ZHU Hai, ZHENG Mengze, JIA Weiwei, et al. Isolation, Purification and Crystallization of Restriction Enzyme Bsa I and Its Preparation of Seleno-derived Derivatives[J]. Science and Technology of Food Industry, 2023, 44(22): 110−116. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022110215.
Citation: ZHU Hai, ZHENG Mengze, JIA Weiwei, et al. Isolation, Purification and Crystallization of Restriction Enzyme Bsa I and Its Preparation of Seleno-derived Derivatives[J]. Science and Technology of Food Industry, 2023, 44(22): 110−116. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022110215.

Isolation, Purification and Crystallization of Restriction Enzyme Bsa I and Its Preparation of Seleno-derived Derivatives

More Information
  • Received Date: November 20, 2022
  • Available Online: September 17, 2023
  • Objective: To screen and optimize the method for preparing three-dimensional structural samples of restriction endonuclease Bsa I. Methods: In this study, the Escherichia coli expression system was employed to express the Bsa I protein and its selenomethionine derivative. Firstly, a recombinant expression vector pBAD-Bsa I was constructed and transformed into Escherichia coli (E.coli) ER2566 for expression. Purification was carried out using affinity chromatography and anion exchange chromatography. Subsequently, selenomethionine derivation situation was assessed using mass spectrometry and circular dichroism spectroscopy, followed by enzyme activity determination. Lastly, preliminary crystal growth studies were conducted using the sitting-drop method. Results: Through a two-step purification approach, recombinant Bsa I and Se-Bsa I selenomethionine derivative with a purity exceeding 90% were obtained. Mass spectrometry analysis revealed that all 11 methionine residues in the recombinant Se-Bsa I protein were selenomethionine-incorporated. Circular dichroism spectroscopy and enzyme activity testing confirmed that the selenomethionine incorporation had no significant impact on the structure and activity of Bsa I protein. Crystallization experiments demonstrated that the recombinant Bsa I protein could not only form granular crystals under conditions of 0.2 mol/L magnesium acetate tetrahydrate at pH6.5, 0.1 mol/L sodium cacodylate trihydrate with 20% polyethylene glycol 8000, but also form spherical structures under conditions of 0.1 mol/L sodium acetate trihydrate at pH4.6 and 2 mol/L ammonium sulfate. Conclusion: This study successfully achieved the recombinant expression of Bsa I and Se-Bsa I selenomethionine derivative, conducted preliminary screening of protein crystal conditions, aiming to provide valuable insights for deciphering the three-dimensional structure of Bsa I protein.
  • [1]
    郭晓强. 酶的研究与生命科学(三):分子生物学酶的发现和应用[J]. 自然杂志, 2015, 37(5):369-380

    GUO Xiaoqiang. Enzymes and life sciences (Ⅲ):Discovery and utilization of molecular biology enzymes[J]. Chinese Journal of Nature, 2015, 37(5):369−390.
    [2]
    SHEN B W, DOYLE L, BRADLEY P, et al. Structure, subunit organization and behavior of the asymmetric type IIT restriction endonuclease BbvCI[J]. Nucleic Acids Research,2019,47(1):450−467. doi: 10.1093/nar/gky1059
    [3]
    PINGOUD A, FUXREITER M, PINGOUD V, et al. Type II restriction endonucleases:Structure and mechanism[J]. Cellular and Molecular Life Sciences,2005,62(6):685−707. doi: 10.1007/s00018-004-4513-1
    [4]
    DI F F, MICHELI G, CAMILLONI G. Restriction enzymes and their use in molecular biology:An overview[J]. Journal of Biosciences,2019,44(2):1−8.
    [5]
    LIPPOW S M, AHA P M, PARKER M H, et al. Creation of a type IIS restriction endonuclease with a long recognition sequence[J]. Nucleic Acids Research,2009,37(9):3061−3073. doi: 10.1093/nar/gkp182
    [6]
    PINGOUD A, WILSON G G, WENDE W. Type II restriction endonucleases—a historical perspective and more[J]. Nucleic Acids Research,2014,42(12):7489−7527. doi: 10.1093/nar/gku447
    [7]
    ZHU Z, XU S. Method for cloning and expression of Bsa I restriction endonuclease and Bsa I methylase in E. coli:US, 09933313[P]. 2003-02-04.
    [8]
    BHADRA S, NGUYEN V, TORRES J A, et al. Producing molecular biology reagents without purification[J]. PLoS one,2021,16(6):e0252507. doi: 10.1371/journal.pone.0252507
    [9]
    言普. 基于Golden Gate技术的载体构建新方法[D]. 北京:中国科学院大学, 2012

    YAN Pu. A new approach to carrier construction based on Golden Gate technology[D]. Beijing:University of Chinese Academy of Sciences, 2012.
    [10]
    LIEMAY M L, RENAUD A, ROUSSEAU G, et al. Targeted genome editing of virulent phages using crispr-Cas9[J]. Bio-Protocol,2018,7(1):e2674.
    [11]
    TIAN S W, JIANG L J, GAO Q, et al. Efficient CRISPR/Cas9-based gene knockout in watermelon[J]. Plant Cell Reports,2017,36(3):399−406. doi: 10.1007/s00299-016-2089-5
    [12]
    LEE J, CHUNG J H, KIM H M, et al. Designed nucleases for targeted genome editing[J]. Plant Biotechnol,2016,14(2):448−462. doi: 10.1111/pbi.12465
    [13]
    CARDI T, NEAL S C J. Progress of targeted genome modification approaches in higher plants[J]. Plant Cell Reports,2016,35(7):1401−1416. doi: 10.1007/s00299-016-1975-1
    [14]
    YANG G, MITON CM, TOKURIKI N. A mechanistic view of enzyme evolution[J]. Protein Science,2020,29(8):1724−1747. doi: 10.1002/pro.3901
    [15]
    SCHIERLING B, NOËL A J, WENDE W, et al. Controlling the enzymatic activity of a restriction enzyme by light[J]. Proceedings of the National Academy of Sciences,2010,107(4):1361−1366. doi: 10.1073/pnas.0909444107
    [16]
    BLUNDELL T L. The first resolution revolution in protein structure analysis:X-ray diffraction of polypeptide conformations and globular protein folds in 1950s and 1960s[J]. Progress in Biophysics and Molecular Biology,2021,167:32−40. doi: 10.1016/j.pbiomolbio.2021.09.002
    [17]
    闫创业. 蛋白质晶体学中的相位解析优化以及三维模型构建的研究[D]. 北京:清华大学, 2014

    YAN C Y. Research on phase resolution optimisation in protein crystallography and three-dimensional model construction[D]. Beijing:Tsinghua University, 2014.
    [18]
    范海福, 梁栋材. 结构基因组学中的衍射相位问题[J]. 生命科学,2003(2):65−69 doi: 10.3969/j.issn.1004-0374.2003.02.002

    FAN Haifu, LIANG Dongcai. Diffraction phase problems in structural genomics[J]. Life Science,2003(2):65−69. doi: 10.3969/j.issn.1004-0374.2003.02.002
    [19]
    BLUNDELL T L, CHAPLIN A K. The resolution revolution in X-ray diffraction, Cryo-EM and other Technologies[J]. Progress in Biophysics and Molecular Biology,2021,160:2−4. doi: 10.1016/j.pbiomolbio.2021.01.003
    [20]
    PABST G, KUCERKA N, NIEH M P, et al. Applications of neutron and X-ray scattering to the study of biologically relevant model membranes[J]. Chemistry and Physics of Lipids,2010,163(6):460−479. doi: 10.1016/j.chemphyslip.2010.03.010
    [21]
    CHEN Y H, ZHANG M. Preparation and crystal growth of Arabidopsis thaliana VSP1 selenium protein derivatives[J]. Journal of Biology,2011,28(6):23−25.
    [22]
    SHOEMAKER S C, ANDO N. X-rays in the cryo-electron microscopy era:Structural biology’s dynamic future[J]. Biochemistry,2018,57(3):277−285. doi: 10.1021/acs.biochem.7b01031
    [23]
    卢圣栋. 现代分子生物学实验技术[M]. 2版. 北京:中国协和医科大学出版社, 1999

    LU S D. Modern experimental techniques of molecular biology [M]. 2 (Ed.). Beijing:China Union Medical University Press, 1999.
    [24]
    DAVIES A M, TATA R, AGHA R, et al. Crystal structure of a putative phosphinothricin acetyltransferase (PA 4866) from Pseudomonas aeruginosa PAC 1[J]. Proteins:Structure, Function & Bioinformatics, 2005, 61(3):677-679.
    [25]
    程艺, 马超, 陈晓雨, 等. 限制性内切酶Nco Ⅰ的高效重组表达、硒代与结晶条件初步筛选[J]. 食品与生物技术学报,2021,40(7):81−88 doi: 10.3969/j.issn.1673-1689.2021.07.010

    CHENG Yi, MA Chao, CHEN Xiaoyu, et al. Efficient recombinant expression, selenogeneration and preliminary screening of selenobacteria and crystallization conditions of restriction enzyme Nco I[J]. Journal of Food and Biotechnology,2021,40(7):81−88. doi: 10.3969/j.issn.1673-1689.2021.07.010
    [26]
    SMITH J H, THOMPSON A, et al. Reactivity of selenomethionine--dents in the magic bullet?[J]. Structure, 1998, 6(7):815.
    [27]
    邵钰晨, 马燕燕, 谷庆花, 等. 限制性内切酶Mlu Ⅰ蛋白及其硒代衍生物的制备[J]. 微生物学通报,2021,48(5):1528−1537

    SHAO Yuchen, MA Yanyan, GU Qinghua, et al. Preparation of restriction enzyme Mlu I protein and its selenoderivative[J]. Microbiology China,2021,48(5):1528−1537.
    [28]
    RYAN D J, SPRAGGINS J M, CAPRIOLI R M. Protein identification strategies in MALDI imaging mass spectrometry:A brief review[J]. Current Opinion in Chemical Biology,2019,48:64−72. doi: 10.1016/j.cbpa.2018.10.023
    [29]
    王铮, 郭新秋, 朱邦尚, 等. 利用圆二色光谱检测计算血清白蛋白二级结构[J]. 实验室研究与探索,2013,32(10):294−296

    WANG Zheng, GUO Xinqiu, ZHU Bangshang, et al. Calculation of serum albumin secondary structure by circular dichroic spectroscopy[J]. Laboratory Research and Exploration,2013,32(10):294−296.
    [30]
    蒋君梅, 杜巧丽, 陈美晴, 等. 马铃薯Y病毒衣壳蛋白的表达、纯化与结晶条件筛选[J]. 植物保护学报,2022,49(2):508−514

    JIANG Junmei, DU Qiaoli, CHEN Meiqing, et al. Expression, purification and crystallization criteria screening of potato Y virus capsid protein[J]. Journal of Plant Protection,2022,49(2):508−514.
    [31]
    曹汝菲, 李泽轩, 许欢, 等. 脆弱拟杆菌Pif1解旋酶的表达纯化与晶体生长[J]. 生物技术通报,2021,37(9):180−190

    CAO Rufei, LI Zexuan, XU Huan, et al. Expression purification and crystal growth of bacteroides fragilis Pif1 helicase[J]. Biotechnology Bulletin,2021,37(9):180−190.
    [32]
    郭元亨, 吕哲, 丁子元, 等. 乙醇体系中D-阿洛酮糖的结晶工艺优化[J]. 食品工业科技,2019,40(24):185−189,198

    GUO Yuanheng, LÜ Zhe, DING Ziyuan, et al. Crystallization process optimization of D-psicose in ethanol system[J]. Science and Technology of Food Industry,2019,40(24):185−189,198.
    [33]
    马晓慧. 非洲猪瘟病毒四种蛋白的表达纯化及结晶条件初步探索[D]. 哈尔滨:哈尔滨工业大学, 2021

    MA X H. Preliminary study on expression, purification and crystallization conditions of four proteins of african swine fever virus[D]. Harbin:Harbin Institute of Technology, 2021.
    [34]
    程焯, 王业民, 郑舰艇, 等. 雷可肽生物合成中LxmX的表达纯化与结晶[J]. 基因组学与应用生物学,2021,40(Z3):3027−3034

    CHENG Zhuo, WANG Yemin, ZHENG Jianting, et al. Expression purification and crystallization of LxmX in raclopeptide biosynthesis[J]. Genomics and Applied Biology,2021,40(Z3):3027−3034.
    [35]
    HOFMANN M, WINZER M, WEBER C, et al. Limitations of polyethylene glycol-induced precipitation as predictive tool for protein solubility during formulation development[J]. Journal of Pharmacy and Pharmacology,2018,70(5):648−654. doi: 10.1111/jphp.12699
    [36]
    戚宝杰, 刘清海, 谢静莉, 等. 变形杆菌属脂肪酶LipK107的分离纯化、结晶及初步晶体学分析[J]. 食品工业科技,2012(19):201−204

    QI Baojie, LIU Qinghai, XIE Jingli, et al. Purification, crystallization and preliminary crystallographic analysis of a lipase from Proteus sp. K107[J]. Science and Technology of Food Industry,2012(19):201−204.
    [37]
    颜俊杰, 李玉洁, 郑迎迎, 等. 黑曲霉内切葡聚糖酶AnCel5A的表达纯化与晶体优化[J]. 食品与生物技术学报,2019,38(2):81−86

    YAN Junjie, LI Yujie, ZHENG Yingying, et al. Expression purification and crystal optimization of endoglucanase AnCel5A from Aspergillus niger[J]. Journal of Food and Biotechnology,2019,38(2):81−86.
  • Cited by

    Periodical cited type(6)

    1. 孙小玉,陈丽,高瑞芳,周群明,康嘉桐,于慧,靳敏. 香青兰总黄酮灌胃对博来霉素诱导大鼠肺纤维化的抑制作用及其机制. 山东医药. 2024(23): 41-46 .
    2. 侯润庚,丁骁,杨力颖,张曼,张颖君,赵平. 西印度醋栗枝叶提取物的α-葡萄糖苷酶抑制活性及安全性评价. 食品工业科技. 2023(07): 252-259 . 本站查看
    3. 李想,刘庆,高晨,周红兵,常虹,王佳,白万富,石松利. 苦杏仁苷对肾纤维化大鼠的保护作用及其机制. 医药导报. 2022(09): 1282-1289 .
    4. 郝瑞敏,贺晓慧,朱丽,谢婧妍,张秀凤. 蒙古扁桃在内蒙古的潜在地理分布及未来适生区预测. 湖北农业科学. 2022(16): 121-126 .
    5. 吴桐,周红兵,王佳,常虹,白万富,权博文,郝海梅,白迎春,石松利. 蒙古扁桃不同极性部位对肝纤维化大鼠的保护作用及机制研究. 食品工业科技. 2021(14): 348-355 . 本站查看
    6. 李倩,白万富,周红兵,郝海梅,李想,常虹,石松利. 蒙古扁桃油对肺纤维化大鼠的保护作用研究. 中药药理与临床. 2021(04): 90-96 .

    Other cited types(2)

Catalog

    Article Metrics

    Article views PDF downloads Cited by(8)

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return