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>&#215;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&#176;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 &#181;L &#215;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 &#176;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&#8217;-CCAAGATATCACTAGnnnnnnnnnnnnnnnnnnnnnnnn-3&#8217; 
			Gene specific primer, forward. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&#8230;. (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&#39;-CCATGGGACGTCGACnnnnnnnnnnnnnnnnnnnnnnnn-3&#8217; 
			Gene specific primer, reverse. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&#8230;. (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&#39;-GTCGACGTCCCATGGTTTTGTATAGAAT-3&#39; 
			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&#39;-CTAGTGATATCTTGGTGATGTAGATAGGTG-3&#39; 
			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&#8217;-CAGTAAGCCAGATGCTACAC-3&#8217; 
			Sequencing primer, forward</td>
		</tr>
		<tr>
			<td>Primer 6</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom oligo synthesis</td>
			<td>5&#8217;- CCTGCGCTGGGAAGATAAAC-3&#8217; 
			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 
			Other seamless cloning reagents are also avairable.</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase &#38; 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 (&#215;40) stock solution (S1, S2, S3, S4). 
			Prepare &#215;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. 
			We preparaed ultrapure water by using Milli-Q Reference and Elix10 system. 
			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

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

科研

生物化学

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

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 ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

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.