酿脓链球菌Cas9核酸酶的重组表达、分离纯化及酶学特性

Expression, purification and enzymatic characterization of recombinant Streptococcus pyogenes Cas9 nuclease

摘要:

【背景】 Cas9核酸酶是一种RNA引导的核酸内切酶，可与单链向导RNA (single-guide RNA，sgRNA)形成稳定的核糖核蛋白复合物，识别和切割特定的核苷酸片段。由于其具备高灵活性和高效率的特点，目前已经成为基础科学研究领域和临床治疗方法中使用最广泛的基因编辑工具。【目的】为Cas9核酸酶的合理开发和利用提供理论依据。【方法】利用大肠杆菌表达系统表达野生型酿脓链球菌(Streptococcus pyogenes) Cas9核酸酶，经硫酸铵沉淀和镍柱亲和层析两步纯化获得较高纯度表达产物，并对其热稳定性、pH稳定性、金属离子的影响等酶学特性进行研究。【结果】经高密度发酵后，大肠杆菌湿菌重达191.0 g/L。纯化后酿脓链球菌Cas9核酸酶的比酶活达641.29 U/mg，纯化倍数为16.02，收率为46.40%。Cas9核酸酶在25?42 °C保温2 h后剩余酶活保持在65%以上，而在45 °C保温15 min后全部失活；其在pH 6.0?10.0范围内稳定性较高，剩余酶活大于68%，在pH 9.0时稳定性最高；0.5?20.0 mmol/L浓度范围内的Mg2+对该酶有激活作用，10.0 mmol/L的Mg2+可使该酶酶活力提高约23%；Ba2+、Co2+、Ca2+、Mn2+、Cu2+、Fe2+、Zn2+对该酶有不同程度的抑制作用，其中0.5 mmol/L的Cu2+和Fe2+对Cas9核酸酶有完全抑制作用。【结论】异源表达并纯化出具有较高纯度和较高酶活力的酿脓链球菌Cas9核酸酶，并对其酶学特性进行了初步研究，该结果对CRISPR/Cas9技术的进一步推广和应用有一定的指导意义。

Abstract:

[Background] Cas9 nuclease is a site-specific RNA-guided endonuclease that can form a stable ribonucleoprotein complex with single guide RNA (sgRNA), which recognizes and cleaves target DNA molecules. Due to its high flexibility and efficiency, Cas9 is the most widely used gene-editing tool in both basic research and clinical treatment. [Objective] To provide a theoretical basis for the rational development and utilization of Cas9 nuclease. [Methods] The wild type Cas9 nuclease from Streptococcus pyogenes was expressed in the Escherichia coli system. The expressed enzyme was then purified by ammonium sulfate precipitation and Ni2+-affinity chromatography. Finally, the purified Cas9 nuclease was characterized for its thermal stability, pH stability and the influence of metal ions. [Results] The results showed that the wet weight of the bacteria was 191.0 g/L after high-density fermentation. The specific activity was 641.29 U/mg and the bacteria was purified up to 16.02 times with a recovery of 46.40% after purification. Cas9 nuclease retained over 65% of its initial activity after incubation between 25 and 42 °C for 2 h, but it was completely deactivated after treatment at 45 °C for 15 min. The enzyme was stable between pH 6.0 and 10.0 with residual enzyme activity more than 68%, and especially the highest stability at pH 9.0. Mg2+ activated the enzyme between 0.5 and 20.0 mmol/L, and 10.0 mmol/L Mg2+ increased the enzyme activity by 23%. Besides, this enzyme was inhibited by some metal ions such as Ba2+, Co2+, Ca2+, Mn2+, Cu2+, Fe2+ and Zn2+, wherein Cu2+ and Fe2+ completly inhibited Cas9 nuclease at a concentration of 0.5 mmol/L. [Conclusion] Cas9 nuclease from Streptococcus pyogenes was heterologously expressed and purified with high purity and high activity. The purified Cas9 nuclease were characterized, serving as reference for further promotion and application of this enzyme in CRISPR/Cas9 technology.