠ- SAD can recognize cruciform structure, but it loses the capability to introduce two nicked sites simultaneously. In buffer with Mg2＋, the specific nicking activity of T7EⅠ-SAD to pUC（AT） and random nicking activity to pUC19 are close to that of wide-type enzyme. In buffer with Mn2＋, the random nicking activity of T7EⅠ-SAD is higher than wide-type T7EⅠ significantly. Results of stepwise denaturing and elution experiment showed that the subunits of T7E Ⅰ-SAD can disassociate in 5～6 mol/L urea or 1.75～2.0 mol/L guanidine hydrochloride. Gel-shift assay showed T7EⅠ-SAD can binding to four-way junction substrate Junction3 specifically. This study provides an efficient strategy to prepare cytotoxic protein, it provides a sample intuitive approach to probe the stability of protein dimerization too.