新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能

doi:10.12034/j.issn.1009-606X.219121

过程工程学报 ›› 2019, Vol. 19 ›› Issue (6): 1197-1203.DOI: 10.12034/j.issn.1009-606X.219121

新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能

陶娇丽^1,2，黄永东^2*，赵岚²，朱凯²，吴学星²，周丹妮¹，苏志国²，马光辉^2,3，刘红缨^1*?

1. 中国矿业大学(北京)化学与环境工程学院，北京 100083 2. 中国科学院过程工程研究所生化工程国家重点实验室，北京 100190 3. 中国科学院大学化工学院，北京 100049

收稿日期:2019-01-21 修回日期:2019-03-27 出版日期:2019-12-22 发布日期:2019-12-22
通讯作者: 陶娇丽 3107971055@qq.com

Controllable preparation of novel charged nanodisc and its binding with cytochrome P450

Jiaoli TAO^1,2, Yongdong HUANG^2*, Lan ZHAO², Kai ZHU², Xuexing WU², Danni ZHOU¹, Zhiguo SU², Guanghui MA^2,3, Hongying LIU^1*

1. School of Chemical & Environmental Engineering, China University of Mining & Technology, Beijing 100083, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 3. School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China

Received:2019-01-21 Revised:2019-03-27 Online:2019-12-22 Published:2019-12-22

摘要/Abstract

摘要： 分别采用氮气吹干法和旋转蒸发法制备由磷脂和膜支架蛋白组成的电荷型纳米盘，用凝胶过滤色谱对其尺寸分级，分析了其性能，考察了其与肝微粒体细胞色素P450的结合能力。结果表明，纳米盘外观澄清透明，微观呈圆盘状，平均直径10 nm，在pH 7.4下Zeta电位为?19.86 mV；肝微粒体破碎液与纳米盘能很好结合，CO差示光谱在450 nm出现明显吸收峰，细胞色素P450含量为0.10 nmol/mg，比活比未经纳米盘处理时提高13.0倍，较传统方法提升1.5倍，且操作时间由数日缩短至数小时。电荷型纳米盘在结合膜蛋白细胞色素P450的同时，活性保持良好，在膜蛋白研究领域极具应用潜力。

关键词: 纳米盘, 电荷, 膜蛋白, 纯化, 重构, 细胞色素P450

Abstract: Membrane proteins are associated with the phospholipid bilayers as integral membrane and have multiple and important functions, such as responsible for carrying out import and export of molecules and communication with the surrounding environment. It has been up to 60% of all currently used drugs target for therapeutic purposes and always been the emphasis of research in the fields of biology, medicine and material science. Membrane proteins are experiencing the bottlenecks due to their low contents, strong hydrophobicity and difficulty in purification. The pretreatment method of membrane proteins is to solubilize the membrane for isolation the membrane proteins using large amounts of detergents firstly, and then purified by reconstitution to restore their functions. Novel charged nanodiscs with both membrane protein binding and protecting were prepared with phospholipid and membrane scaffold protein using both nitrogen blow-drying method and rotary evaporation, respectively in this work. The nanodiscs had clear and transparent appearance, and they were uniform and disc-shaped with an average particle size of 10 nm and a charge of ?19.86 mV under pH 7.4, further size fractionation by gel filtration chromatography. By adjusting the preparation conditions, the properties of nanodiscs could be controlled well. The nanodiscs had a good binding to cytochrome P450 in liver microsome. The CO difference spectrum analysis results showed that the protein?nanodisc system exhibited a strong absorption peak at 450 nm. The content of cytochrome P450 was about 0.10 nmol/mg and the specific activity was improved 13.0 fold, 1.5 times higher than that of the traditional method, the operation time reduced from several days to several hours. It demonstrated that cytochrome P450 can be bound and protected simultaneously.

Key words: nanodisc, charge, membrane protein, purification, reconstitution, cytochrome P450

陶娇丽黄永东赵岚朱凯吴学星周丹妮苏志国马光辉刘红缨. 新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能[J]. 过程工程学报, 2019, 19(6): 1197-1203.

Jiaoli TAO Yongdong HUANG Lan ZHAO Kai ZHU Xuexing WU Danni ZHOU Zhiguo SU Guanghui MA Hongying LIU. Controllable preparation of novel charged nanodisc and its binding with cytochrome P450[J]. Chin. J. Process Eng., 2019, 19(6): 1197-1203.

参考文献

[1]Joh N H, Min A, Faham S, et al.Modest Stabilization by Most Hydrogen-Bonded Side-chain Interactions in Membrane Proteins[J].Nature, 2008, 453(7199):1266-1270 [2]White S H.Biophysical Dissection of Membrane Proteins[J].Nature, 2009, 459(7245):344-346 [3] Zhekova H R, Ngo V, da Silva M C, et al.Selective Ion Binding and Transport by Membrane Proteins – A Computational Perspective [J]. Coordination Chemistry Reviews, 2017, 345: 108-136.[J].Coordination Chemistry Reviews, 2017, 345:108-136 [4]Sackmann E.Supported Membranes: Scientific and Practical Applications[J].Science, 1996, 271(5245):43-48 [5]Misawa N, Osaki T, Takeuchi S.Membrane Protein-based Biosensors[J].Journal of the Royal Society Interface, 2018, 15(141):20170952- [6]Zuo C, Tang S, Zheng J S.Chemical Synthesis and Biophysical Applications of Membrane Proteins[J].Journal of Peptide Science, 2015, 21(7):540-549 [7]Baker M.Making Membrane Proteins for Structures: A Trillion Tiny Tweaks[J].Nature Methods, 2010, 7(6):429-434 [8]Au A E, Josefsson E C.Regulation of Platelet Membrane Protein Shedding in Health and Disease[J].Platelets, 2017, 28(4):342-353 [9]Mayerhofer P U.Targeting and Insertion of Peroxisomal Membrane Proteins: ER Trafficking versus Direct Delivery to Peroxisomes[J].Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2016, 1863(5):870-880 [10]Veneziano R, Rossi C, Chenal A, et al.Synthesis and Characterization of Tethered Lipid Assemblies for Membrane Protein Reconstitution (Review)[J].Biointerphases, 2017, 12(4):04-E301 [11]Hagan C L, Kim S, Kahne D.Reconstitution of Outer Membrane Protein Assembly from Purified Components[J].Science, 2010, 328(5980):890-892 [12]Wiedmer S K, Jussila M S, Riekkola M L.Phospholipids and Liposomes in Liquid Chromatographic and Capillary Electromigration Techniques[J].Trac-Trends in Analytical Chemistry, 2004, 23(8):562-582 [13]Whiles J A, Deems R, Vold R R, et al.Bicelles in Structure-Function Studies of Membrane-associated Proteins[J].Bioorganic Chemistry, 2002, 30(6):431-442 [14]Jamshad M, Lin Y P, Knowles T J, et al.Surfactant-free Purification of Membrane Proteins with Intact Native Membrane Environment[J].Biochemical Society Transactions, 2011, 39(3):813-818 [15] Mclean M A, Gregory M C, Sligar S G.Nanodiscs: A Controlled Bilayer Surface for the Study of Membrane Proteins [J]. Annual Review of Biophysics, 2018, 47:107-124.[J].ANNUAL REVIEW OF BIOPHYSICS, 2018, 47:107-124 [16]Hong W X, Baker K A, Ma X Q, et al.Design,Synthesis,and Properties of Branch-chained Maltoside Detergents for Stabilization and Crystallization of Integral Membrane Proteins: Human Connexin 26[J].Langmuir, 2010, 26(11):8690-8696 [17]Prive G G.Detergents for the Stabilization and Crystallization of Membrane Proteins[J].Methods, 2007, 41(4):388-397 [18]Chae P S, Rasmussen S G F, Rana R R, et al.Maltose-neopentyl glycol (MNG) Amphiphiles for Solubilization,Stabilization and Crystallization of Membrane Proteins[J].Nature Methods, 2010, 7(2):1003-1008 [19]Bazzacco P, Sharma K S, Durand G, et al.Trapping and Stabilization of Integral Membrane Proteins by Hydrophobically Grafted Glucose-Based Telomers[J].Biomacromolecules, 2009, 10(12):3317-3326 [20]Denisov I G, Grinkova Y V, Lazarides A A, et al.Directed Self-Assembly of Monodisperse Phospholipid Bilayer Nanodiscs with Controlled Size[J].Journal of the American Chemical Society, 2004, 126(11):3477-3487 [21] Zhao L, Wei W, Huang Y D, et al.A Novel rProtein A Chromatographic Media for Enhancing Cleaning-in-Place Performance [J]. Journal of Immunological Methods, 2018, 460: 45-50.[J].JOURNAL OF IMMUNOLOGICAL METHODS, 2018, 460:45-50 [22]Zhang H W, Zhao L, Huang Y D, et al.Uniform Polysaccharide Composite Microspheres with Controllable Network by Microporous Membrane Emulsification Technique[J].Analytical and Bioanalytical Chemistry, 2018, 410(18):4331-4341 [23]Denisov I G, Sligar S G.Cytochromes P450 in Nanodiscs[J].Biochimica et Biophysica Acta - Proteins and Proteomics, 2011, 1814(1):223-229 [24] Duan H, Schuler M A.Heterologous Expression and Strategies for Encapsulation of Membrane-localized Plant P450s [J]. Phytochemistry Reviews, 2006, 5:507-523.[J].Phytochemistry Reviews, 2006, 5:507-523 [25]Duan H, Civjan N R, Sligar S G, et al.Co-incorporation of Heterologously Expressed Arabidopsis Cytochrome P450 and P450 Reductase into Soluble Nanoscale Lipid Bilayers[J].Archives of Biochemistry and Biophysics, 2004, 424(2):141-153

新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能

Controllable preparation of novel charged nanodisc and its binding with cytochrome P450

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	杨帆温良英赵岩徐健张生富杨仲卿. TiO₂(100)表面C和Cl₂吸附反应的第一性原理计算[J]. 过程工程学报, 2020, 20(5): 569-575.
[2]	葛佳那向明吴学星杨维兴郝冬霞马光辉. 用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能[J]. 过程工程学报, 2020, 20(1): 84-90.
[3]	尤星力杨延丽苏志国张媛张松平. 鸡白痢沙门氏菌兔抗血清多抗IgG的纯化与稳定[J]. 过程工程学报, 2019, 19(6): 1204-1211.
[4]	韦巍黄永东赵岚吴学星朱天孝李冬雪靳海波张荣月苏志国马光辉. 新型耐碱蛋白A介质的制备与性能[J]. 过程工程学报, 2019, 19(3): 609-616.
[5]	赵春龙孙峙郑晓洪高文芳张延玲林晓. 碳酸锂的制备及其纯化过程的研究进展[J]. 过程工程学报, 2018, 18(1): 20-28.
[6]	朱凯赵岚黄永东李强邱晗王启宝苏志国马光辉. 葡聚糖接枝型耐碱Protein A介质的层析性能提升研究[J]. 过程工程学报, 2016, 16(5): 856-861.
[7]	王浩聪陈芳芳王海洋李梦寒黄慧明张永强. L35-硫酸铵双水相体系萃取水溶液中Cr(VI)[J]. 过程工程学报, 2015, 15(5): 788-794.
[8]	张晨旭李强黄永东赵岚张荣月巩方玲宋翠苏志国袁其朋马光辉. 聚二乙烯基苯微球介质制备及其在奥曲肽层析分离中的应用[J]. 过程工程学报, 2015, 15(3): 482-488.
[9]	李敏堵国成. 聚乙烯醇脱氢酶的分离纯化及其酶学性质[J]. , 2014, 14(6): 1015-1019.
[10]	杨益金陆俊佳覃霞畅荣妮赖青鸟华荣方玲吴华裕侯伟吴飞翔谢彤袁志刚舒伟. 人与鼠源内皮抑制素不同标签重组蛋白的表达纯化及活性分析[J]. , 2014, 14(4): 649-654.
[11]	周亚娟吴剑荣朱莉郑志永詹晓北周文臣. 阴离子交换层析法纯化聚唾液酸工艺[J]. , 2012, 12(5): 822-827.
[12]	马宇峰吕丕平岳占国魏炜马光辉余蓉. 以壳聚糖为基质的智能电荷翻转体系用于紫杉醇的高效输送[J]. , 2012, 12(3): 460-465.
[13]	张晓鸥宋茂谦王长海 Jan-Christer Janson 顾铭. 疏水相互作用与亲水相互作用组合色谱纯化缩宫素[J]. , 2010, 10(3): 598-602.
[14]	张培昆王立刘桂芹李正强杨秉彪郭桂彬. 空分装置三吸附器TSA纯化系统及其节能效果分析[J]. , 2009, 9(5): 932-939.
[15]	周进银鹏. 葡萄糖苷酶在毕赤酵母中的重组表达及一步纯化[J]. , 2009, 9(5): 981-986.