限制性内切酶的无细胞快速制备研究

doi:10.12211/2096-8280.2022-048

摘要/Abstract

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EcoRⅠ、BamHⅠ和BsaⅠ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（EcoRⅠ 3.7 × 10⁵~3.7 × 10⁶ U/mg，BamHⅠ 8.3 × 10²~4.1 × 10³ U/mg，BsaⅠ 4.4 × 10⁵~ 4.4 × 10⁶ 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 × 10⁵ ~ 5.7 × 10⁸ 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。

关键词: 限制性内切酶, 无细胞蛋白合成, 酶蛋白制备, 酶活检测, 合成生物学

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 EcoRⅠ, BamHⅠ,and BsaⅠ were successfully synthesized and the specific DNA cleavage activities were tested (EcoRⅠ 3.7 × 10⁵ ~ 3.7 × 10⁶ U/mg, BamHⅠ8.3 × 10² ~ 4.1 × 10³ U/mg, and BsaⅠ 4.4 × 10⁵~ 4.4 × 10⁶ 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 × 10⁵ ~ 5.7 × 10⁸ U/L cell-free reaction), which will provide new insights to the discovery, engineering, and production of restriction endonucleases in the future.

Key words: restriction endonuclease, cell-free protein synthesis, enzyme protein production, catalytic activity, synthetic biology

中图分类号:

Q814.1

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.

图/表 7

图1 无细胞体系表达与制备限制性内切酶的流程示意图

Fig. 1 Schematic diagram for cell-free production of restriction endonucleases

表1 本研究中用到的引物序列

Table 1 Oligonucleotide primers used in this study

引物名称	序列	用途
linearPro_F	CCTACAGCGTGAGCATTG	扩增启动子片段
linearPro_R	CATATGGTGATGATGATG	扩增启动子片段
linearTer_F	GTCGACCGGCTGCTAACA	扩增终止子片段
linearTer_R	CGGATTCAGTCGTCACTCA	扩增终止子片段
SUMOPro_F	GGATCTCGACGCTCTCCCT	扩增启动子+SUMO标签片段
SUMOPro_R	AGGTCCCTGAAACAGGACCTCTAAACCACCAATCTGTTCTCTG	扩增启动子+SUMO标签片段
SUMO_mut_F	TACGACGGTATTCGTATTCAAGCTGATCAGAC	去除pSUMO质粒中的EcoRⅠ酶切位点
SUMO_mut_R	CAGCTTGAATACGAATACCGTCGTACAAGAATC	去除pSUMO质粒中的EcoRⅠ酶切位点
EcoRⅠ_SUMO_F	TTAGAGGTCCTGTTTCAGGGACCTAGCAACAAAAAACAGAGC	扩增EcoRⅠ基因片段
EcoRⅠ_SUMO_R	CATCATCATCACCATATGAGCAACAAAAAACAGAGC	扩增EcoRⅠ基因片段
sfGFP_SUMO_F	ATTGGTGGTACCGAGCTCATGAGCAAAGGTGAAGAA	构建质粒pSUMO-sfGFP
sfGFP_SUMO_R	GAGTGCGGCCGCAAGCTTTTATTTTTCGAACTGCGG	构建质粒pSUMO-sfGFP
BamHⅠ_F	CATCATCATCACCATATGAAAGTGGAAAAAGA	扩增BamHⅠ基因片段
BamHⅠ_R	TGTTAGCAGCCGGTCGACTTATTTGTTTTCCACTTTATC	扩增BamHⅠ基因片段
BsaⅠ_F	TTAGAGGTCCTGTTTCAGGGACCTATGGCAAAAAAGCGGAA	扩增BsaⅠ基因片段
BsaⅠ_R	TGTTAGCAGCCGGTCGACTTAATCCAGATCCGCAAA	扩增BsaⅠ基因片段

图2 限制性内切酶EcoRⅠ的无细胞制备（Western blot和SDS-PAGE检测无细胞表达、纯化的限制性内切酶EcoRⅠ）CE—cell extract，以BL21 Star （DE3）制备的细胞提取物；CE+ —含有分子伴侣（pG-KJE8）的BL21 Star（DE3）细胞提取物；T—全菌蛋白；S—可溶蛋白；M—蛋白标准样品；FT—纯化上样穿出液；W—洗杂流出液；Eluate—洗脱液

Fig. 2 Cell-free production of restriction endonuclease EcoRⅠ(Western blot and SDS-PAGE analyses of cell-free expressed and purified restriction endonuclease EcoRⅠ) CE—cell extract of BL21 Star (DE3); CE+ —cell extract of BL21 Star (DE3) with chaperone (pG-KJE8); T—total protein; S—soluble protein; M—marker; FT—flow throughout sample; W—washed sample; Eluate—eluted sample

图3 无细胞体系中限制性内切酶EcoRⅠ酶活的实时检测

Fig. 3 Real-time catalytic activity detection of restriction endonuclease EcoRⅠ in CFPS system

图4 无细胞制备限制性内切酶EcoRⅠ的酶活测定NC—阴性对照；PC—阳性对照

Fig. 4 Catalytic activity determination of cell-free produced restriction endonuclease EcoRⅠNC—negative control; PC—positive control

图5 限制性内切酶BamHⅠ和BsaⅠ的无细胞制备（Western blot和SDS-PAGE检测无细胞表达纯化的限制性内切酶BamHⅠ和BsaⅠ）

Fig. 5 Cell-free production of restriction endonucleases BamHⅠ and BsaⅠ(Western Blot and SDS-PAGE analyses of cell-free expressed and purified restriction endonucleases BamHⅠ and BsaⅠ)

图6 无细胞制备限制性内切酶BamHⅠ和BsaⅠ的酶活分析

Fig. 6 Catalytic activity assay of cell-free produced restriction endonucleases BamHⅠ and BsaⅠ

参考文献 39

1	LURIA S E, HUMAN M L. A nonhereditary, host-induced variation of bacterial viruses[J]. Journal of Bacteriology, 1952, 64(4): 557-569.
2	BERTANI G, WEIGLE J J. Host controlled variation in bacterial viruses[J]. Journal of Bacteriology, 1953, 65(2): 113-121.
3	ARBER W. Host-controlled modification of bacteriophage[J]. Annual Review of Microbiology, 1965, 19: 365-378.
4	DANNA K, NATHANS D. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae [J]. Proceedings of the National Academy of Sciences of the United States of America, 1971, 68(12): 2913-2917.
5	KELLY T J JR, SMITH H O. A restriction enzyme from Hemophilus influenzae.Ⅱ[J]. Journal of Molecular Biology, 1970, 51(2): 393-409.
6	WILLIAMS R J. Restriction endonuclease[J]. Molecular Biotechnology, 2003, 23(3): 225-243.
7	PINGOUD A M. Restriction Endonucleases[M]. Berlin, Heidelberg: Springer Berlin Heidelberg, 2004.
8	PINGOUD A, WILSON G G, WENDE W. TypeⅡrestriction endonucleases—a historical perspective and more[J]. Nucleic Acids Research, 2014, 42(12): 7489-7527.
9	ROBERTS R J. How restriction enzymes became the workhorses of molecular biology[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(17): 5905-5908.
10	KNIZEWSKI L, KINCH L N, GRISHIN N V, et al. Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches[J]. BMC Structural Biology, 2007, 7: 40.
11	GUPTA R, CAPALASH N, SHARMA P. Restriction endonucleases: natural and directed evolution[J]. Applied Microbiology and Biotechnology, 2012, 94(3): 583-599.
12	DI FELICE F, MICHELI G, CAMILLONI G. Restriction enzymes and their use in molecular biology: an overview[J]. Journal of Biosciences, 2019, 44(2): 38.
13	CHENG S C, KIM R, KING K, et al. Isolation of gram quantities of EcoRⅠ restriction and modification enzymes from an overproducing strain[J]. Journal of Biological Chemistry, 1984, 259(18): 11571-11575.
14	HOCHULI E, BANNWARTH W, DÖBELI H, et al. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent[J]. Nature Biotechnology, 1988, 6(11): 1321-1325.
15	WATANABE N, TAKASAKI Y, SATO C, et al. Structures of restriction endonuclease HindⅢ in complex with its cognate DNA and divalent cations[J]. Acta Crystallographica Section D, 2009, 65(12): 1326-1333.
16	朱化星, 邹媛华, 汤玉洁, 等. 一种制备限制性内切酶类产品的方法: CN113652412A[P]. 2021-11-16.
	ZHU H X, ZOU Y H, TANG Y J, et al. Method for preparing restriction enzyme product: CN113652412A[P]. 2021-11-16.
17	于博文, 李建辉, 单永超. 一种SalⅠ限制性内切酶的制备方法: CN112813087A[P]. 2021-05-18.
	YU B W, LI J H, SHAN Y C. Preparation method of SalⅠ restriction endonuclease: CN112813087A[P]. 2021-05-18.
18	ORLOWSKI J, BUJNICKI J M. Structural and evolutionary classification of type Ⅱ restriction enzymes based on theoretical and experimental analyses[J]. Nucleic Acids Research, 2008, 36(11): 3552-3569.
19	LIU W Q, ZHANG L, CHEN M, et al. Cell-free protein synthesis: recent advances in bacterial extract sources and expanded applications[J]. Biochemical Engineering Journal, 2019, 141: 182-189.
20	LIU W Q, WU C Z, JEWETT M C, et al. Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system[J]. Biotechnology and Bioengineering, 2020, 117(12): 4001-4008.
21	XU H L, YANG C, TIAN X T, et al. Regulatory part engineering for high-yield protein synthesis in an all-Streptomyces-based cell-free expression system[J]. ACS Synthetic Biology, 2022, 11(2): 570-578.
22	JI X Y, LIU W Q, LI J. Recent advances in applying cell-free systems for high-value and complex natural product biosynthesis[J]. Current Opinion in Microbiology, 2022, 67: 102142.
23	LI J, LAWTON T J, KOSTECKI J S, et al. Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase[J]. Biotechnology Journal, 2016, 11(2): 212-218.
24	MARTIN R W, DES SOYE B J, KWON Y C, et al. Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids[J]. Nature Communications, 2018, 9: 1203.
25	ZHANG L Y, GUO W, LU Y. Advances in cell-free biosensors: principle, mechanism, and applications[J]. Biotechnology Journal, 2020, 15(9): 2000187.
26	SILVERMAN A D, KARIM A S, JEWETT M C. Cell-free gene expression: an expanded repertoire of applications[J]. Nature Reviews Genetics, 2020, 21(3): 151-170.
27	SI Y Y, KRETSCH A M, DAIGH L M, et al. Cell-free biosynthesis to evaluate lasso peptide formation and enzyme-substrate tolerance[J]. Journal of the American Chemical Society, 2021, 143(15): 5917-5927.
28	RASOR B J, VÖGELI B, LANDWEHR G M, et al. Toward sustainable, cell-free biomanufacturing[J]. Current Opinion in Biotechnology, 2021, 69: 136-144.
29	ZAWADA J F, BURGENSON D, YIN G, et al. Cell-free technologies for biopharmaceutical research and production[J]. Current Opinion in Biotechnology, 2022, 76: 102719.
30	BERNARDI A, BERNARDI G. Cloning of all EcoRⅠ fragments from phage λ in E. coli [J]. Nature, 1976, 264(5581): 89-90.
31	BROOKS J E, BENNER J S, HEITER D F, et al. Cloning the BamHⅠ restriction modification system[J]. Nucleic Acids Research, 1989, 17(3): 979-997.
32	ZHU Z Y, SAMUELSON J C, ZHOU J, et al. Engineering strand-specific DNA nicking enzymes from the type ⅡS restriction endonucleases BsaⅠ, BsmBⅠ, and BsmAⅠ[J]. Journal of Molecular Biology, 2004, 337(3): 573-583.
33	CASINI A, STORCH M, BALDWIN G S, et al. Bricks and blueprints: methods and standards for DNA assembly[J]. Nature Reviews Molecular Cell Biology, 2015, 16(9): 568-576.
34	KHORASANIZADEH S, PETERS I D, RODER H. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues[J]. Nature Structural Biology, 1996, 3(2): 193-205.
35	CREIGHTON T E. How important is the molten globule for correct protein folding?[J]. Trends in Biochemical Sciences, 1997, 22(1): 6-10.
36	ENGLANDER S W. Protein folding intermediates and pathways studied by hydrogen exchange[J]. Annual Review of Biophysics and Biomolecular Structure, 2000, 29: 213-238.
37	MARBLESTONE J G, EDAVETTAL S C, LIM Y, et al. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO[J]. Protein Science, 2006, 15(1): 182-189.
38	高嵩, 张坤晓, 许恒皓, 等. 一种高效重组表达限制性内切酶的方法: CN107058260A[P]. 2017-08-18.
	GAO S, ZHANG K X, XU H H, et al. Method for efficient recombinant expression of restriction endonuclease: CN107058260A[P]. 2017-08-18.
39	于博文, 李建辉, 单永超. 重组NcoⅠ限制性内切酶的制备方法: CN112662647A[P]. 2021-04-16.
	YU B W, LI J H, SHAN Y C. Preparation method of recombinant NcoⅠ restriction endonuclease: CN112662647A[P]. 2021-04-16.

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