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.
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]. The Chinese Journal of Process Engineering, 2019
, 19(6)
: 1197
-1203
.
DOI: 10.12034/j.issn.1009-606X.219121
[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
