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

BAO Ling-Na1 WU Yong1,2 LIN Jun1 DONG Yuan-Zhen1,2 HUANG Zong-Qing2

(1.Shanghai Institute of Pharmaceutical Industry, China Institute of Pharmaceutical Industry, Shanghai, China 201203)
(2.Shanghai Domray Biotechnology Co., Ltd., Shanghai, China 201203)

【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 folds with a recovery of 46.40% after purification. Cas9 nuclease retained over 65% of its initial activity after incubation at 25–42 °C for 2 h, but it was completely deactivated after treatment at 45 °C for 15 min. The nuclease was stable between pH 6.0 and 10.0 with residual nuclease activity more than 68%, and especially the highest stability at pH 9.0. Mg2+ at concentration of 0.5–20.0 mmol/Lactivated the nuclease, and 10.0 mmol/L Mg2+ increased the nuclease activity by 23%. Besides, this nuclease was inhibited by some metal ions such as Ba2+, Co2+, Ca2+, Mn2+, Cu2+, Fe2+and Zn2+, wherein Cu2+ and Fe2+ completely 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 was characterized, serving as a reference for further promotion and application of this nuclease in CRISPR/Cas9 technology.

【Keywords】 Streptococcus pyogenes Cas9 nuclease; Purification; Ammonium sulfate precipitation; Affinity chromatography; Enzymatic characteristics;

【DOI】

【Funds】 National Key Research and Development Program of China (2017YFD0501404)

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This Article

ISSN:0253-2654

CN: 11-1996/Q

Vol 47, No. 07, Pages 2003-2011

July 2020

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Abstract

  • 1 Material
  • 2 Method
  • 3 Results and analysis
  • 4 Discussion and conclusion
  • References