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

我们要感谢德国研究协会（DFG）资助BE1911/8-1、LOEWE罗意威项目GLUE和跨膜运输与通信合作研究中心（SFB807）的财政支持。

1. S30裂解物的制备
<ol><li>第一天： 从LB琼脂平板上的甘油储备液中划出细胞，并在37°C孵育过夜。</li>
	<li>第二天： 用琼脂平板上的细胞接种200mLLB培养基，并在37°C下孵育12-14小时。</li>
	<li>第三天：接种10 L无菌YPTG培养基（10 g / L酵母提取物，16 g / L胰蛋白胨，5 g / L NaCl，19.8 g / L葡萄糖，4.4 mM KH 2 PO 4，8 mM K2HPO4）在15 L搅拌釜式反应器...

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描述了优化CF表达和功能性MPs共翻译插入纳米盘的设置和策略。所需设备包括生物反应器、法式压榨装置或类似设备、紫外可见分光光度计和荧光读数器、适用于两室配置设置的 CF 反应容器以及温控培养箱。其他标准设备是用于收获大肠杆菌细胞的离心机以及用于制备S30裂解物的台式离心机，其重量至少为30，000 x g。如果要制备S80-S100裂解物，则需要超速离心机。所列设备定期在生?...

膜蛋白（MPs）由于其在水环境中的不溶性，是蛋白质生产研究中具有挑战性的靶标。传统的MP生产平台包括基于细胞的系统，如大肠杆菌、酵母或真核细胞。合成的重组MP要么从细胞膜中提取，要么从包涵体1中重新折叠。洗涤剂溶解后，MPs可以通过建立的体外复溶方案转移到合适的膜环境中。除了囊泡和脂质体外，MP以纳米盘2或salipro3颗粒的形式重建为平面膜已成为最近常规技术。然而，所有这些策略都意味着洗涤剂与MP接触可能导致不稳定，低聚物解离，甚至蛋白质结构和活性丧失4。因此，筛选最佳的洗涤剂增溶和复溶条件可能既繁琐又耗时5。
无细胞（CF）系统的开放性允许表达反应直接提供具有确定脂质组成的预制膜。在这种基于脂质的表达模式（L-CF）中，合成的MP有机会共平移插入到提供的双层6，7 中（图1）。由围绕平面脂质双层盘8 的膜支架蛋白（MSP）组成的纳米盘似乎特别适合于此策略9，10。纳米盘可以常规地与各种不同的脂质在体外组装，它们非常稳定，并且储备液可以浓缩到1mM。然而， 大肠杆菌 中的MSP表达及其纯化是必要的。作为一种替代策略，MSP可以在脂质体11，12，13提供的CF反应中与目标MP共同表达。将MSP和MP的DNA模板添加到反应中，并且在表达时可以形成MP /纳米盘。虽然避免了MSP的产生，但共表达策略需要仔细微调最终的DNA模板浓度，并且可以预期样品生产效率的变化更大。
MPs共翻译插入预成型纳米盘的膜是一种非生理的，并且在很大程度上仍然未知的机制，独立于易位机器和信号序列13，14，15，16。插入效率的主要决定因素是膜蛋白的类型以及所提供膜的脂质组成，通常首选带负电荷的脂质15，17。由于纳米盘膜的尺寸相对受限，因此在MP插入18时释放了大量的脂质。纳米盘尺寸的变化使得插入和调谐更高的低聚MP配合物15，18。其中，离子通道KcsA，离子泵蛋白视质（PR）和多药转运蛋白EmrE15，18显示了同源低聚复合物的正确组装。MPs可能以相对相等的频率从两侧进入对称纳米盘膜。因此，应该考虑到插入一个纳米盘的不同单体或低聚物可能具有相反的方向。然而，方向偏差可能是由PR六聚体和KcsA四聚体18形成的合作插入机制引起的。MP取向的进一步偏差可能是由于插入的MP的方向开关可能在纳米盘膜的边缘。
从大肠杆菌菌株生产CF裂解物是一种可靠的常规技术，几乎可以在任何生化实验室中进行19，20。应该考虑到，除了二硫键形成外，如果使用大肠杆菌裂解物合成MP，则大多数其他翻译后修饰都不存在。虽然这可能会为结构研究产生更均匀的样本，但可能有必要排除对单个MP靶标功能的潜在影响。然而，在大肠杆菌CF裂解物中有效生产G蛋白偶联受体（GPCR）14，21，22，真核转运蛋白23或大型异构体组装体24的高质量样品表明它们甚至适用于复杂的靶标。制备级量（≈ 1 mg/mL）的样品可以通过由反应混合物（RM）和进料混合物（FM）室组成的双室连续交换无池（CECF）配置获得。FM体积超过RM体积15至20倍，并提供低分子量能量化合物和前体的储存库19。因此，表达反应延长数小时，MP靶标的最终产率增加。RM和FM隔室由截止时间为10-14 kDa的透析膜隔开。两个隔室需要CECF反应容器的特殊设计（图1）。作为RM容器的商用透析盒与固定FM的定制有机玻璃容器相结合就是合适的例子。MP产量可以通过修改RM：FM比率或在一定孵育一段时间后交换FM来进一步操纵。
MP的产量和质量经常需要对反应参数进行严格优化。CF表达的一个重要优点是可以根据MP的个性化要求修改和微调几乎任何化合物。一个简单的策略是首先通过建立基本的生产方案来提高MP的产量，然后通过微调反应和折叠条件来优化MP质量。CF裂解物中没有生理过程，其复杂性降低，因此MPs25的有效生产成功率很高。在大多数情况下，DNA模板设计和优化Mg 2+离子浓度的常规考虑因素足以获得令人满意的产率26。根据表达模式的不同，MP质量的优化可能会变得非常耗时，因为需要筛选更多种类的参数14，17，22。
为了建立所描述的CF表达平台，需要生产 大肠杆菌 CF裂解物（i），T7 RNA聚合酶（ii），纳米盘（iii）和碱性CECF反应化合物（iv）（图1）的方案。 大肠杆菌 K12 菌株 A1927 或 BL21 衍生物经常用于制备高效的 S30（以 30，000 x g 离心）裂解物。除 S30 裂解物外，还可以使用在其他 g 力（例如 S12）下离心的相应裂解物。裂解物在表达效率和蛋白质组复杂性方面有所不同。对所述方案制备的S30裂解物的蛋白质组进行了详细研究28。S30蛋白质组仍然含有一些残留的外膜蛋白，这些蛋白可能会在离子通道的表达和分析时产生背景问题。对于此类靶标，建议使用 S80-S100 裂解物。裂解物制备过程中有价值的修饰是在细胞的对数生长中期通过热休克和乙醇供应相结合来诱导SOS反应。所得的HS30裂解物富含伴侣，可与S30裂解物共混，以改善MP折叠22。 大肠杆菌 裂解物中的CF表达作为偶联转录/翻译过程操作，DNA模板含有由T7 RNA聚合酶（T7RNAP）控制的启动子。该酶可以在携带pAR1219质粒29的BL21（DE3）星形细胞中高效生产。
MSP1E3D1的两个拷贝组装成一个直径为10-12nm的纳米盘，但下面描述的协议也可能适用于其他MSP衍生物。然而，比用MSP1E3D1形成的纳米盘大的纳米盘往往不太稳定，而用MSP衍生物（如MSP1）形成的较小的纳米盘可能无法容纳较大的MP或MP复合物。MSP1E3D1纳米盘可以在体外与各种不同的脂质或脂质混合物组装。预制纳米盘通常在-80°C下稳定>1年，而不同脂质成分的稳定性可能有所不同。为了筛选脂质对MP折叠和稳定性的影响，有必要制备一套由代表性的脂质/脂质混合物组装而成的纳米盘。以下脂质可以给出良好的起始选择：1，2-二肉豆蔻酰-sn-甘油-3-磷酸-（1'-rac-甘油）（DMPG）、1，2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱（DMPC）、1，2-二油酰基-sn-甘油-3-磷酸-rac-（1-甘油）（DOPG）、1，2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸-（1'-rac-甘油）（POPG）和1-棕榈酰基-2-油酰基甘油-3-磷酸胆碱（POPC）。
描述了制备3mL CECF反应的方案。可以以 1：1 的比例进一步向上或向下缩放。对于双室CECF配置，必须制备包含所有化合物的RM和仅包含低分子量化合物的FM。具有 10-14 kDa MWCO 的商用 3 mL 透析设备可用作 RM 容器，然后将其放入装有 FM 的定制有机玻璃容器中（图 1D）30。RM：FM的比例为1：20，因此必须为3 mL RM制备60 mL FM。 几种组分的质量、浓度或类型对于合成MP的最终产量和/或质量至关重要（表1）。DNA模板应根据已发表的指南制备，并对靶标的阅读框进行密码子优化，可进一步显著提高产物收率26。对于制备级CECF反应，描述了用于生产PR的既定方案。为了建立新MP的表达方案，通常需要对某些化合物进行优化筛选（表1）以提高产量和质量。对于Mg2+ 离子，确实存在聚焦良好的最佳浓度，该浓度经常显示不同DNA模板的显着差异。其他CF反应化合物，如新批次的T7RNAP或S30裂解物，可能会进一步改变最佳Mg2+ 浓度。作为示例，描述了在14-24mM浓度范围内并以2mM步长的典型Mg2+ 筛选。每种浓度一式两份和分析规模的CECF反应中筛选。作为CECF反应容器，定制的Mini-CECF Plexiglas容器30 与容纳FM的标准24孔微孔板结合使用（图1B）。或者，可以使用商业透析柱与96深孔微孔板或其他适当设置的商用透析器装置的组合（图1C）。

<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

无细胞表达系统允许对反应环境进行定制设计，以支持即使是复杂蛋白质（如膜蛋白）的功能折叠。演示了将膜蛋白共翻译插入和折叠到作为纳米盘提供的预成型和定义的膜中的实验程序。该方案完全不含洗涤剂，可在一天内产生毫克纯化样品。所得的膜蛋白/纳米盘样品可用于各种功能研究和结构应用，如结晶、核磁共振或电子显微镜。描述了基本关键组分的制备，例如无细胞裂解物、具有设计膜的纳米盘、关键储备溶液以及双室无细胞表达反应的组装。由于膜蛋白的折叠要求可能高度多样化，因此该协议的主要重点是调节对样品质量很重要的参数和反应步骤，例如关键碱性反应化合物，纳米盘的膜组成，氧化还原和伴侣环境或DNA模板设计。整个过程通过蛋白视紫红质和G蛋白偶联受体的合成得到证明。

举例说明了微调反应化合物对合成MP的最终产率或质量的影响。一种常见的常规方法是通过表达绿色荧光蛋白（GFP）来调整CF反应中的最佳Mg2+浓度，作为系统效率的便捷监测器。例如，GFP是由Mg 2+浓度在14至24mM之间的pET-21a（+）载体合成的（图2）。SDS-PAGE分析确定了20mM处的最佳Mg 2+浓度（图2A），这与CF反应上清液的互补荧光测量（485nm处激发，535nm处发射测量?...

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

共翻译插入到预先形成的纳米盘中，使得在定义的脂质环境中研究无细胞合成的膜蛋白成为可能，而无需与去垢剂接触。该协议描述了基本系统组分的制备以及提高表达效率和样品质量的关键参数。

膜蛋白共翻译插入预成型纳米盘

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

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

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

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

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

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

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.

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 ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

膜蛋白对洗涤剂敏感相互作用的检测

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

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

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

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

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

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

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

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 ...

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

阿西尔-PEGyl交换凝胶移位测定，用于脑膜蛋白棕榈酸化定量测定

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