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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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&#160;</td>
			<td>Andor</td>
			<td>DU-897E-CS0&#160;</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&#160;</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&#160;</td>
			<td>Olympus</td>
			<td>IX21</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus PlanApo N 60x Oil Microscope Objective&#160;</td>
			<td>Olympus</td>
			<td>1-U2B933&#160;</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&#160;</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。

Bottom-up synthetic biology has gained increasing interest over the past decade as an emerging field with numerous potential applications in bioengineering, drug delivery, and regenerative medicine1,2. The development of synthetic cells as a cornerstone of bottom-up synthetic biology, in particular, has attracted a wide range of scientific communities due to the promising applications of synthetic cells as well as their cell-like physical and biochemical properties that facilitate in vitro biophysical studies3,4,5,6. Synthetic cells are often engineered in cell-sized giant unilamellar vesicles (GUVs) in which different biological processes are recreated. Reconstitution of cell cytoskeleton7,8, light-dependent energy regeneration9, cellular communication10,11, and biosensing12 are examples of efforts made to reconstruct cell-like behaviors in synthetic cells.
While some cellular processes rely on soluble proteins, many characteristics of natural cells, such as sensing and communication, often utilize membrane proteins, including ion channels, receptors, and transporters. A major challenge in synthetic cell development is the reconstitution of membrane proteins. Although traditional methods of membrane protein reconstitution in lipid bilayers rely on detergent-mediated purification, such methods are laborious, ineffective for proteins that are toxic to the expression host, or are often not suited for membrane protein reconstitution in GUVs13.
An alternative method for protein expression is cell-free expression (CFE) systems. CFE systems have been a powerful tool in synthetic biology that allows in vitro expression of various proteins using either cell lysate or purified transcription-translation machinery14. CFE systems can also be encapsulated in GUVs, thus allowing compartmentalized protein synthesis reactions that can be programmed for various applications, such as the creation of light-harvesting synthetic cells9 or mechanosensitive biosensors15,16. Analogous to recombinant protein expression methods, membrane protein expression is challenging in CFE systems17. Aggregation, misfolding, and lack of post-translational modification in CFE systems are major bottlenecks that hinder successful membrane protein synthesis using CFE systems. The difficulty of bottom-up membrane protein reconstitution using CFE systems is due in part to the absence of a complex membrane protein biogenesis pathway that relies on signal peptides, signal recognition particles, translocons, and chaperoning molecules. However, recently, multiple studies have suggested that the presence of membranous structures such as microsomes or liposomes during translation promotes successful membrane protein expression18,19,20,21. Additionally, Eaglesfield et al. and Steink&#252;her et al. have found that the inclusion of specific hydrophobic domains known as signal peptides in the N-terminus of the membrane protein can significantly improve its expression22,23. Altogether, these studies suggest that the challenge of membrane protein reconstitution in synthetic cells can be overcome if the protein translation occurs in the presence of the GUV membrane and if proper N-terminal signal peptide is utilized.
Here, a protocol for encapsulation of the protein synthesis using recombinant elements (PURE) CFE reactions for membrane protein reconstitution in GUVs is presented. Bacterial glutamate receptor24 (GluR0) is selected as the model membrane protein, and the effect of its N-terminal signal peptide on its membrane reconstitution is studied. The effect of proteorhodopsin signal peptide, which was shown to improve membrane protein reconstitution efficiency by Eaglesfield et al.22, is investigated by constructing a mutated variant of GluR0 denoted as PRSP-GluR0 and its expression and membrane localization with wild-type GluR0 (referred to as WT-GluR0 hereafter) that harbors its native signal peptide is compared. This protocol is based on the inverted emulsion method25 with modifications that make it robust for CFE encapsulation. In the presented method, the CFE reactions are first emulsified using a lipid-in-oil solution that generates micron-sized droplets that contain the CFE system and are stabilized by the lipid monolayer. The emulsion droplets are then layered on top of an oil-water interface that is saturated with another lipid monolayer. The emulsion droplets are then forced to travel across the oil-water interface via centrifugal force. Through this process, the droplets obtain another monolayer, thus generating a bilayer lipid vesicle. The GUVs containing the CFE reaction are then incubated, during which the membrane protein is expressed and incorporated into the GUV membrane. Although this protocol is specified for cell-free expression of GluR0, it can be used for cell-free synthesis of other membrane proteins or different synthetic cell applications such as cytoskeleton reconstitution or membrane fusion studies26.

作者聚焦：合成细胞中的膜蛋白重构

通过封装无细胞表达系统重建细菌谷氨酸受体通道

Our protocol addresses the reconstitution of a membrane protein within the lipid bilayer of a synthetic cell made using the inverted droplet emulsion method. The advantage of using the inverted droplet emulsion method is its ability to generate synthetic cells that provide a stable environment for the cell-free expression of membrane proteins. Our protocol offers a robust method for researchers in the field to synthesize membrane proteins inside synthetic cells using cell-free expression system.My lab is working on reconstituting various ion channels in synthetic cells through the reconstitution of the action potential by a well-defined set of ion channels.

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

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。

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

787 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

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

科研

生物工程

Bulk Cell-Free Expression Reactions with Small Unilamellar Vesicles (SUVs) for Synthetic Cell Applications

To begin, blow a gentle stream of Argonne into a glass vial containing POPC lipids while gently rotating the vial. When the lipids have dried as a film at the bottom of the vial, loosely cap it, then transfer it to a desiccater. After one hour add 0.5 milliliters of ultrapure deionized water to dissolve the lipid film.Vortex the solution for two minutes. Next, soak two filter supports and deionized water. Place them on each of the internal membrane supports.Then place a 100 nanometer polycarbonate filter between the two internal membrane supports, held together by the extruder outer casing and retainer nut. Place the setup in the extruder stand to finish setting up the mini extrusion apparatus. Next, load the lipid water mix into a one milliliter gast tight syringe.Use swing arm clips to secure the syringe into one end of the mini extruder. Insert the second syringe into the other end of the mini extruder, making sure that it is fully depressed. Now pass the lipid water mix from the original syringe into the empty one through the apparatus.After repeating the extrusion 11 times, transfer the SUVs to a 1.5 milliliter micro centrifuge tube. To assemble the cell-free expression protocol, pipette solutions one to 3 DNA plasmid encoding for soluble SFGFP or variants of glutamate receptor SFGFP, murine RNAse, and SUV solution into a vial. Add ultrapure deionized water to bring the reaction volume to 20 microliters.Transfer the cell-free expression solution to a 96 well conical V bottom plate. Incubate the plate in a plate reader at 37 degrees Celsius for four to five hours. Set up the plate reader with a gain of 100, excitation of 485 nanometers, emission of 528 nanometers, a runtime of five hours, and an interval measurement of one read for every two minutes.Measure the GFP signal every two minutes to monitor the cell-free reaction in real time. Soluble SFGFP had the highest expression among all three proteins.

用于合成细胞应用的具有小单层囊泡 （SUV） 的大量无细胞表达反应

Cell-Free Reactions in Giant Unilamellar Vesicles (GUVs) for Synthetic Cell Applications

To begin, prepare the GUV outer buffer solution in a 1.5-milliliter microcentrifuge tube. Mixed together spermidine, ATP, GTP, CTP, UTP, creatine phosphate, magnesium acetate, potassium glutamate, HEPES, potassium hydroxide, folinic acid. glucose, and amino acid stock.Add ultra purified deionized water to bring the final volume of the solution to one milliliter. Next, in a fume hood, add 17.3 microliters of POPC stock solution to a 15-milliliter glass vial. To this, pipette 1.08 microliters of PEO-b-PBD copolymer.Carefully blow a gentle stream of argon gas into the glass vial while rotating the vial to evaporate the chloroform. Then pipette 1.2 milliliters of light mineral oil into the glass vial. Vortex the lipid and oil at maximum speed for 10 to 20 seconds.Place the glass vial in an oven at around 50 degrees Celsius for 20 minutes before vortexing for an additional 10 to 20 seconds at maximum speed. Next, pipette 270 microliters of the GUV outer solution into a 1.5-milliliter microcentrifuge tube. To this, pipette 15 microliters each of five molar sodium chloride and 4.5 molar potassium chloride.Gently pipette 300 microliters of lipid and oil mixture on top of the GUV outer solution. After incubation, assemble a CFE reaction by pipetting Solutions 1 to 3, DNA plasmid encoding for soluble sfGFP or variants of glutamate receptor sfGFP, murine RNAse, and one molar sucrose into a vial. Add ultrapure deionized water to bring the reaction volume to 20 microliters.Add 600 microliters of the lipid-oil mixture to the microcentrifuge tube containing the 20-microliter reaction mixture. Pipette up and down vigorously for one minute to emulsify the reaction. Gently layer the inner emulsion on top of the oil layer in the tube.Centrifuge for 10 minutes at 2000 g at four degrees Celsius. Next, carefully pipette out the excess oil and outer solution from the microcentrifuge tube. Gently mix the GUV pellet in the remaining solution to resuspend it.Transfer the GUV solution to a clean 96-well clear flat bottom plate for incubation. Transfer the sealed plate into a plate reader to incubate at 37 degrees Celsius for five to six hours. After incubation, place the plate on an inverted confocal microscope.Focus on a region of interest containing the GUVs, then capture the images at an excitation wavelength of 488 nanometers. Save the images in the TIFF format. Open the images in an image processing software.Click on the Brightness/Contrast setting panel to open it. Then adjust the brightness and contrast to make the fluorescent proteins visible. The wild-type glutamate receptor cell-free expression reactions encapsulated in GUV demonstrated excellent expression and membrane localization.PRSP-glutamate receptor was prone to aggregation and punctate formation. The soluble sfGFP was expressed in GUVS and stayed in the GUV lumen.