Name: cell-free production of proteoliposomes for functional analysis and antibody development targeting membrane proteins
Uploaded: 2020-09-22T15:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins at JoVE.com

本研究得到了AMED支持药物发现和生命科学研究的平台项目（支持创新药物发现和生命科学研究（BINDS）的基础）的支持，资助号为JP20am0101077。这项工作也得到了JSPS KAKENHI授权号20K05709的部分支持。

1. pEU表达质粒的制备
注意:pEU表达质粒应包括起始密码子，靶膜蛋白的开放阅读框和片段中的终止密码子（见 图1）。需要时，在适当的位置添加检测/纯化标签序列。限制性内切酶切或无缝克隆均适用于亚克隆。在这里，我们描述了一种使用无缝克隆方法的协议。
<ol><li>准备插入片段。<ol><li>使用 cDNA 模板、引物 1 和引物 2 通过 PCR 扩增目的基因。引物1和引物2分别包含15 bp重叠，用于无缝克隆（参见 材料表）。 注意:不包括要处理并从细胞中的成熟蛋白中去除的序列（例如，信号序列）。合成蛋白质的加工不在小麦无细胞系统中进行。不要添加科扎克序列。pEU-E01-MCS载体具有E01翻译增强剂。</li>
		<li>向PCR产物中加入1/25体积的 DpnI限制性内切酶以去除模板质粒DNA。在37°C孵育30分钟。</li>
		<li>使用 PCR 纯化试剂盒纯化 PCR 产物并将浓度调整为 20–50 ng/μL。</li>
	</ol></li>
	<li>线性化 pEU-E01-MCS 载体。<ol><li>使用 pEU-E01-MCS、引物 3 和引物 4 进行反向 PCR。</li>
		<li>向反向PCR产物中加入1/25体积的 DpnI限制性内切酶。在37°C孵育30分钟。</li>
		<li>根据制造商的建议，使用PCR纯化试剂盒纯化PCR产物。将浓度调整为 20–50 ng/μL。</li>
	</ol></li>
	<li>混合 2 μL 插入片段、2 μL 线性化载体和 4 μL 2x 无缝克隆酶混合物。</li>
	<li>用无缝克隆产品转化 大肠杆菌 菌株 JM109。使用撒布器，将细菌悬浮液散布在LB-氨苄西林琼脂平板上。 注意:pEU载体具有氨苄青霉素耐药标志物。</li>
	<li>确认pEU质粒中分别从MCS的5'和3'侧使用引物5和引物6构建的表达质粒的序列。</li>
	<li>扩增和纯化表达质粒。<ol><li>将质粒转化的大 肠杆菌 菌株JM109在150mL的LB-氨苄青霉素培养基中以37°C和每分钟125次振作振荡过夜。</li>
		<li>使用市售质粒制备中型试剂盒提取和纯化质粒。将质粒溶解在 500 μL 的 TE 缓冲液中。 注意:请勿使用小型制备试剂盒进行质粒提取。它不能提供足够质量和数量的质粒。</li>
		<li>加入 500 μL 苯酚/氯仿/异戊醇 （25:24:1）。剧烈混合5分钟，并在17，800×g 和室温下离心5分钟。将上层质粒溶液转移到新管中。 注意:戴上一次性手套，以保护皮肤免受苯酚和氯仿的侵害。 注意:为了去除质粒提取试剂盒中RNase的污染，请使用苯酚-氯仿纯化纯化来纯化质粒。</li>
		<li>加入 500 μL 氯仿以完全去除苯酚。剧烈混合5分钟，并在17，800 ×g 和室温下离心5分钟。将上层质粒溶液转移到新管中。</li>
		<li>加入2.5体积的乙醇和1/8体积的7.5M乙酸铵，并在-30°C下储存1小时。</li>
		<li>在4°C下以17，800 ×g 离心10分钟。用 500 μL 70% 乙醇洗涤沉淀。小心地除去上清液，让沉淀干燥5分钟。</li>
		<li>将表达质粒完全溶解在 100 μL 超纯水中。测量在260nm处具有吸光度的质粒的浓度。将浓度调节至1毫克/毫升。</li>
	</ol></li>
</ol>2. 体外转录
注意:在转录和翻译步骤中使用脱氧核糖核酸酶和无核酸酶的塑料管和吸头。避免高压灭菌塑料器皿以防止污染。
<ol><li>在新的塑料管中收集超纯水。 注意:不要使用经过DEPC处理的水，因为残留的DEPC会强烈抑制反应。使用新鲜纯化的超纯水进行转录和翻译。</li>
	<li>通过混合 115.2 μL 超纯水、40 μL 转录缓冲液 LM、20 μL NTP 混合物、2.4 μL 80 U/μL RNase 抑制剂、2.4 μL 80 U/μL SP6 聚合酶和 20 μL 1 mg/mL pEU 表达质粒来制备转录反应混合物。通过倒置轻轻混合试剂。执行快速旋转。</li>
	<li>将转录反应在37°C孵育6小时。</li>
	<li>通过倒置轻轻混合反应并快速旋转。立即用于翻译，否则冷冻并储存在-80°C。</li>
	<li>通过电泳确认转录产物。<ol><li>混合 100 mL 的 1x TAE 缓冲液和 1 g 琼脂糖。在微波炉中加热悬浮液以制备1%琼脂糖TAE凝胶。</li>
		<li>取 1 μL 转录反应，与 3 μL 水和 4 μL 2x 上样染料混合。 注意:不需要对RNA进行变性。</li>
		<li>将 4 μL 混合物和 2 μL DNA 分子量标准标记物加载到琼脂糖 TAE 凝胶中。</li>
		<li>在100V下电泳20分钟。</li>
		<li>将凝胶在溴化乙锭中染色30分钟。使用紫外透照仪和凝胶成像仪检查mRNA的梯形带模式。 注意:当观察到小于500 bp的拖尾条带时，怀疑mRNA降解。</li>
	</ol></li>
</ol>3. 准备翻译材料
<ol><li>准备翻译缓冲区。<ol><li>在 50 mL 管中分别混合 27 mL 新鲜制备的超纯水和 0.75 mL 各 40x 储备溶液，用于 S1、S2、S3 和 S4。 注意:根据最终所需的 1x 翻译缓冲区量调节材料量。 注意:使用后请勿存储或重新冻结多余的 1x 翻译缓冲区。</li>
	</ol></li>
	<li>准备肌酸激酶储备溶液。将冻干肌酸激酶溶解在超纯水中至终浓度为 20 mg/mL。将溶液少量（每 10 至 50 μL）分装在 0.2 mL 8 条联管中。将试管冷冻在液氮中，并储存在-80°C。 注意:解冻后不要重新冷冻肌酸激酶溶液。</li>
	<li>清洗透析杯（0.1 mL 大小）以去除透析膜中的甘油。 注意:有几种不同尺寸的透析杯。小型杯（0.1 mL）分别用于小规模测试（第5.4节），大尺寸杯（2 mL）分别用于大规模生产（第5.5节）。大尺寸杯子透析膜的清洗步骤是可以避免的。<ol><li>将 1 mL 超纯水放入新的 1.5 mL 管中。将小型透析杯 （0.1 mL） 插入试管中。在杯中加入 0.5 mL 超纯水。</li>
		<li>在室温下孵育30分钟以上。</li>
	</ol></li>
</ol>4. 脂质体的制备
注意:在这里，我们描述了两种制备脂质体的方案。一种使用即用型冻干脂质体（第4.1节），而另一种通过水合薄膜脂质体产生脂质体（第4.2节）。
<ol><li>使用冻干脂质体制备脂质体。 注意:生产蛋白脂质体的一种更简单的方法是使用市售的阿索凝集素脂质体。阿索凝集素是一种从大豆中提取的天然脂质。<ol><li>打开含有10mg冻干凝集素脂质体的小瓶（参见 材料表），并向小瓶底部加入200μL翻译缓冲液（第3.1节）。密封小瓶并孵育10分钟。</li>
		<li>通过将小瓶放在涡旋混合器上1分钟来剧烈混合。</li>
		<li>将小瓶插入 50 mL 管中。通过以500 x g 离心1分钟来旋转管。</li>
		<li>使用移液器将阿索凝集素脂质体悬浮液（50 mg 脂质/mL）转移到新的 1.5 mL 管中。立即使用脂质体进行翻译，否则在液氮中冷冻并储存在-80°C。</li>
	</ol></li>
	<li>通过水合薄脂质膜来制备脂质体。<ol><li>如果脂质以粉末形式出售，请溶解在氯仿或适当的有机溶剂中至10-100 mg/mL浓度。 注意:可以使用纯化和/或合成的两亲性脂质制备薄脂膜。阿索凝集素的纯化方法如前所述38。功能修饰的脂质，如生物素化脂质、荧光脂质和佐剂脂质，可以添加到基础脂质中以产生功能性脂质体。</li>
		<li>将含有50mg脂质的脂质溶液转移到蒸发瓶中。</li>
		<li>使用旋转蒸发器，蒸发溶剂并将脂质均匀地铺在烧瓶底部的壁上，形成一层脂质薄膜。</li>
		<li>将烧瓶放入真空干燥器中，在负压下放置过夜以完全除去溶剂。</li>
		<li>向蒸发瓶中加入 1 mL 翻译缓冲液。旋转烧瓶以将缓冲液铺在脂质薄膜上。孵育5分钟以水合薄膜。</li>
		<li>用超声波均质器或超声波清洗机对烧瓶进行超声处理。偶尔改变烧瓶的角度，使溶液彻底接触薄膜。确保薄膜脂质膜从底部剥离并完全均匀地乳化。 注意:含有脂质体的生物素化脂质的电子显微照片如图 1所示。</li>
		<li>将脂质体悬浮液（50 mg 脂质/mL）转移到新的 1.5 mL 管中。如果不立即使用，请将脂质体冷冻在液氮中，并储存在-80°C。</li>
	</ol></li>
</ol>5. 体外翻译
<ol><li>通过将试管在室温下漂浮在水面上几分钟来快速解冻小麦胚芽提取物。解冻后，立即倒置试管轻轻混合，向下旋转，在冰上冷却直至使用。 注意:使用后将小麦胚芽提取物冷冻在液氮中，并储存在-80°C。 它经受住了几次冻融循环。</li>
	<li>解冻 20 mg/mL 肌酸激酶储备溶液。混合 5 μL 储备溶液和 45 μL 翻译缓冲液，制备 2 mg/mL 肌酸激酶工作溶液。 注意:不建议重新冷冻肌酸激酶。</li>
	<li>需要时解冻脂质体或mRNA。</li>
	<li>进行小规模的蛋白质翻译。<ol><li>从试管和透析杯（0.1mL）中取出水，如步骤3.3.2中制备的那样。</li>
		<li>分别在管和透析杯中注入 1 mL 和 300 μL 翻译缓冲液。 注意:如果透析杯底部未到达 1.5 mL 管中的翻译缓冲液表面，请向管中额外注入 50–100 μL 缓冲液。</li>
		<li>通过混合 15.6 μL 翻译缓冲液、2.4 μL 2 mg/mL 肌酸激酶、12 μL 50 mg/mL 脂质体、15 μL 小麦胚芽提取物和 15 μL mRNA 来制备翻译反应混合物。通过倒置试管轻轻混合，然后向下旋转。</li>
		<li>使用 200 μL 移液器吸出 60 μL 平移反应混合物。</li>
		<li>将移液器吸头插入透析杯中翻译缓冲液的下表面。缓慢而轻柔地移出反应混合物。用盖子盖住透析杯以防止蒸发。 注意:反应混合物自然沉入杯子底部并形成双层。不要通过混合或摇晃杯子来干扰双层。</li>
	</ol></li>
	<li>进行大规模翻译（图1）。<ol><li>将 22 mL 翻译缓冲液倒入 25 mL 管中。将大尺寸透析杯 （2 mL） 插入管中，并在杯中加入 2 mL 翻译缓冲液。</li>
		<li>通过混合 130 μL 翻译缓冲液、20 μL 2 mg/mL 肌酸激酶、100 μL 50 mg/mL 脂质体、125 μL 小麦胚芽提取物和 125 μL mRNA 制备翻译反应混合物。通过移液轻轻混合。</li>
		<li>使用 1，000 μL 移液器吸出所有平移反应混合物 （500 μL）。以与步骤5.4.5中描述的相同方式将平移反应混合物注入透析杯中。用盖子盖住透析杯以防止蒸发。</li>
	</ol></li>
	<li>将反应物在15°C孵育24小时。</li>
	<li>通过移液在透析杯中充分混合反应。将粗蛋白脂质体悬浮液转移到新管中。 注意:建议使用平底 1.5 mL 管从小规模翻译中收集蛋白脂质体。离心后，脂质体在管的底角形成紧凑且易于看到的沉淀。</li>
</ol>6. 蛋白脂质体的纯化
<ol><li>将含有粗蛋白脂质体悬浮液的管在4°C下以17，800 ×g 离心10分钟。</li>
	<li>除去上清液。通过移液将蛋白脂质体沉淀悬浮在PBS（小规模:1mL，大型:10mL）中。</li>
	<li>重复蛋白脂质体的离心和洗涤另外两圈。</li>
	<li>洗涤后，加入少量PBS，并通过移液重新悬浮蛋白脂质体沉淀。使用微量移液器测量悬浮液的体积。添加 PBS 以将体积调整为 60 μL（小规模）或 500 μL（大规模）。将悬浮液转移到新的 1.5 mL 管中。</li>
	<li>将 10 μL 蛋白脂质体悬浮液转移到新的 PCR 管中以进行 SDS-PAGE。将其余样品分成较小的部分，以便在必要时使用。在液氮中冷冻并储存在-80°C。</li>
</ol>7. SDS-PAGE 和 CBB 染色
<ol><li>将 70 μL 水和 40 μL 3x SDS-PAGE 样品缓冲液加入 10 μL 蛋白脂质体悬浮液中。 注意:不要煮沸SDS-PAGE样品，因为膜蛋白会聚集，并且在电泳中几乎不会渗透到丙烯酰胺凝胶中。此外，向SDS-PAGE样品缓冲液中添加足够的还原剂（例如，终浓度为3%的2-巯基乙醇）以防止氧化。</li>
	<li>在电泳室中放置5%–20%梯度SDS-PAGE凝胶。同时加载 3 μL、6 μL、12 μL 蛋白脂质体样品、2 μL 蛋白质大小标记物和 BSA 标准系列。</li>
	<li>在52mA，400V下电泳30分钟。</li>
	<li>用CBB染料染色凝胶1小时。在热水中脱色并扫描凝胶图像。</li>
	<li>使用NIH Image J软件（https://imagej.nih.gov/ij/），量化每个泳道中膜蛋白的条带强度。使用BSA标准系列估计合成膜蛋白的量。</li>
</ol>

作者没有什么可透露的。

所提出的协议提供了一种以高成功率生产膜蛋白的方法。该协议简单、可重复且易于扩展。它还有可能减少消耗大量膜蛋白的实验的时间和成本。与双层法或透析法相比，双层透析方法将生产率提高了4-10倍（图2B）45。在极端情况下，双层透析法的离子通道和转运蛋白的产率分别比双层法提高了30倍和20倍（数据未显示）。该方案的高生产率是免疫抗原生产?...

膜蛋白是诊断和治疗中最重要的药物靶点之一。事实上，一半的小复合药物靶标是膜蛋白，例如G蛋白偶联受体（GPCR）和离子通道1。多年来，研究人员一直致力于膜蛋白的生物化学、生物物理和结构研究，以阐明其结构和功能2，3。还积极开发针对膜蛋白的单克隆抗体，以加速功能和结构研究，并开发治疗和诊断应用4，5，6，7，8，9。所有这些研究都需要大量的高质量膜蛋白10。例如，抗体开发需要几毫克具有自然构象的纯化膜蛋白。X射线晶体学需要大量高度纯化的膜蛋白。然而，膜蛋白的大规模生产仍然是膜蛋白研究的瓶颈11。膜蛋白具有具有一个或多个跨膜螺旋的复杂结构，在细胞稳态中起重要作用。膜蛋白的异源过表达导致多种障碍，例如膜蛋白聚集在高局部浓度下积累或干扰细胞信号通路。即使表达成功，后续的样品制备步骤也面临困难。例如，蛋白脂质体的制备需要膜蛋白溶解、纯化和稳定的高水平技能和专业经验，并且需要花费大量的精力和时间12，13。
另一方面，近几十年来出现了一些先进技术，可以在不使用活细胞的情况下生产蛋白质14，15，16，17，18。无细胞蛋白质合成技术在试管中重建翻译反应。由于细胞表达系统没有限制，因此无细胞系统有可能合成各种难以表达或在细胞中显示毒性的蛋白质。纯化的细胞提取物或重组的翻译机制与模板mRNA、氨基酸和能量源混合，并在短时间内合成重组蛋白。关于膜蛋白的合成，某些由脂质或两亲分子组成的支架，例如脂质体、双细胞、纳米盘或共聚物被添加到无细胞反应19，20，21，22，23，24中。合成的膜蛋白与支架相互作用，可以在水中稳定。无细胞合成的膜蛋白广泛用于功能研究和抗体生产25，26，27，28，29，30，31。
在该协议中，我们描述了一种使用小麦无细胞系统和脂质体生产蛋白石体的有效无细胞方法。小麦无细胞蛋白质合成系统是一种强大的体外翻译系统，使用小麦胚芽15，32，33的提取物。小麦胚芽含有大量的翻译机制和很少的翻译抑制剂。作为真核生物成员的小麦中的翻译机制适用于翻译真核蛋白质，其翻译效率几乎不受模板mRNA的密码子使用的影响。使用小麦无细胞系统，我们合成了多种蛋白质，包括蛋白激酶34，35，泛素连接酶36，转录因子37和膜蛋白，成功率很高。对于膜蛋白生产，我们将脂质囊泡脂质体作为支架19，38 添加到翻译混合物中。膜蛋白的疏水结构域与脂质双层相互作用，并与脂质体自发整合。密度梯度离心用于从内源性小麦蛋白中严格分离蛋白石质体，即使翻译反应混合物的普通离心足以简单纯化蛋白石质体20。利用小麦无细胞系统合成了多种完整的膜蛋白，并应用于各种研究和开发25，38，39，40，41，42，43，44。此外，我们还开发了用于大规模生产的"双层透析法"45，46.该方法中，将杯式透析装置浸入底物进料缓冲液中，并在杯中形成两层平移反应混合物和底物进料缓冲液，如图1所示。连续供应底物和去除副产物可以在反应混合物的顶部和底部长时间有效地进行，从而获得出色的转化效果（图2A和图2B）45。

<table><tbody><tr><td>&amp;times;3 SDS-PAGE sample buffer</td><td></td><td></td><td>Containing 10% 2-mercaptoethanol</td></tr><tr><td>5-20% gradient SDS-PAGE gel</td><td>ATTO</td><td>E-D520L</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td>Diluted ethanol by ultrapure water.</td></tr><tr><td>Agarose</td><td>Takara Bio</td><td></td><td></td></tr><tr><td>Ammonium acetate</td><td>Nakalai tesque</td><td>02406-95</td><td>As this reagent is deliquescent, dissolve all of it in water once opened and store it at -30&amp;deg;C.</td></tr><tr><td>Ampicillin Sodium</td><td>Nakalai tesque</td><td>02739-74</td><td></td></tr><tr><td>Asolectin Liposome, lyophilized</td><td>CellFree Sciences</td><td>CFS-PLE-ASL</td><td>A vial contains 10 mg of lyophilized liposomes.</td></tr><tr><td>BSA standard</td><td></td><td></td><td>1000 ng, 500 ng, 250 ng, 125 ng BSA / 10 &amp;micro;L &amp;times;1 SDS-PAGE sample buffer</td></tr><tr><td>CBB gel stain</td><td></td><td></td><td></td></tr><tr><td>cDNA clone of interest</td><td></td><td></td><td>Plasmid harboring cDNA clone or synthetic DNA fragment</td></tr><tr><td>Chloroform</td><td>Nakalai tesque</td><td>08402-84</td><td></td></tr><tr><td>Cooled incubator</td><td></td><td></td><td>Temperature ranging from 0 to 40 &amp;deg;C or wider.</td></tr><tr><td>Creatine kinase</td><td>Roche Diagnostics</td><td>04524977190</td><td></td></tr><tr><td>Dialysis cup (0.1 mL)</td><td>Thermo Fisher Scientific</td><td>69570</td><td>Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 0.1 mL</td></tr><tr><td>Dialysis cup (2 mL)</td><td>Thermo Fisher Scientific</td><td>88404</td><td>Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 2 mL</td></tr><tr><td>DNA ladder marker</td><td>Thermo Fisher Scientific</td><td>SM0311</td><td>GeneRuler 1 kb DNA Ladder</td></tr><tr><td>DpnI</td><td>Thermo Fisher Scientific</td><td>FD1703</td><td>FastDigest DpnI</td></tr><tr><td>E. coli strain JM109</td><td></td><td></td><td></td></tr><tr><td>Electrophoresis chamber</td><td>ATTO</td><td></td><td></td></tr><tr><td>Ethanol (99.5%)</td><td>Nakalai tesque</td><td>14713-95</td><td></td></tr><tr><td>Ethidium bromide</td><td></td><td></td><td></td></tr><tr><td>Evaporation flask, 100 mL</td><td></td><td></td><td></td></tr><tr><td>Gel imager</td><td></td><td></td><td></td></tr><tr><td>Gel scanner</td><td></td><td></td><td>We use document scanner and LED immuninator as a substitute.</td></tr><tr><td>LB broth</td><td></td><td></td><td></td></tr><tr><td>Lipids of interest</td><td>Avanti Polar Lipids</td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td>TOMY</td><td>MX-307</td><td></td></tr><tr><td>NTP mix</td><td>CellFree Sciences</td><td>CFS-TSC-NTP</td><td>Mixture of ATP, GTP, CTP, UTP, at 25 mM each</td></tr><tr><td>Nuclease-free 25 mL tube</td><td>IWAKI</td><td>362-025-MYP</td><td></td></tr><tr><td>Nucrease-free plastic tubes</td><td>Watson bio labs</td><td></td><td>Do not autoclave. Use them separately from other experiments.</td></tr><tr><td>Nucrease-free tips</td><td>Watson bio labs</td><td></td><td>Do not autoclave. Use them separately from other experiments.</td></tr><tr><td>PBS buffer</td><td></td><td></td><td></td></tr><tr><td>PCR purification kit</td><td>MACHEREY-NAGEL</td><td>740609</td><td>NucleoSpin Gel and PCR Clean-up</td></tr><tr><td>pEU-E01-MCS vector</td><td>CellFree Sciences</td><td>CFS-11</td><td></td></tr><tr><td>Phenol/chloroform/isoamyl alcohol (25:24:1)</td><td>Nippon Gene</td><td>311-90151</td><td></td></tr><tr><td>Plasmid prep Midi kit</td><td>MACHEREY-NAGEL</td><td>740410</td><td>NucleoBond Xtra Midi</td></tr><tr><td>Primer 1</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&amp;rsquo;-CCAAGATATCACTAGnnnnnnnnnnnnnnnnnnnnnnnn-3&amp;rsquo;&lt;br/&gt; Gene specific primer, forward. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&amp;hellip;. (20-30 bp) shows gene specific sequence.</td></tr><tr><td>Primer 2</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&#039;-CCATGGGACGTCGACnnnnnnnnnnnnnnnnnnnnnnnn-3&amp;rsquo;&lt;br/&gt; Gene specific primer, reverse. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&amp;hellip;. (20-30 bp) shows gene specific sequence.</td></tr><tr><td>Primer 3</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&#039;-GTCGACGTCCCATGGTTTTGTATAGAAT-3&#039;&lt;br/&gt; Forward primer for vector linearization. Underline works as overlap in seamless cloning.</td></tr><tr><td>Primer 4</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&#039;-CTAGTGATATCTTGGTGATGTAGATAGGTG-3&#039;&lt;br/&gt; Reverse primer for vector linearization. Underline works as overlap in seamless cloning.</td></tr><tr><td>Primer 5</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&amp;rsquo;-CAGTAAGCCAGATGCTACAC-3&amp;rsquo;&lt;br/&gt; Sequencing primer, forward</td></tr><tr><td>Primer 6</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&amp;rsquo;- CCTGCGCTGGGAAGATAAAC-3&amp;rsquo;&lt;br/&gt; Sequencing primer, reverse</td></tr><tr><td>Protein size marker</td><td>Bio-Rad</td><td>1610394</td><td>Precision Plus Protein Standard</td></tr><tr><td>Rotary evaporator</td><td></td><td></td><td></td></tr><tr><td>seamless cloning enzyme mixture</td><td>New England BioLabs</td><td>E2611L</td><td>Gibson Assembly Master Mix&lt;br/&gt; Other seamless cloning reagents are also avairable.</td></tr><tr><td>SP6 RNA Polymerase &amp;amp; RNase Inhibitor</td><td>CellFree Sciences</td><td>CFS-TSC-ENZ</td><td></td></tr><tr><td>Submarine Electrophoresis system</td><td></td><td></td><td></td></tr><tr><td>TAE buffer</td><td></td><td></td><td></td></tr><tr><td>Transcription Buffer LM</td><td>CellFree Sciences</td><td>CFS-TSC-5TB-LM</td><td></td></tr><tr><td>Translation buffer</td><td>CellFree Sciences</td><td>CFS-SUB-SGC</td><td>SUB-AMIX SGC (&amp;times;40) stock solution (S1, S2, S3, S4).&lt;br/&gt; Prepare &amp;times;1 translation buffer before use by mixing stock S1, S2, S3, S4 stock and ultrapure water.</td></tr><tr><td>Ultrapure water</td><td></td><td></td><td>We recommend to prepare ultrapure water by using ultrapure water production system every time you do experiment. Do not autoclave.&lt;br/&gt; We preparaed ultrapure water by using Milli-Q Reference and Elix10 system.&lt;br/&gt; Commercially available nuclease-free water (not DEPC-treated water) can be used as a substitute. Take care of contamination after open the bottle.</td></tr><tr><td>Ultrasonic homogenizer</td><td>Branson</td><td>SONIFIER model 450D-Advanced</td><td>Ultrasonic cleaner can be used as a substitute.</td></tr><tr><td>UV transilluminator</td><td></td><td></td><td></td></tr><tr><td>Vacuum desiccator</td><td></td><td></td><td></td></tr><tr><td>Wheat germ extract</td><td>CellFree Sciences</td><td>CFS-WGE-7240</td><td>WEPRO7240</td></tr></tbody></table>

cell-free production of proteoliposomes for functional analysis and antibody development targeting membrane proteins

膜蛋白在各种细胞过程中起着至关重要的作用，并发挥着至关重要的功能。膜蛋白在药物发现中具有医学意义，因为它们是一半以上药物的靶标。对膜蛋白进行生化、生物物理和结构研究以及抗体开发的一个障碍是难以生产大量具有正确构象和活性的高质量膜蛋白。在这里，我们描述了一种"双层透析方法"，使用小麦无生殖细胞系统，脂质体和透析杯在短时间内高效合成膜蛋白并制备纯化的蛋白脂质体，成功率高。膜蛋白可以产生多达几毫克，例如GPCR，离子通道，转运蛋白和四跨蛋白。这种无细胞方法有助于减少制备高质量蛋白水产酶体的时间、成本和精力，并为膜蛋白的功能分析、药物靶标筛选和抗体开发提供了合适的手段。

使用该协议，可以在短时间内获得部分纯化的蛋白脂质体。代表性结果如图 2A所示。采用双层透析法（小规模）成功合成了25个A、B、C类GPCR，并通过离心和缓冲液洗涤进行了部分纯化。虽然合成蛋白质的量因蛋白质类型而异，但当使用大型透析杯时，每次反应通常可以合成50至400μg膜蛋白。由于小麦无细胞系统的高可扩展性，通过增加反应次数可以很容易地产生几毫克的膜?...

Watch this Scientific Journal Video about Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins at JoVE.com

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

该协议描述了一种有效的无细胞方法，通过使用小麦无细胞系统和脂质体的双层透析方法生产高质量的蛋白水产体。该方法为膜蛋白的功能分析、药物靶点筛选和抗体开发提供了合适的手段。

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

cell-free-production-proteoliposomes-for-functional-analysis-antibody

Research

JoVE Journal

Biochemistry

3.7K 次观看.  Ehime University. 该协议描述了一种有效的无细胞方法，通过使用小麦无细胞系统和脂质体的双层透析方法生产高质量的蛋白水产体。该方法为膜蛋白的功能分析、药物靶点筛选和抗体开发提供了合适的手段。

视频: Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

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

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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

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

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

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 ...

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.

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 ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

生物工程

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

抗原脂质体用于生成特定于疾病的抗体

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

免疫与感染

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

生物学

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

用于疫苗开发的无细胞规模生产和对来自 穆里达鲁姆衣原 体的重组主要外膜蛋白的佐剂添加

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

摘要

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使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用

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

膜蛋白共翻译插入预成型纳米盘

含脂肪酸脂质体的制备、纯化及应用

抗原脂质体用于生成特定于疾病的抗体

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

巨脂质体制剂成像和膜片钳电

用于疫苗开发的无细胞规模生产和对来自穆里达鲁姆衣原体的重组主要外膜蛋白的佐剂添加

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

摘要

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使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用

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

膜蛋白共翻译插入预成型纳米盘

含脂肪酸脂质体的制备、纯化及应用

抗原脂质体用于生成特定于疾病的抗体

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

巨脂质体制剂成像和膜片钳电

用于疫苗开发的无细胞规模生产和对来自 穆里达鲁姆衣原 体的重组主要外膜蛋白的佐剂添加

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

用于疫苗开发的无细胞规模生产和对来自穆里达鲁姆衣原体的重组主要外膜蛋白的佐剂添加