Name: co-translational insertion of membrane proteins into preformed nanodiscs
Uploaded: 2020-11-19T14:30:00
Description: Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

Мы хотели бы поблагодарить грант Deutsche Forschungsgemeinschaft (DFG) BE1911/8-1, проект LOEWE GLUE и совместный исследовательский центр Transport and Communication across Membranes (SFB807) за финансовую поддержку.

1. Приготовление лизата S30
<ol><li>День 1: Удалите клетки из запасов глицерина на агаровой пластине LB и инкубируйте при 37 °C в течение ночи.</li>
	<li>День 2: Инокулируют 200 мл LB-среды клетками из агаровой пластины и инкубируют при 37 °C в течение 12-14 ч.</li>
	<li>День 3: Инокулир?...

<ol>
	<li>Pandey, A., Shin, K., Patterson, R. E., Liu, X. Q., Rainey, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+strategies+for+protein+production+and+purification+enabling+membrane+protein+structural+biology.">Current strategies for protein production and purification enabling membrane protein structural biology.</a> Biochemistry and Cell Biology. 94 (6), 507-527 (2016).</li><li>Ritchie, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+in+phospholipid+bilayer+nanodiscs.">Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.</a> Methods in Enzymology. 464 (09), 211-231 (2009).</li><li>Frauenfeld, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+lipoprotein+nanoparticle+system+for+membrane+proteins.">A novel lipoprotein nanoparticle system for membrane proteins.</a> Nature Methods. 13 (4), 345-351 (2016).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+stability+can+be+compromised+by+detergent+interactions+with+the+extramembranous+soluble+domains.">Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.</a> Protein Science. 23 (6), 769-789 (2014).</li><li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins,+lipids+and+detergents:+not+just+a+soap+opera.">Membrane proteins, lipids and detergents: not just a soap opera.</a> Biochimica et Biophysica Acta. 1666 (1-2), 105-117 (2004).</li><li>Junge, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+cell-free+protein+synthesis+for+the+functional+and+structural+analysis+of+membrane+proteins.">Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins.</a> New Biotechnology. 28 (3), (2011).</li><li>Smolskaya, S., Logashina, Y. A., Andreev, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+extract-based+cell-free+expression+system+as+an+alternative+for+difficult-to-obtain+protein+biosynthesis.">Escherichia coli extract-based cell-free expression system as an alternative for difficult-to-obtain protein biosynthesis.</a> International Journal of Molecular Sciences. 21 (928), 1-21 (2020).</li><li>Bayburt, T. H., Grinkova, Y. V., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+discoidal+phospholipid+bilayer+nanoparticles+with+membrane+scaffold+proteins.">Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins.</a> Nano Letters. 2 (8), 853-856 (2002).</li><li>Katzen, F., Peterson, T. C., Kudlicki, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+expression:+no+cells+required.">Membrane protein expression: no cells required.</a> Trends in Biotechnology. 27 (8), 455-460 (2009).</li><li>Roos, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+co-translationally+formed+nanodisc+complexes+with+small+multidrug+transporters,+proteorhodopsin+and+with+the+E.+coli+MraY+translocase.">Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase.</a> Biochimica et Biophysica Acta-Biomembranes. 1818 (12), 3098-3106 (2012).</li><li>Cappuccio, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+co-expression+of+functional+membrane+proteins+and+apolipoprotein,+forming+soluble+nanolipoprotein+particles.">Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles.</a> Molecular and Cellular Proteomics. 7 (11), 2246-2253 (2008).</li><li>Patriarchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodelivery+of+a+functional+membrane+receptor+to+manipulate+cellular+phenotype.">Nanodelivery of a functional membrane receptor to manipulate cellular phenotype.</a> Scientific Reports. 8 (1), 1-11 (2018).</li><li>Shelby, M. L., He, W., Dang, A. T., Kuhl, T. L., Coleman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+co-translational+approaches+for+producing+mammalian+receptors:+expanding+the+cell-free+expression+toolbox+using+nanolipoproteins.">Cell-free co-translational approaches for producing mammalian receptors: expanding the cell-free expression toolbox using nanolipoproteins.</a> Frontiers in Pharmacology. 10 (744), 1-12 (2019).</li><li>Rues, R. B., D&#246;tsch, V., Bernhard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-translational+formation+and+pharmacological+characterization+of+beta1-adrenergic+receptor/nanodisc+complexes+with+different+lipid+environments.">Co-translational formation and pharmacological characterization of beta1-adrenergic receptor/nanodisc complexes with different lipid environments.</a> Biochimica et Biophysica Acta-Biomembranes. 1858 (6), 1306-1316 (2016).</li><li>Henrich, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+native+membrane+protein+assembly+in+nanodiscs+by+combined+non-covalent+mass+spectrometry+and+synthetic+biology.">Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology.</a> eLife. 6, 1-19 (2017).</li><li>Harris, N. J., Pellowe, G. A., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+expression+tools+to+study+co-translational+folding+of+alpha+helical+membrane+transporters.">Cell-free expression tools to study co-translational folding of alpha helical membrane transporters.</a> Scientific Reports. 10 (1), 1-13 (2020).</li><li>Henrich, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+requirements+for+the+enzymatic+activity+of+MraY+translocases+and+in+vitro+reconstitution+of+the+lipid+II+synthesis+pathway.">Lipid requirements for the enzymatic activity of MraY translocases and in vitro reconstitution of the lipid II synthesis pathway.</a> Journal of Biological Chemistry. 291 (5), 2535-2546 (2016).</li><li>Peetz, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+co-translational+membrane+protein+insertion+by+combined+LILBID-mass+spectrometry+and+NMR+spectroscopy.">Insights into co-translational membrane protein insertion by combined LILBID-mass spectrometry and NMR spectroscopy.</a> Analytical Chemistry. 89 (22), 12314-12318 (2017).</li><li>Kigawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Escherichia+coli+cell+extract+for+highly+productive+cell-free+protein+expression.">Preparation of Escherichia coli cell extract for highly productive cell-free protein expression.</a> Journal of Structural and Functional Genomics. 5 (1-2), 63-68 (2004).</li><li>Schwarz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparative+scale+expression+of+membrane+proteins+in+Escherichia+coli-based+continuous+exchange+cell-free+systems.">Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.</a> Nature Protocols. 2 (11), 2945-2957 (2007).</li><li>Yang, J. P., Cirico, T., Katzen, F., Peterson, T. C., Kudlicki, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+synthesis+of+a+functional+G+protein-coupled+receptor+complexed+with+nanometer+scale+bilayer+discs.">Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs.</a> BMC Biotechnology. 11 (57), (2011).</li><li>Rues, R. B., Dong, F., D&#246;tsch, V., Bernhard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+optimization+of+cell-free+synthesized+human+endothelin+B+receptor+folding.">Systematic optimization of cell-free synthesized human endothelin B receptor folding.</a> Methods. 147 (1), 73-83 (2018).</li><li>Keller, T., Gorboulev, V., Mueller, T. D., D&#246;tsch, V., Bernhard, F., Koepsell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+organic+cation+transporter+1+contains+three+binding+sites+for+substrate+1-methyl-4-phenylpyridinium+per+monomer.">Rat organic cation transporter 1 contains three binding sites for substrate 1-methyl-4-phenylpyridinium per monomer.</a> Molecular Pharmacology. 95 (2), 169-182 (2019).</li><li>Matthies, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+expression+and+assembly+of+ATP+synthase.">Cell-free expression and assembly of ATP synthase.</a> Journal of Molecular Biology. 413 (3), 593-603 (2011).</li><li>Schwarz, D., Daley, D., Beckhaus, T., D&#246;tsch, V., Bernhard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+expression+profiling+of+E.+coli+inner+membrane+proteins.">Cell-free expression profiling of E. coli inner membrane proteins.</a> Proteomics. 10 (9), 1762-1779 (2010).</li><li>Haberstock, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+to+increase+the+efficiency+of+membrane+protein+production+in+cell-free+expression+systems.">A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems.</a> Protein Expression and Purification. 82, 308-316 (2012).</li><li>Falkinham, J. O., Clark, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+a+double+male+strain+of+Escherichia+coli+K12.">Genetic analysis of a double male strain of Escherichia coli K12.</a> Genetics. 78 (2), 633-644 (1974).</li><li>Foshag, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+E.+coli+S30+lysate+proteome:+A+prototype+for+cell-free+protein+production.">The E. coli S30 lysate proteome: A prototype for cell-free protein production.</a> New Biotechnology. 40, 245-260 (2018).</li><li>Davanloo, P., Rosenberg, A. H., Dunn, J. J., Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+the+gene+for+bacteriophage+T7+RNA+polymerase.">Cloning and expression of the gene for bacteriophage T7 RNA polymerase.</a> Proceedings of the National Academy of Sciences of the United States of America. 81 (7), 2035-2039 (1984).</li><li>Reckel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+cell-free+expression+of+membrane+proteins.">Strategies for the cell-free expression of membrane proteins.</a> Methods in Molecular Biology. 607, 187-212 (2010).</li><li>Denisov, I. G., Baas, B. J., Grinkova, Y. V., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperativity+in+cytochrome+P450+3A4:+Linkages+in+substrate+binding,+spin+state,+uncoupling,+and+product+formation.">Cooperativity in cytochrome P450 3A4: Linkages in substrate binding, spin state, uncoupling, and product formation.</a> Journal of Biological Chemistry. 282 (10), 7066-7076 (2007).</li><li>Scholz, O., Thiel, A., Hillen, W., Niederweis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+gene+expression+with+an+improved+green+fluorescent+protein.">Quantitative analysis of gene expression with an improved green fluorescent protein.</a> European Journal of Biochemistry. 267 (6), 1565-1570 (2000).</li><li>Henrich, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gene+to+function+cell-free+electrophysiological+and+optical+analysis+of+ion+pumps+in+nanodiscs.">From gene to function cell-free electrophysiological and optical analysis of ion pumps in nanodiscs.</a> Biophysical Journal. 113 (6), 1331-1341 (2017).</li><li>Shen, H. H., Lithgow, T., Martin, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+model+membranes:+Seeking+better+ways+to+retain+protein+activities.">Reconstitution of membrane proteins into model membranes: Seeking better ways to retain protein activities.</a> International Journal of Molecular Sciences. 14 (1), 1589-1607 (2013).</li></ol>

Описана установка и стратегии оптимизации экспрессии CF и котрансляционной вставки функциональных МП в нанодиски. Требуемое оборудование включает в себя биореактор, френч-прессовое устройство или аналогичное, УФ/ВИС и флуоресцентный считыватель, реакционные сосуды CF, подходящие для д...

<table>
	<tbody>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (DMPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840445P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850345C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (sodium salt) (DOPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DOPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840475C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (POPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840034C</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-propan-1,3-diol (Tris)</td>
			<td>Carl Roth (Germany)</td>
			<td>4855</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>4227</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth (Germany)</td>
			<td>9781</td>
			<td></td>
		</tr>
		<tr>
			<td>[3H]-dihydroalprenolol Hydrochloride</td>
			<td>American Radiolabeled Chemicals (USA)</td>
			<td>ART0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl phosphate lithium potassium salt (ACP)</td>
			<td>Merck (Germany)</td>
			<td>1409</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate (ATP)</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr>
			<td>Alprenolol hydrochloride</td>
			<td>Merck (Germany)</td>
			<td>A0360000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange chromatography column material: Q-sepharose&#174;</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>Q1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Type GE 446EC-1</td>
			<td>Gettinge (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor Type 884 124/1</td>
			<td>B.Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall RC12BP+; Thermo Scientific (Germany); Sorvall RC-5C; Thermo Scientific (Germany); Mikro 22 R; Hettich (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid</td>
			<td>Carl Roth (Germany)</td>
			<td>8137</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R250</td>
			<td>Carl Roth (Germany)</td>
			<td>3862</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks 500 ml baffled flasks, 2 l baffled flasks</td>
			<td>Schott Duran (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine 5&#39;-triphosphate disodium salt</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>C1506</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose monohydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6780</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassiumhydrogen phosphate trihydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing SpectrumTM Labs Spectra/PorTM 12-14 kD MWCO Standard RC tubing</td>
			<td>Fisher Scientific (Germany)</td>
			<td>8700152</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreit</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell disruptor</td>
			<td>SLM; Amico Instruments (USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth (Germany)</td>
			<td>3783</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate di-sodium salt (GTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>K025</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAC column: HiTrap IMAC HP 5 mL</td>
			<td>GE Life Sciences (Germany)</td>
			<td>GE17-5248</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Carl Roth (Germany)</td>
			<td>3899</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td>Carl Roth (Germany)</td>
			<td>2316</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Carl Roth (Germany)</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Carl Roth (Germany)</td>
			<td>3076.1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Carl Roth (Germany)</td>
			<td>HN23</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>T202</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Carl Roth (Germany)</td>
			<td>T203</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>3774</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Carl Roth (Germany)</td>
			<td>3772</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glycine</td>
			<td>Carl Roth (Germany)</td>
			<td>3187</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Carl Roth (Germany)</td>
			<td>3852</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Carl Roth (Germany)</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Carl Roth (Germany)</td>
			<td>1699</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Carl Roth (Germany)</td>
			<td>4207</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Carl Roth (Germany)</td>
			<td>9359</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Carl Roth (Germany)</td>
			<td>1713</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Carl Roth (Germany)</td>
			<td>1709</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Carl Roth (Germany)</td>
			<td>4682</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Carl Roth (Germany)</td>
			<td>1738</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophane</td>
			<td>Carl Roth (Germany)</td>
			<td>7700</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Carl Roth (Germany)</td>
			<td>T207</td>
			<td></td>
		</tr>
		<tr>
			<td>MD100 dialysis units</td>
			<td>Scienova (Germany)</td>
			<td>40077</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2-Hydroxyethylpiperazine-N&#39;-2-ethansulfonic acid (HEPES)</td>
			<td>Carl Roth (Germany)</td>
			<td>6763</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecylphosphocholine</td>
			<td>Antrace (USA)</td>
			<td>F308S</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE chamber: Mini-Protean Tetra Cell</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel casting system: Mini Protean Handcast systems</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel power supply: Power Pac 3000</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone/peptone from caseine</td>
			<td>Carl Roth (Germany)</td>
			<td>6681</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump: ip-12</td>
			<td>Ismatec (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenol pyruvate monopotassium salt</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>860077</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth (Germany)</td>
			<td>P018</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Carl Roth (Germany)</td>
			<td>4986</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate Kinase</td>
			<td>Roche (Germany)</td>
			<td>10109045001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation counter: Hidex 300 SL</td>
			<td>Hidex (Finland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth (Germany)</td>
			<td>8029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>K305</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>P029</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Nanodrop</td>
			<td>Peqlab (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer/fluorescence reader Spark&#174;</td>
			<td>Tecan (Switzerland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA (E. coli)</td>
			<td>Roche (Germany)</td>
			<td>10109550001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra sonificator</td>
			<td>Labsonic U, B. Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine 5&#8217;-triphosphate tri-sodium salt (UTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>U6625</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-30 antifoam</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>A6457</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Carl Roth (Germany)</td>
			<td>2904</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-translational insertion of membrane proteins into preformed nanodiscs

Бесклеточные экспрессионные системы позволяют адаптировать реакционные среды для поддержки функционального сворачивания даже сложных белков, таких как мембранные белки. Показаны экспериментальные процедуры котрансляционной вставки и сворачивания мембранных белков в преформированные и определенные мембраны, поставляемые в виде нанодисков. Протокол полностью не содержит моющих средств и может генерировать миллиграммы очищенных образцов в течение одного дня. Полученные образцы мембранного белка / нанодисков могут быть использованы для различных функциональных исследований и структурных применений, таких как кристаллизация, ядерный магнитный резонанс или электронная микроскопия. Описано получение основных ключевых компонентов, таких как бесклеточные лизаты, нанодиски с конструированными мембранами, критические стоковые растворы, а также сборка двухкамерных бесклеточных реакций экспрессии. Поскольку требования к сворачиванию мембранных белков могут быть весьма разнообразными, основным направлением этого протокола является модуляция параметров и стадий реакции, важных для качества образца, таких как критические основные реакционные соединения, мембранный состав нанодисков, окислительно-восстановительная и шаперонная среда или дизайн шаблона ДНК. Весь процесс демонстрируется синтезом протеорходопсина и рецептора, связанного с G-белком.

Иллюстрируется влияние тонко настраиваемых реакционных соединений на конечный выход или качество синтезированных МП. Частым рутинным подходом является корректировка оптимальной концентрации Mg2+ в реакциях CF путем экспрессии зеленого флуоресцентного белка (GFP) в качестве удобн?...

Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Котрансляционная вставка в предварительно сформированные нанодиски позволяет изучать бесклеточные синтезированные мембранные белки в определенных липидных средах без контакта с моющими средствами. Этот протокол описывает подготовку основных компонентов системы и критических параметров для повышения эффективности экспрессии и качества выборки.

Котрансляционная вставка мембранных белков в предварительно сформированные нанодиски

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Используя строительные леса, липосом Развести липидами проксимальный белок-белковых взаимодействий<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Метод визуализации и анализа Мембранные белков, взаимодействующих с помощью просвечивающей электронной микроскопии

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Кавитация азота и дифференциального центрифугирования позволяет для контроля за распределением периферийных мембранных белков в культивируемых клеток

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Моющее бесплатно сверхскоростной воссоздание мембранных белков в липидных бислоев с помощью дополнительных заряженные Proteoliposomes Fusogenic.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Обнаружение моющих средств чувствительных взаимодействия между мембранные белки

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Флуоресценции колебания спектроскопии Assay белок белковых взаимодействий в ячейке контакты

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Изоляция частиц липопротеинов из куриного яичного желтка для изучения бактериального патогена жирной кислоты Инкорпорации в Мембрана фосфолипиды

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Локализация сумо-модифицированных белков с использованием флуоресцентного сумо-улавливания белков

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Pepti'uick, одноступенчатая инкорпорация мембранных белков в биотинилатированные пептидиски для оптимизированных белковых связывающих анализов

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Acyl-PEGyl Обмен Гель Сдвиг Анализ для количественного определения Palmitoylation мозга Membrane белков

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...