APL 感谢美国国家科学基金会 （EF1935265）、美国国立卫生研究院 （R01-EB030031 和 R21-AR080363） 和陆军研究办公室 （80523-BB） 的支持

用于本研究的试剂和设备在 材料表中提供。
1. 存在小单层囊泡 （SUV） 的本体 CFE 反应
<ol><li>SUV的准备工作 注意:此步骤需要按照使用氯仿的安全说明在通风橱中执行。<ol><li>通过转移 76 μL 溶解在氯仿中的 25 mg/mL POPC 储备溶液，在玻璃小瓶中制备 5 mM 1-棕榈酰基-2-油酰基-甘油-3-磷酸胆碱 （POPC） SUV。</li>
		<li>在轻轻旋转玻璃瓶的同时，将一股温和的氩气吹入玻璃瓶中，在底部形成一层干燥脂质膜。接下来，将玻璃瓶转移到干燥器中，其盖子松动以蒸发多余的氯仿。</li>
		<li>将玻璃瓶保持在干燥器中1小时。然后，加入 0.5 mL 超纯去离子水以溶解脂质膜并涡旋约 2 分钟。</li>
		<li>通过将两个过滤器支架浸泡在去离子水中并将它们放置在每个内部膜支架上来设置微型挤出装置。然后，浸泡一个 100 nm 的聚碳酸酯过滤器，并将其放置在由挤出机外壳和固定螺母固定在一起的两个内部膜支架之间。将此设置放在挤出机支架中。</li>
		<li>用超纯去离子水冲洗两个 1 mL 气密注射器 3 次。</li>
		<li>将脂质水混合物样品装入其中一个 1 mL 气密注射器中，并使用摆臂夹将其放置在小型挤出机的一端，以将注射器固定到位。将第二个注射器插入微型挤出机的另一端，并确保其完全凹陷。 注意:当用脂质水混合物装入注射器时，请确保注射器中没有空气，然后再将其通过微型挤出机。</li>
		<li>轻轻地将脂质水混合物从原始注射器传递到空注射器中，通过微型挤出器装置。重复此步骤 11 次以形成 SUV。将 SUV 转移到 1.5 mL 微量离心管中。 注意:SUV 溶液可在 4 °C 下储存长达 2 周。</li>
	</ol></li>
	<li>CFE反应组装<ol><li>按照制造商提供的无细胞表达方案组装 CFE 反应，并稍作修改，详见下文。</li>
		<li>混合 10 μL 溶液 1（含有氨基酸、NTP、tRNA 和酶底物，以及必要的缓冲液）、1 μL 溶液 2（20% 甘油中的蛋白质）、2 μL 溶液 3（核糖体 （20 μM））、适量的编码可溶性 sfGFP-sfCherry（1-10）27（以下简称可溶性 sfGFP）、WT-GluR0-sfGFP 或 PRSP-GluR0-sfGFP（10-60 ng/1000 碱基对）， 1 μL 鼠 RNAse 抑制剂和 4 μL 5 mM SUV 溶液（用于膜蛋白）或 4 μL 水（用于可溶性蛋白）。</li>
		<li>通过加入超纯去离子水，使反应的最终体积达到 20 μL。 注意: 组装 CFE 系统时，所有组件必须保持在冰上。所有材料都对温度敏感，如果达到室温，它们就会降解。</li>
	</ol></li>
	<li>CFE反应孵育和监测<ol><li>将 CFE 溶液转移到 96 孔锥形 V 形底板中。为防止反应过程中蒸发，请使用密封膜覆盖板。</li>
		<li>将板在37°C下在酶标仪中孵育4-5小时，同时通过每2分钟测量在488nm / 528nm激发/发射波长处增益为100的GFP信号来监测CFE反应。</li>
	</ol></li>
</ol>2. 封装在GUV中的CFE反应
<ol><li>GUV外缓冲溶液的制备<ol><li>在 1.5 mL 微量离心管中，混合 1.5 μL 1 M 亚精胺、37.5 μL 100 mM ATP、25 μL 100 mM GTP、12.5 μL 100 mM CTP、12.5 μL 100 mM UTP、25 μL 1 M 磷酸肌酸、18 μL 1 M 醋酸镁、93.33 μL 3 M 谷氨酸钾， 50 μL 1 M HEPES KOH （pH 7.4）、1.15 μL 332 mM 亚叶酸、100 μL 2 M 葡萄糖和 50 μL 20 种氨基酸中每一种的 6 mM 混合物储备溶液（按照 Sun 等人描述的方案制备28）。</li>
		<li>通过加入超纯去离子水，使溶液的最终体积达到 1 mL。 注意:所有组件必须放在冰上。最后加入氨基酸混合物，以避免氨基酸耗竭28.该溶液可以以330μL等分试样进行分装，并在-20°C下储存直至使用。</li>
	</ol></li>
	<li>油中脂质混合物的制备 注意:此步骤需要按照使用氯仿的安全说明在通风橱中执行。<ol><li>在通风橱中，将 17.3 μL 25 mg/mL POPC 储备溶液和 1.08 μL 50 mg/mL 聚丁二烯-b-聚环氧乙烷 （PEO-b-PBD） 共聚物混合在 15 mL 玻璃瓶中。 注:最终的油中脂质溶液含有 0.5 mM 脂质，分别含有 95% 和 5% POPC 和 PEO-b-PBD。PEO-b-PBD 用于增强蛋白质表达过程中的膜稳定性，但保持低摩尔比以减少共聚物聚集成与脂质分子分离的胶束的倾向29,30。</li>
		<li>小心地将一股温和的氩气吹入玻璃瓶中，同时旋转小瓶以蒸发氯仿。</li>
		<li>将 1.2 mL 轻质矿物油移液到装有干燥脂质的 15 mL 玻璃瓶中。</li>
		<li>通过以最大速度涡旋 10-20 秒混合脂质和油。溶解的脂质-共聚物混合物看起来会浑浊。</li>
		<li>为确保油中所有可能的脂质聚集体完全溶解并分散在整个油中，将玻璃瓶置于50°C左右的烤箱中20分钟，然后以最大速度涡旋10-20秒。</li>
	</ol></li>
	<li>CFE反应组装和封装（图1 总结了以下步骤）。<ol><li>通过混合在步骤 2.1 中制备的 270 μL CFE 外部缓冲溶液、15 μL 5 M NaCl 和 15 μL 4.5 M KCl，在 1.5 mL 微量离心管中制备 300 μL GUV 外溶液。 注:在此步骤中，向 GUV 外溶液中加入 0.45 μL 1,4-二硫苏糖醇 （DTT）。添加 NaCl 和 KCl 的目的是调整外部溶液的渗透压以使其与内部 CFE 溶液相匹配。NaCl 和 KCl 的确切体积取决于所需的渗透压调整。可以添加 NaCl 或 KCl 或两者来调节渗透压。</li>
		<li>轻轻吸取 300 μL 油中脂质混合物，置于 GUV 外溶液顶部。 注意:添加脂质油混合物时，重要的是油不要与外层水溶液混合。添加后，油中脂质混合物和GUV外溶液之间应有一个可见的界面。</li>
		<li>将油水界面在室温下孵育 2 小时，使脂质单层形成并在界面处稳定。</li>
		<li>同时，按照第 1.2.1 节组装包含编码膜蛋白变体或可溶性 GFP 的质粒 DNA 的 CFE 反应。用 4 μL 1 M 蔗糖替换 4 μL 5 mM SUV 溶液或水。该反应将是 GUV 内溶液。 注:渗透压计用于测量内溶液和外溶液的渗透压。然后通过添加NaCl或水来相应地调节外部溶液的渗透压。CFE反应的渗透压通常约为1600 mOsm/Kg。 在反应中加入蔗糖可增加内溶液密度，从而允许囊泡在离心步骤中穿过油水界面。蔗糖的替代品是 Opti-Prep 密度梯度解决方案。</li>
		<li>将 600 μL 脂质油混合物加入含有 CFE 反应的微量离心管中，并上下剧烈移液器 ~1 分钟，以在脂质油溶液中乳化反应并在合成细胞周围形成脂质单层。 注意:最终溶液不应有任何气泡，并且应看起来不透明。</li>
		<li>在设置油水界面的 1.5 mL 微量离心管中，轻轻地将内溶液乳液移至油层顶部。 注意: 注意不要干扰或破坏界面的稳定性。</li>
		<li>在4°C下以2,000× g 离心10分钟。 注意:离心速度已针对此协议进行了优化。Adir等人31 报告了不同的离心速度。</li>
		<li>离心结束后，使用移液器小心地从微量离心管中取出多余的油和外部溶液。取出外部溶液，直到剩余体积约为 100 μL。 注意:在微量离心管的底部通常可以看到 GUV 颗粒。然而，缺乏可见的颗粒并不一定意味着没有 GUV 产量。无需使用移液器，而是可以通过抽吸去除外部溶液顶部的多余油。确保油中脂质溶液被完全去除至关重要。GUV溶液中的油污染会导致图像质量低下。</li>
		<li>轻轻地上下移液，将 GUV 沉淀重悬于剩余的 100 μL 溶液中。接下来，将 GUV 溶液转移到干净的 96 孔透明平底板中进行孵育。</li>
	</ol></li>
</ol>3. 封装的CFE反应孵育和成像
<ol><li>使用密封膜覆盖板以防止蒸发。将板在37°C孵育5-6小时。可以使用酶标仪并按照步骤 1.3.2 准备酶标仪进行孵育步骤。</li>
	<li>孵育结束后，将96孔板放在配备有EMCCD相机（或sCMOS相机），DAQ-MX控制激光器（或集成激光合路器系统）和CSU-X1转盘共聚焦（或激光扫描共聚焦）的倒置显微镜的成像台上。聚焦任何含有 GUV 的 ROI，并使用 Plan-Apochromat 60 x/1.4 NA 物镜在 488 nm 激发波长处捕获图像。</li>
	<li>将 GUV 的图像保存为.tiff格式。</li>
	<li>在图像处理软件（例如 ImageJ 或 Fiji）中打开图像。打开 亮度/对比度 设置面板。将亮度和对比度调整到适当的设置，使荧光蛋白可见。</li>
	<li>如果目标是比较不同表达蛋白质的信号强度，请首先使用位于"图像>堆栈"子菜单下的"图像到堆栈"面板堆叠包含不同蛋白质的 GUV 的单个图像。然后，使用"亮度/对比度"面板调整所有图像的亮度和对比度。</li>
</ol>

膜蛋白在巨型单层囊泡中的无细胞表达

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Y., 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=Calcium-triggered+DNA-mediated+membrane+fusion+in+synthetic+cells.">Calcium-triggered DNA-mediated membrane fusion in synthetic cells.</a> Chemical Communications. 59 (57), 8806-8809 (2023).</li><li>Moghimianavval, H., 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=Engineering+functional+membrane-membrane+interfaces+by+interspy.">Engineering functional membrane-membrane interfaces by interspy.</a> Small. 19 (13), 2202104 (2023).</li><li>Sun, Z. 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=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> J Vis Exp. (79), e50762 (2013).</li><li>Jacobs, M. L., Boyd, M. A., Kamat, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diblock+copolymers+enhance+folding+of+a+mechanosensitive+membrane+protein+during+cell-free+expression.">Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression.</a> PNAS. 116 (10), 4031-4036 (2019).</li><li>Kostarelos, K., Tadros, T. F., Luckham, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+conjugation+of+(tri-)+block+copolymers+to+liposomes+toward+the+construction+of+sterically+stabilized+vesicle+systems.">Physical conjugation of (tri-) block copolymers to liposomes toward the construction of sterically stabilized vesicle systems.</a> Langmuir. 15 (2), 369-376 (1999).</li><li>Adir, 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=Preparing+protein+producing+synthetic+cells+using+cell+free+bacterial+extracts,+liposomes+and+emulsion+transfer.">Preparing protein producing synthetic cells using cell free bacterial extracts, liposomes and emulsion transfer.</a> J Vis Exp. (158), e60829 (2020).</li><li>van de Cauter, L., van Buren, L., Koenderink, G. H., Ganzinger, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+giant+unilamellar+vesicle+production+for+artificial+cells+-+current+challenges+and+future+directions.">Exploring giant unilamellar vesicle production for artificial cells - current challenges and future directions.</a> Small Methods. 7 (12), 2300416 (2023).</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> Biochim Biophys Acta Biomembr. 1666 (1), 105-117 (2004).</li><li>Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent-free+systems+for+structural+studies+of+membrane+proteins.">Detergent-free systems for structural studies of membrane proteins.</a> Biochem Soc Trans. 49 (3), 1361-1374 (2021).</li><li>Bashirzadeh, Y., Wubshet, N., Litschel, T., Schwille, P., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+encapsulation+of+reconstituted+cytoskeleton+inside+giant+unilamellar+vesicles.">Rapid encapsulation of reconstituted cytoskeleton inside giant unilamellar vesicles.</a> J Vis Exp. (177), e63332 (2021).</li><li>Hwang, S. 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=Hybrid+vesicles+enable+mechano-responsive+hydrogel+degradation.">Hybrid vesicles enable mechano-responsive hydrogel degradation.</a> Angew Chemie Int Ed. 62 (41), e202308509 (2023).</li><li>Rideau, E., Dimova, R., Schwille, P., Wurm, F. R., Landfester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+polymersomes:+a+comparative+review+towards+cell+mimicking.">Liposomes and polymersomes: a comparative review towards cell mimicking.</a> Chem Soc Rev. 47 (23), 8572-8610 (2018).</li><li>Tsumoto, K., Hayashi, Y., Tabata, J., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reverse-phase+method+revisited:+Rapid+high-yield+preparation+of+giant+unilamellar+vesicles+(GUVs)+using+emulsification+followed+by+centrifugation.">A reverse-phase method revisited: Rapid high-yield preparation of giant unilamellar vesicles (GUVs) using emulsification followed by centrifugation.</a> Colloids Surf A: Physicochem Eng Asp. 546, 74-82 (2018).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> J Am Chem Soc. 137 (33), 10767-10776 (2015).</li><li>Bailoni, 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=Minimal+out-of-equilibrium+metabolism+for+synthetic+cells:+A+membrane+perspective.">Minimal out-of-equilibrium metabolism for synthetic cells: A membrane perspective.</a> ACS Synth Biol. 12 (4), 922-946 (2023).</li></ol>

作者声明没有利益冲突。

实际上，任何依赖于分子或信息在细胞膜上转移的细胞过程，如细胞信号传导或细胞兴奋，都需要膜蛋白。因此，膜蛋白的重组成为实现不同应用的各种合成细胞设计的主要瓶颈。传统的去垢剂介导的生物膜中膜蛋白重建需要 GUV 生成方法，例如温和溶胀或电形成。溶胀方法通常产生小尺寸的囊泡，当复杂的溶液（在产生合成细胞时通常出现这种情况）被封装时，电形成产量显着下降<sup class="xref"...

在过去十年中，自下而上的合成生物学作为一个新兴领域越来越受到关注，在生物工程、药物递送和再生医学中具有许多潜在应用1,2。特别是，由于合成细胞的前景广阔的应用以及其促进体外生物物理研究的类细胞物理和生化特性，合成细胞作为自下而上的合成生物学的基石的发展吸引了广泛的科学界 3,4,5,6 .合成细胞通常被设计在细胞大小的巨型单层囊泡 （GUV） 中，其中重建了不同的生物过程。细胞骨架的重建 7,8、光依赖性能量再生9、细胞通讯10,11 和生物传感12 是在合成细胞中重建细胞样行为的例子。
虽然一些细胞过程依赖于可溶性蛋白质，但天然细胞的许多特性，如感应和通信，通常利用膜蛋白，包括离子通道、受体和转运蛋白。合成细胞发育的一个主要挑战是膜蛋白的重建。尽管脂质双层中膜蛋白重建的传统方法依赖于洗涤剂介导的纯化，但这些方法费力，对于对表达宿主有毒的蛋白质无效，或者通常不适合 GUV 中的膜蛋白重建13。
蛋白表达的另一种方法是无细胞表达 （CFE） 系统。CFE 系统一直是合成生物学中的强大工具，它允许使用细胞裂解物或纯化的转录翻译机器体外表达各种蛋白质14。CFE 系统也可以封装在 GUV 中，从而允许区室化的蛋白质合成反应，这些反应可以针对各种应用进行编程，例如创建光捕获合成细胞9 或机械敏感生物传感器15,16。与重组蛋白表达方法类似，膜蛋白表达在 CFE 系统中具有挑战性17。CFE 系统中的聚集、错误折叠和缺乏翻译后修饰是阻碍使用 CFE 系统成功合成膜蛋白的主要瓶颈。使用 CFE 系统进行自下而上的膜蛋白重建的困难部分是由于缺乏依赖于信号肽、信号识别颗粒、转位子和伴侣分子的复杂膜蛋白生物发生途径。然而，最近，多项研究表明，在翻译过程中微粒体或脂质体等膜结构的存在促进了膜蛋白的成功表达 18,19,20,21。此外，Eaglesfield 等人和 Steinküher 等人发现，在膜蛋白的 N 末端包含称为信号肽的特定疏水结构域可以显着改善其表达22,23。总而言之，这些研究表明，如果蛋白质翻译在GUV膜存在的情况下发生，并且使用适当的N端信号肽，则可以克服合成细胞中膜蛋白重建的挑战。
在这里，提出了一种使用重组元件（PURE）CFE反应封装蛋白质合成的方案，用于GUVs中的膜蛋白重建。选取细菌谷氨酸受体24 （GluR0）作为模型膜蛋白，研究其N端信号肽对其膜重建的影响。Eaglesfield等[22]通过构建GluR0的突变变体（称为PRSP-GluR0）来研究蛋白hododrein信号肽可提高膜蛋白重建效率的作用，并比较其表达和膜定位与野生型GluR0（以下简称WT-GluR0）携带其天然信号肽。该协议基于倒置乳液方法25 ，并进行了修改，使其对于CFE封装具有鲁棒性。在所提出的方法中，CFE反应首先使用脂质油溶液乳化，该溶液产生微米大小的液滴，这些液滴包含CFE系统并由脂质单层稳定。然后，将乳液滴分层在油水界面的顶部，该界面被另一个脂质单层饱和。然后，乳液液滴通过离心力被迫穿过油水界面。通过这个过程，液滴获得另一个单层，从而产生双层脂质囊泡。然后将含有 CFE 反应的 GUV 孵育，在此过程中膜蛋白表达并掺入 GUV 膜中。尽管该协议被指定用于GluR0的无细胞表达，但它可用于其他膜蛋白的无细胞合成或不同的合成细胞应用，如细胞骨架重建或膜融合研究26。

<table><tbody><tr><td>100 nm polycarbonate filter</td><td>STERLITECH</td><td>1270193</td><td></td></tr><tr><td>96 Well Clear Bottom Plate</td><td>ThermoFisher Scientific</td><td>165305</td><td></td></tr><tr><td>BioTek Synergy H1M Hybrid Multi-Mode Reader</td><td>Agilent</td><td>11-120-533</td><td></td></tr><tr><td>Creatine phosphate</td><td>Millipore Sigma</td><td>10621714001</td><td></td></tr><tr><td>CSU-X1 Confocal Scanner Unit</td><td>Yokogawa</td><td>CSU-X1&amp;nbsp;</td><td></td></tr><tr><td>Density gradient medium (Optiprep)</td><td>Millipore Sigma</td><td>D1556</td><td>Optional to switch with sucrose in inner solution</td></tr><tr><td>Filter supports</td><td>Avanti</td><td>610014</td><td></td></tr><tr><td>Fisherbrand microtubes (1.5 mL)</td><td>Fisher Scientific</td><td>05-408-129&amp;nbsp;</td><td></td></tr><tr><td>Folinic acid calcium salt hydrate</td><td>Millipore Sigma</td><td>F7878</td><td></td></tr><tr><td>Glucose</td><td>Millipore Sigma</td><td>158968</td><td></td></tr><tr><td>HEPES</td><td>Millipore Sigma</td><td>H3375</td><td></td></tr><tr><td>iXon X3 camera&amp;nbsp;</td><td>Andor</td><td>DU-897E-CS0&amp;nbsp;</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate</td><td>Millipore Sigma</td><td>G1501</td><td></td></tr><tr><td>Light mineral oil</td><td>Millipore Sigma</td><td>M5904</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate&amp;nbsp;</td><td>Millipore Sigma</td><td>M5661</td><td></td></tr><tr><td>Mini-extruder kit (including syringe holder and extruder stand)</td><td>Avanti</td><td>610020</td><td></td></tr><tr><td>Olympus IX81 Inverted Microscope&amp;nbsp;</td><td>Olympus</td><td>IX21</td><td></td></tr><tr><td>Olympus PlanApo N 60x Oil Microscope Objective&amp;nbsp;</td><td>Olympus</td><td>1-U2B933&amp;nbsp;</td><td></td></tr><tr><td>PEO-b-PBD</td><td>Polymer Source</td><td>P41745-BdEO</td><td></td></tr><tr><td>pET28b-PRSP-GluR0-sfGFP plasmid DNA</td><td>Homemade</td><td>N/A</td><td></td></tr><tr><td>pET28b-sfGFP-sfCherry(1-10) plasmid DNA</td><td>Homemade</td><td>N/A</td><td></td></tr><tr><td>pET28b-WT-GluR0-sfGFP plasmid DNA</td><td>Homemade</td><td>N/A</td><td></td></tr><tr><td>POPC lipid in chloroform&amp;nbsp;</td><td>Avanti</td><td>850457C</td><td></td></tr><tr><td>Potassium chloride</td><td>Millipore Sigma</td><td>P9541</td><td></td></tr><tr><td>PUREfrex 2.0</td><td>Cosmo Bio USA</td><td>GFK-PF201</td><td></td></tr><tr><td>Ribonucleotide Solution Set</td><td>New England BioLabs</td><td>N0450</td><td></td></tr><tr><td>RNase Inhibitor, Murine</td><td>New England BioLabs</td><td>M0314S</td><td></td></tr><tr><td>RTS Amino Acid Sampler</td><td>Biotechrabbit</td><td>BR1401801</td><td></td></tr><tr><td>Sodium chloride</td><td>Millipore Sigma</td><td>S9888</td><td></td></tr><tr><td>Spermidine</td><td>Millipore Sigma</td><td>S2626</td><td></td></tr><tr><td>Sucrose</td><td>Millipore Sigma</td><td>S0389</td><td></td></tr><tr><td>VAPRO Vapor Pressure Osmometer Model 5600</td><td>ELITechGroup</td><td>VAPRO 5600</td><td></td></tr></tbody></table>

reconstitution of the bacterial glutamate receptor channel by encapsulation of a cell-free expression system

无细胞表达 （CFE） 系统是合成生物学中的强大工具，允许在合成细胞中进行细胞功能的仿生，如生物传感和能量再生。然而，重建广泛的细胞过程需要成功地将膜蛋白重建到合成细胞的膜中。虽然可溶性蛋白的表达通常在常见的CFE系统中是成功的，但合成细胞脂质双层中膜蛋白的重建已被证明具有挑战性。本文展示了一种在巨型单层囊泡（GUV）中重建模型膜蛋白（细菌谷氨酸受体（GluR0）作为模型合成细胞的方法，该方法基于在合成细胞内封装和孵育CFE反应。利用该平台，证明了用蛋白视紫红质信号肽替换GluR0的N端信号肽对GluR0成功共翻译转位到杂交GUVs膜中的作用。该方法提供了一种稳健的程序，允许在合成细胞中无细胞重建各种膜蛋白。

在包封CFE反应之前，含有天然和蛋白视紫红质信号肽的GluR0-sfGFP的两种变体（信号肽序列见 补充表1）和可溶性sfGFP在本体反应中单独表达，并通过使用酶标仪检测sfGFP信号来监测其表达（图2A).膜蛋白在不存在或存在 100 nm SUV 的情况下表达。此外，使用将sfGFP信号与其浓度相关联的校准曲线（补充图1），估计合成蛋白质的浓度（补?...

Watch this Scientific Journal Video about Membrane Protein Reconstitution in Synthetic Cells at JoVE.com

作者聚焦:合成细胞中的膜蛋白重构

该协议描述了用于在巨大的单层囊泡（GUV）内封装无细胞表达（CFE）系统的倒置乳液方法，用于研究模型膜蛋白的合成和掺入脂质双层。

通过封装无细胞表达系统重建细菌谷氨酸受体通道

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

我们的方案解决了使用倒滴乳液方法制成的合成细胞脂质双层内膜蛋白的重建。使用倒滴乳液方法的优点是它能够产生合成细胞，为膜蛋白的无细胞表达提供稳定的环境。我们的方案为该领域的研究人员提供了一种强大的方法，可以使用无细胞表达系统在合成细胞内合成膜蛋白。我的实验室正在研究通过一组明确定义的离子通道重建动作电位来重建合成细胞中的各种离子通道。

reconstitution-bacterial-glutamate-receptor-channel-encapsulation

Research

JoVE Journal

Bioengineering

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

1.0K 次观看.  University of Michigan, Ann Arbor. 我们的方案解决了使用倒滴乳液方法制成的合成细胞脂质双层内膜蛋白的重建。使用倒滴乳液方法的优点是它能够产生合成细胞，为膜蛋白的无细胞表达提供稳定的环境。我们的方案为该领域的研究人员提供了一种强大的方法，可以使用无细胞表达系统在合成细胞内合成膜蛋白。我的实验室正在研究通过一组明确定义的离子通道重建动作电位来重建合成细胞中的各种离?...

视频: 作者聚焦:合成细胞中的膜蛋白重构

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

化学

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

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 ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

生物工程

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

生物学

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

神经科学

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...