Name: detergent-assisted reconstitution of recombinant drosophila atlastin into liposomes for lipid-mixing assays
Uploaded: 2019-07-03T13:00:00
Description: Watch this Scientific Journal Video about Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays at JoVE.com

我们感谢迈克尔·斯特恩博士和他的实验室对阿克拉松相关项目的见解和反馈。这项工作得到了国家普通医学研究所[R01GM101377]和国家神经疾病和中风研究所[R01NS102676]的支持。

1. 消费税-达特尔-赫8的净化
<ol><li>
蛋白质表达和酸酶制剂
	<ol>
<li>在 pGEX4-T32中使用 GST-DAtl-His8 结构将 BL21 (DE3)大肠杆菌转换为大肠杆菌,并在阿霉素板上进行选择。</li>
		<li>在14 mL培养管中选择单一转化剂,接种5 mL的LB+阿霉素(5μL,100mg/mL安西林),在25°C下孵育,在200rpm下摇动6-8小时。 注:由于表达漏水,不建议在较高温度下孵育。在25°C下生长会减少蛋白质在此...

<ol>
	<li>Chernomordik, L. V., Kozlov, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+membrane+fusion.">Mechanics of membrane fusion.</a> Nature Structural and Molecular Biology. 15, 675-683 (2008).</li><li>Orso, G., 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=Homotypic+fusion+of+ER+membranes+requires+the+dynamin-like+GTPase+Atlastin.">Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin.</a> Nature. 460, 978-983 (2009).</li><li>McNew, J. A., Sondermann, H., Lee, T., Stern, M., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP-dependent+membrane+fusion.">GTP-dependent membrane fusion.</a> Annual Review of Cell and Developmental Biology. 29, 529-550 (2013).</li><li>Rigaud, J. L., Pitard, B., Levy, D. <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+liposomes:+application+to+energy-transducing+membrane+proteins.">Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1231, 223-246 (1995).</li><li>D&#252;zg&#252;ne&#351;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Assays+for+Liposome+Fusion.">Fluorescence Assays for Liposome Fusion.</a> Methods in Enzymology. 372, 260-274 (2003).</li><li>Wilschut, J., Duzgunes, N., Fraley, R., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+membrane+fusion:+kinetics+of+calcium+ion+induced+fusion+of+phosphatidylserine+vesicles+followed+by+a+new+assay+for+mixing+of+aqueous+vesicle+contents.">Studies on the mechanism of membrane fusion: kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents.</a> Biochemistry. 19, 6011-6021 (1980).</li><li>Ellens, H., Bentz, J., Szoka, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-+and+calcium-induced+fusion+and+destabilization+of+liposomes.">Proton- and calcium-induced fusion and destabilization of liposomes.</a> Biochemistry. 24, 3099-3106 (1985).</li><li>Meers, P., Ali, S., Erukulla, R., Janoff, A. 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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=Small-volume+extrusion+apparatus+for+preparation+of+large,+unilamellar+vesicles.">Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.</a> Biochimica Et Biophysica Acta. 1061, 297-303 (1991).</li><li>Paternostre, M. T., Roux, M., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+membrane+protein+insertion+into+liposomes+during+reconstitution+procedures+involving+the+use+of+detergents.+1.+Solubilization+of+large+unilamellar+liposomes+(prepared+by+reverse-phase+evaporation)+by+Triton+X-100,+octyl+glucoside,+and+sodium+cholate.">Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by Triton X-100, octyl glucoside, and sodium cholate.</a> Biochemistry. 27, 2668-2677 (1988).</li><li>Scott, B. L., 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=Liposome+Fusion+Assay+to+Monitor+Intracellular+Membrane+Fusion+Machines.">Liposome Fusion Assay to Monitor Intracellular Membrane Fusion Machines.</a> Methods in Enzymology. 372, 274-300 (2003).</li><li>Zhang, W., 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=Crystal+structure+of+an+orthomyxovirus+matrix+protein+reveals+mechanisms+for+self-polymerization+and+membrane+association.">Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.</a> PNAS. 114, 8550-8555 (2017).</li><li>Parlati, 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=Rapid+and+efficient+fusion+of+phospholipid+vesicles+by+the+&#945;-helical+core+of+a+SNARE+complex+in+the+absence+of+an+N-terminal+regulatory+domain.">Rapid and efficient fusion of phospholipid vesicles by the &#945;-helical core of a SNARE complex in the absence of an N-terminal regulatory domain.</a> PNAS. 96, 12565-12570 (1999).</li><li>Sugiura, S., Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+lipid+composition+is+vital+for+homotypic+ER+membrane+fusion+mediated+by+the+dynamin-related+GTPase+Sey1p.">Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p.</a> Scientific Reports. 6, 20407 (2016).</li><li>Powers, R. 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本文的方法为重组lastin的净化、重组和测量融合活性提供了一种有效的方法。为了确保高产量的功能lastlastin一些关键步骤必须考虑。在低温(16°C)下必须表达阿列酮,以避免聚集,并且一种应针对0.4~1.5mg/mL的最终浓度。非常稀蛋白不会以1:400蛋白质与脂质比进行最佳重组。通过此处描述的浮点测定法(图 2),可以有选择地分析重组效率。脂质体浮化测定的优点之一是,可以扩展分析?...

膜融合是许多生物反应的关键过程。在生物条件下,膜融合不是自发的,需要专门的融合蛋白来催化这种反应1。ER同质膜融合在动物中由与发电机相关GTPase在lastin2中介导。阿克拉斯丁在同质融合中的作用对于外围ER中的三向结至关重要,它构成了一个贯穿整个细胞的大型互联小管网络。阿克拉斯林有一个保守的域形态,包括一个大的GTPase,一个三螺旋束中间域,一个疏水膜锚,和一个短的细胞质C端尾部3。体外研究用重组果蝇对骨素表明,当重组为脂质体时,它保持其富源性。其他的lastin,包括人类同源物,未能在体外重述融合。我们在这里描述一种方法,用于纯化GST和多肽标记重组果蝇对lastin,将其重组为脂体,并分析融合。
研究体外膜融合是一个挑战,因为富源蛋白通常具有膜锚。为了研究它们,有必要将它们重新组成模型脂质双层。大型单片囊泡(LUV)是研究脂质蛋白相互作用的有用工具。我们在这里提出了一个系统,使不同脂质成分的LUV蛋白质重组和融合测定。通过多种方法将整体蛋白质重组到LUV中,可以实现,包括有机溶剂介导的重组、机械机制或洗涤剂辅助重组4。我们这里提出了一种通过洗涤剂去除将果蝇在骨素中重组成预制脂体的方法。这种重组方法的优点包括高再生产量和脂质双层中甲苯的适正定向。此外,通过这种方法,蛋白质不干燥或暴露于有机溶剂,从而保持结构和功能。其缺点之一是,洗涤剂的存在可能并不适用于所有蛋白质,而最终的蛋白酶体在脂质双层中可能有一些结合的洗涤剂。进一步透析可用于消除更多的洗涤剂。然而,透析可能需要很长时间,因此可能导致蛋白质活性的丧失。
评估lastin的聚变活性可以通过脂质混合测定,如前所述2。在这里,我们划定了一种通过N-(7-硝基苯-2-oxa-1,3-二氮-4-yl)/利萨明罗达明-B磺胺(罗达明)标记脂质测量lastin介导聚变的方法。这种测定需要融合供体(标记)蛋白酶体和受体(未标记)蛋白酶体。FRET释放可以在反应过程中测量为从"标记"脂质体稀释的供体受体对到"未标记"脂质体,这是膜融合过程中脂质混合的结果(图1)5。虽然这种测定是膜融合的代理,但它在区分膜融合和血合液方面受到限制,这种状态只有外层传单混合。为了解决这个问题,另一种选择是用二硝基石对NBD进行外部包装。遵循与NBD/罗达明脂质混合测定相同的方法,在淬火外单子时,任何NBD FRET通过融合释放的,都将是由于内部传单混合8。
替代融合测定由内部水含量混合地址完全融合只有5。这方面的例子有二聚氰胺(Tb)/二恶酸(DPA)测定和氨基苯三硫酸(ANTS)/p-二甲苯二(二甲苯)溴化物(DPX)测定。在 Tb/DPA 测定中,封装 Tb 的脂体池与封装 DPA 的脂体混合并融合在一起;融合后,荧光通过内部能量从DPA转移到Tb在[Tb(DPA)3+3-结合复合物6内增加。相比之下,对于ANTS/DPX测定,ANTS荧光由DPX7淬火。虽然这些系统解决了内部内容混合问题,但需要更深入地制备脂体,以去除非封装试剂,以及荧光荧光的意外相互作用。

<table>
	<tbody>
		<tr>
			<td>10 mL poly-prep chromatography columns</td>
			<td>Biored</td>
			<td>731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>10 x 75 mm Flint glass tubes</td>
			<td>VWR</td>
			<td>608225-402</td>
			<td></td>
		</tr>
		<tr>
			<td>47 mm diameter, 0.45um pore whatman sterile membrane filters</td>
			<td>Whatman</td>
			<td>7141 104</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well white plate</td>
			<td>NUNC</td>
			<td>437796</td>
			<td></td>
		</tr>
		<tr>
			<td>Anapoe X-100</td>
			<td>Anatrace</td>
			<td>9002-931-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell disrupter</td>
			<td>Avestin</td>
			<td>Avestin Emulsiflex C3</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt))</td>
			<td>Avanti</td>
			<td>840035P-10mg</td>
			<td>DOPS</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Research organics inc.</td>
			<td>6381-92-6</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Complete protease inhibitor</td>
		</tr>
		<tr>
			<td>Extruder</td>
			<td>Sigma Aldrich</td>
			<td>Z373400&#160;</td>
			<td>Liposofast Basic Extruder</td>
		</tr>
		<tr>
			<td>GE Akta Prime liquid chromatography system</td>
			<td>GE Pharmacia</td>
			<td>8149-30-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione agarose beads</td>
			<td>Sigma aldrich</td>
			<td>G4510-50ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma Aldrich</td>
			<td>36051-31-7</td>
			<td>Guanosine 5&#39; triphosphate sodium salt hydrate</td>
		</tr>
		<tr>
			<td>HEPES, acid free</td>
			<td>Omnipur</td>
			<td>5330</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>fluka</td>
			<td>5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized metal affinity chromatography (IMAC) resin column</td>
			<td>GE Healthcare</td>
			<td>17040801</td>
			<td>1 mL HiTrap Chelating HP immobilized metal affinity chromatography columns</td>
		</tr>
		<tr>
			<td>Iohexol</td>
			<td>Accurate chemical and scientific corporation</td>
			<td>AN 7050 BLK</td>
			<td>Accudenz/Nycodenz</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Research products international corp.</td>
			<td>I56000-100.0</td>
			<td>IPTG, dioxane free</td>
		</tr>
		<tr>
			<td>L-Glutathione reduced</td>
			<td>Sigma-Aldrich</td>
			<td>G4251-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Omnisolv</td>
			<td>MX0488-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NBD-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt))</td>
			<td>Avanti</td>
			<td>236495</td>
			<td>NBD-DPPE</td>
		</tr>
		<tr>
			<td>n-Dodecyl &#946;-D-maltoside</td>
			<td>Chem-Impex International</td>
			<td>21950</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonpolar polystyrene adsorbent beads</td>
			<td>BioRad</td>
			<td>152-3920</td>
			<td>SM2 Biobeads</td>
		</tr>
		<tr>
			<td>Nuclepore track-etch polycarbonate 19 mm 0.1 um pore membrane</td>
			<td>Whatman</td>
			<td>800309</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima LE80K Ultra centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylcholine, L-&#945;-dipalmitoyl [choline methyl-3H]</td>
			<td>ARC</td>
			<td>ART0284</td>
			<td>Titriated lipids</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>TECAN</td>
			<td>TECAN infinite M200 plate reader</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)</td>
			<td>Avanti</td>
			<td>850457C-25mg</td>
			<td>POPC</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>MP</td>
			<td>151944</td>
			<td></td>
		</tr>
		<tr>
			<td>Rh-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt))</td>
			<td>Avanti</td>
			<td>236495</td>
			<td>Rh-DPPE</td>
		</tr>
		<tr>
			<td>Scintillation Cocktail</td>
			<td>National Diagnostics</td>
			<td>LS-272</td>
			<td>Ecoscint XR Scintillation solution for aqueous or non-aqueous samples</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Beckman</td>
			<td>592928</td>
			<td>Fast turn cap Mini Poly-Q Vial</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T1063-1kU</td>
			<td>Thrombin from human plasma</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes 5 x 41 mm</td>
			<td>Beckman</td>
			<td>344090</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex 9 to 13mm Tube Holder</td>
			<td>VWR</td>
			<td>58816-138</td>
			<td>Insert for vortexing flint glass tubes</td>
		</tr>
		<tr>
			<td>Vortex Insert Retainer</td>
			<td>VWR</td>
			<td>58816-132</td>
			<td>Retainer needed for vortex tube holder</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>VWR</td>
			<td>2.235074</td>
			<td>Vortex Genie 2 model G560</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol molecular biology grade</td>
			<td>Calbiochem</td>
			<td>444203</td>
			<td></td>
		</tr>
	</tbody>
</table>

detergent-assisted reconstitution of recombinant drosophila atlastin into liposomes for lipid-mixing assays

膜融合是真核细胞的关键过程。特殊蛋白质是催化融合所必需的。阿普拉斯是内质视网膜 (ER) 居民蛋白,与 ER 的同质融合有关。在这里,我们详细介绍了通过两轮亲和色谱法来纯化谷胱甘肽 S-转移酶 (GST) 和多组氨酸标记果蝇蛋白的方法。研究体外融合反应需要将纯化融合蛋白插入脂质双层。脂质体是理想的模型膜,因为脂质成分和大小可以调整。为此,我们描述了一种重组方法,通过去除果蝇在骨细胞素到预先成型的脂体。虽然有几种重组方法可用,但通过洗涤剂去除进行重组有几个优点,使其适用于蛋白条和其他类似蛋白质。该方法的优点包括高重组产量和重组蛋白的正确方向。该方法可扩展到其他膜蛋白和其他需要蛋白体的其他应用。此外,我们描述了用于测量膜融合的蛋白石体体基于FRET的脂质混合测定。

图2给出了对正感重组的效率。重组的蛋白酶体在碘醇不连续梯度中漂浮。未结合的蛋白质沉积在底层 (B) 或中间层 (M) 中。重组后的蛋白质会浮到顶层(T)。梯度样本由SDS-PAGE和库马西染色采集和分析。通过密度测定对凝胶进行定量,表明重建效率很高,损失可忽略不计;96%的总蛋白质被发现为浮到顶层(T)的蛋白酶体。不到1%的蛋白质未重组,在中间层(M)中发现,只有3%未重组或聚合并...

Watch this Scientific Journal Video about Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays at JoVE.com

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

生物膜融合由专用融合蛋白催化。通过脂质混合测定,可以测量蛋白质的富源性。我们提出了一种纯化重组果蝇蛋白的方法,这是一种蛋白质,可以调节ER的同质融合,将其重组为预制脂质体,并测试融合能力。

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

此处详述的重组和融合协议是研究模样脂质双层和融合蛋白混合脂质混合膜结合蛋白的一种有效方法。在这里，我们描述了一个洗涤剂辅助重组重组的果蝇图马汀，ER融合蛋白，成预制磷脂酰胆碱脂质体。然后，通过基于F FRET的脂质混合测定来测量亚拉他汀蛋白体融合。洗涤剂辅助重组的主要优势之一是洗涤剂中的蛋白质不会暴露在干燥或溶剂中。我们还在这里表明，地图集的重组方向是非常有效的。基于F FRET的脂质混合测定的另一个优点是，脂质体不需要加载任何染料，也不需要任何额外的透析步骤。为了开始这一程序，请准备氯仿中的脂质混合物，如文本协议中所述。每毫升的 DPPC 添加一个微库到脂质混合物中，确保至少保留 8 微升的这种库存。将所需量的脂质混合物转移到火石玻璃管中。然后在温和的氮气流下干燥脂质混合物约10分钟，直到不再可见氯仿。通过吸尘 30 分钟，在干燥器中进一步干燥产生的脂质膜。在此之后，加入足够的 A100 与 10%甘油，2 毫摩尔 2-二甲醇，和一毫摩尔 EDTA 到脂质膜，使浓度回到 10 毫摩尔。在室温下轻轻涡旋15分钟，重新暂停脂质膜。在液氮中冷冻水合脂。要解冻脂质体，让溶液在室温下坐30秒，然后将它们转回水中，以更快地解冻。重复此冻结解冻循环共 10 次。在此之后，使用迷你挤出机通过聚碳酸酯过滤器传递脂质，孔径为 100 纳米，19 倍。要确定脂质体的总脂质浓度，在三毫升闪烁鸡尾酒中加入四微升脂量和裂糖。测量股票和脂质体每分钟的平均计数，并计算脂质体浓度，如文本协议中所述。将脂质体储存在摄氏四度，长达一周。首先，在0.5毫升管中混合适当数量的缓冲液、额外的洗涤剂和蛋白质，如文本协议中所述。快速添加脂质体，并立即涡旋五秒钟，使混合物均匀。在摄氏四度下孵育一小时。在孵育过程中，称出0.2克聚苯乙烯吸附珠，并转移到微离心管。在管中加入一毫升甲醇，混合一分钟，使珠子脱气。然后，用21规格或更高的针头吸气甲醇。要开始清洗珠子，请给珠子加水。让珠子与水混合五分钟，然后吸水。重复此水洗过程四次。在此之后，加入水，将聚苯乙烯吸附珠浆带到一毫升的体积，最终浓度为每毫升0.2克。计算吸附每个样品中所有洗涤剂所需的聚苯乙烯吸附珠的数量，如文本协议中所述。切一个移液器尖端来收集珠浆。将计算的珠浆量转移到0.5毫升管中，吸水。将样品加入含有珠子的管子，在摄氏四度下孵育一小时。将样品转移到含有新珠子的新鲜管子中，确保将旧珠子留在后面。使用相同条件再次孵育。重复转移和孵育珠子两次的过程。之后，将样品转移到含有新鲜珠子的新管子中，并在摄氏四度下过夜孵育。第二天，从珠子上取出样品，在16，000倍g和4摄氏度的离心机中10分钟，对不溶性蛋白质集料进行颗粒。恢复上清液，并确定最终脂质浓度通过液体闪烁计数，如文本协议中概述。首先，将供体和接受方蛋白糖体在 A100 中各浓度为 0.15 毫摩尔，其中含有甘油、β-甲基苯丙酸、EDTA 和氯化镁。将每个反应转移到适合荧光读数的扁平 96 井板中的井中，确保至少准备四种反应，包括三酯和无 GTP 负对。在 37 摄氏度下将板放入预热板读卡器中。每分钟测量五分钟 NBD 荧光。然后，加入五毫摩尔GTP诱导聚变。使用与以前相同的激发和排放，每分钟测量一小时 NBD 荧光。在此之后，在井中加入5个2.5%n-Dodecyl beta-D-麦芽糖的微升，溶解蛋白糖体，并使用相同的激发和排放，每分钟测量最大NBD荧光量15分钟。在这项研究中，重组的果蝇图马汀被净化并重组成预制的脂质体。地图集重建的效率由在 iohexol 不连续梯度中浮动重组的蛋白体决定。顶部、中间和底部梯度层的样本由 SDS-PAGE 和库马西染色采集和分析。通过密度测定对凝胶进行定量测量，显示重组效率非常高，损失可以忽略不计，96%的总蛋白质被发现为浮附到顶层的蛋白体。不到1%的蛋白质未形成，发现在中间层，只有3%是未构成或聚合和沉积到底层。重组后图图丁的方向通过血栓蛋白裂解测定进行量化。用SDS-PAGE和库马西染色分析样品，通过密度测定进行量化。大多数重组的蛋白质是裂开的，只有7%被保护免受蛋白酶的。通过脂质混合测定，分析了图马汀中位蛋白体融合的动力学和范围。在零时间点诱导与GTP融合之前，进行五分钟的孵育。一小时后，n-Dodecyl beta-D-麦芽糖被添加，以溶解蛋白糖体，并得到最大的荧光共振转移释放。在运行中融合最大值被视为最大荧光的 11%。脂质溶解在氯仿中。在准备脂质混合物时，请确保快速在化学罩上和冰上进行，以避免任何蒸发。通过显微镜分析图铂蛋白体，可以可视化膜锚对脂质体形状和尺寸的影响。这种方法已扩展至研究其他膜锚化蛋白，以及模型脂质双层层的行为方式。该协议使用滴定脂质来量化脂质混合物。处理放射性物质时，请务必采取必要的预防措施。

Research

JoVE Journal

Biochemistry

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

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

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

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

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

科研

生物化学

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

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

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

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

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

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

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

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

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

完全加工重组KRAS4b：分离和描述脱脂和甲基化蛋白

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...