限制性内切酶的无细胞快速制备研究

刘晚秋; 季向阳; 许慧玲; 卢屹聪; 李健

doi:10.12211/2096-8280.2022-048

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限制性内切酶的无细胞快速制备研究

研究论文 | 更新时间：2025-03-20

- 限制性内切酶的无细胞快速制备研究
- Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases
- 合成生物学 2023年4卷第4期页码：840-851
- 作者机构：
  
  上海科技大学物质科学与技术学院，上海 201210
- 作者简介：
  
  [ "刘晚秋（1986—），女，博士，副研究员。研究方向为无细胞合成生物学。E-mail：liuwq@shanghaitech.edu.cn" ]
  [ "李健（1982—），男，博士，研究员，博士生导师。研究方向为无细胞合成生物学。E-mail：lijian@shanghaitech.edu.cn" ]
- 基金信息：
  
  国家自然科学基金(32171427;31971348)
- DOI：10.12211/2096-8280.2022-048
  中图分类号： Q814.1
- 收稿：2022-09-06，
  
  修回：2022-12-01，
  
  纸质出版：2023-08-31
- 稿件说明：
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刘晚秋，季向阳，许慧玲，卢屹聪，李健. 限制性内切酶的无细胞快速制备研究［J］. 合成生物学， 2023， 4（4）： 840-851

LIU Wanqiu， JI Xiangyang， XU Huiling， LU Yicong， LI Jian. Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases［J］. Synthetic Biology Journal， 2023， 4（4）： 840-851
刘晚秋，季向阳，许慧玲，卢屹聪，李健. 限制性内切酶的无细胞快速制备研究［J］. 合成生物学， 2023， 4（4）： 840-851 DOI： 10.12211/2096-8280.2022-048.

LIU Wanqiu， JI Xiangyang， XU Huiling， LU Yicong， LI Jian. Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases［J］. Synthetic Biology Journal， 2023， 4（4）： 840-851 DOI： 10.12211/2096-8280.2022-048.

摘要

限制性内切酶在分子生物学研究中是一类重要的工具酶，目前主要由异源生物合成的方式进行表达与生产，由于它们对特定的DNA序列（即酶切位点）具有切割活性，在异源表达时会对宿主产生较高的细胞毒性。而无细胞生物合成体系具有操作快捷、灵活高效、无细胞毒性等优势，因此，本研究利用无细胞蛋白合成（cell-free protein synthesis， CFPS）技术进行限制性内切酶的表达制备。本课题组选择3种限制性内切酶

Eco

RⅠ、

Bam

HⅠ和

Bsa

Ⅰ作为研究对象，构建线性DNA为表达模板，无需甲基化酶对宿主的保护，在6 h内即可完成蛋白表达。经亲和色谱与凝胶色谱两步纯化，得到了纯度高（95%左右）、酶活相当（

Eco

RⅠ 3.7 × 10

~3.7 × 10

U/mg，

Bam

HⅠ 8.3 × 10

~4.1 × 10

U/mg，

Bsa

Ⅰ 4.4 × 10

~ 4.4 × 10

U/mg）的目标蛋白。同时，建立了限制性内切酶的实时酶活检测方法，将有助于限制性内切酶的催化和快速筛选研究。本研究所开发的限制性内切酶无细胞表达制备体系，从基因模板构建到纯化蛋白所需时间短（1~2 d）、蛋白产量高（32.5~130 mg/L无细胞反应）、制备效率高（1.3 × 10

~ 5.7 × 10

U/L无细胞反应），具有较好的普适性，为限制性内切酶的研发与制备生产提供了新的思路。

Abstract

Restriction endonucleases are a large family of endonucleases characterized with high specificity of DNA sequence recognition and cleavage catalysis

and are vital tools extensively applied in biology studies. Restriction endonucleases are usually produced through heterologous co-expression with the methylases to protect the hosts from cytotoxicity. The recently developed cell-free protein synthesis (CFPS) technology is attractive as the advantages of easy manipulation

high efficiency and lower cytotoxicity. In this study

we applied CFPS technology to produce restriction endonucleases. We first constructed the linear DNA template (LDT) of restriction endonucleases and conducted protein expression in

E. coli

-based cell-free reactions without methylases protection for 6 hours. Then

restriction endonucleases were isolated through two steps purification of affinity chromatography and gel chromatography

. By using this strategy

three restriction endonucleases

Eco

RⅠ

Bam

HⅠ

and

Bsa

Ⅰ were successfully synthesized and the specific DNA cleavage activities were tested (

Eco

RⅠ 3.7 × 10

~ 3.7 × 10

U/mg

Bam

HⅠ

8.3 × 10

~ 4.1 × 10

U/mg

and

Bsa

Ⅰ 4.4 × 10

~ 4.4 × 10

U/mg). Furthermore

we developed a real-time catalytic activity detection method

which will facilitate the study on catalytic mechanism and screening of restriction endonucleases. Our CFPS-based restriction endonucleases production system established in this study exhibits advantageous properties such as time-efficient (1~2 days for the overall process)

high protein yield (32.5~130 mg/L cell-free reaction)

and high catalytic activity (1.3 × 10

~ 5.7 × 10

U/L cell-free reaction)

which will provide new insights to the discovery

engineering

and production of restriction endonucleases in the future.

关键词

Keywords

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