Name: co-translational insertion of membrane proteins into preformed nanodiscs
Uploaded: 2020-11-19T14:30:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

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

1. S30裂解物的制备
<ol><li>第一天: 从LB琼脂平板上的甘油储备液中划出细胞，并在37°C孵育过夜。</li>
	<li>第二天: 用琼脂平板上的细胞接种200mLLB培养基，并在37°C下孵育12-14小时。</li>
	<li>第三天:接种10 L无菌YPTG培养基（10 g / L酵母提取物，16 g / L胰蛋白胨，5 g / L NaCl，19.8 g / L葡萄糖，4.4 mM KH 2 PO 4，8 mM K2HPO4）在15 L搅拌釜式反应器中回火至37°C，并加入100 mL预培养物（1:100）。在37°C，500rpm和高曝气（每分钟~3个空气量）下培养。为防止过度起泡，请添加无菌消泡剂。</li>
	<li>以固定的时间间隔测量 600 nm 处的光密度 （OD）。大约2小时后，培养物将进入对数阶段。</li>
	<li>HS30裂解物的改性: 当细胞处于对数中期（OD 600≈3.6-4.2）时，向培养基中加入300mL乙醇，并在42°C，500rpm和高通气（每分钟约3个空气体积）下再培养45分钟。然后按照步骤1.6中的说明继续冷却和收获细胞培养物。对于标准S30裂解物的生产，请跳过此步骤并继续执行步骤1.6。</li>
	<li>当细胞处于对数中期（OD 600≈3.6-4.2）时，在30分钟内将发酵罐冷却至20°C以下<。最终的OD 600应该在4.5-5.5左右。在4°C下以4，500×g收获细胞20分钟，并弃去上清液。在以下步骤中将细胞保持在4°C。 注意:在冷却过程中，可能会发生过多的泡沫。在这种情况下，添加消泡剂或降低搅拌速度和/或减少生物反应器中的气流。</li>
	<li>使用刮刀将细胞完全悬浮在 300 mL S30-A 缓冲液（10 mM Tris-HCl，pH 8.2、14 mM Mg（OAc）2、60 mM KCl、6 mM 2-巯基乙醇）中，然后上下移液悬浮液直至均匀。在4°C下以8，000× g 离心10分钟。 重复此步骤两次。 注意:2-巯基乙醇有毒。避免接触皮肤或呼吸道。如果可能的话，在引擎盖下工作。在最后一个洗涤步骤之前称量空离心杯。离心后，称量湿细胞沉淀，然后继续步骤1.8或储存沉淀直至进一步使用。 注意:湿细胞沉淀的重量为每1升生物反应器培养物5-7克。此时，方案可以暂停，沉淀可以在液氮中冷冻并在-80°C下储存4-8周。</li>
	<li>将细胞彻底悬浮在 1.1 体积 （1 g = 1 mL） 的 S30-B 缓冲液（10 mM Tris-HCl，pH 8.2、14 mM Mg（OAc）2、60 mM KOAc、1 mM DTT、1 片 cOmplete 蛋白酶抑制剂）中。将悬浮液填充到预冷的法国压力单元中，并在 1，000 psig 下破碎细胞。将单元格通过法式印刷机一次。</li>
	<li>将裂解物在4°C下以30，000× g 离心30分钟。 将上清液转移到新管中并重复离心步骤。 注意:第一次离心后，沉淀顶部可能存在松散和粘稠的层，不应与上清液一起转移。</li>
	<li>应用高盐和加热步骤去除不稳定的裂解物成分并从核糖体中释放mRNA。将裂解物调节至0.4M NaCl，并在42°C下在水浴中孵育45分钟。 注意:此步骤将导致显着的沉淀形成。在孵育期间不时翻转或倒置试管。</li>
	<li>将浑浊悬浮液（通常为 50-80 mL）转移到截止浓度为 10-14 kDa 的透析管中，并用 5 L S30-C 缓冲液（10 mM Tris-HCl，pH 8.2，14 mM Mg（OAc）2，60 mM KOAc，0.5 mM DTT）透析。2-3小时后更换S30-C透析缓冲液，再透析12-14小时。</li>
	<li>第四天: 将透析悬浮液转移到离心管中，并在4°C下以30，000× g 离心30分钟。 将上清液（S30裂解物）转移到新鲜的50 mL管中并轻轻混合。裂解物的蛋白质浓度应约为30-40mg / mL。在液氮中冲击冷冻等分试样（例如0.5mL，1mL），并储存在-80°C。 等分试样稳定>1年。</li>
</ol>2. T7 RNA聚合酶的表达和纯化
<ol><li>第一天: 用新鲜接种的BL21（DE3）星形x pAR1219细胞接种含有100μg/ mL氨苄青霉素的200mL LB培养基，并在37°C和180rpm下孵育12-16小时。</li>
	<li>第二天: 将 1 L 极好的肉汤（12 g/L 胰蛋白胨、24 g/L 酵母提取物、4 mL/L 甘油）接种在装有 10 mL 预培养物的 2 L 挡板烧瓶中，并在 37 °C 和 180 rpm 搅拌下孵育。起始 OD 600 应在 0.1 左右。</li>
	<li>当OD 600达到0.6-0.9时，加入IPTG至终浓度为1mM以诱导T7RNAP表达并继续培养5小时。</li>
	<li>通过在4°C下以4，500× g 离心20分钟来收获细胞。 将细胞冷冻在液氮中并储存在-80°C。 注意:协议可能在此处暂停。</li>
	<li>第三天: 向冷冻细胞沉淀中加入 30 mL 冰冷悬浮缓冲液（30 mM Tris-HCl，pH 8.0，含一片 cOmplete 蛋白酶抑制剂），并在室温下在水浴中解冻沉淀。然后，通过上下移液悬浮沉淀直至均匀。</li>
	<li>如上一节所述，使用法式压榨机进行细胞破碎，或根据制造商的建议使用超声设备。随后，在4°C下以20，000× g 离心30分钟，并将上清液转移到新管中。</li>
	<li>为了沉淀基因组DNA，缓慢地将硫酸链霉素添加到上清液中，并在轻轻搅拌下，直到达到3%（w / v）的终浓度。在4°C下轻轻搅拌孵育5-10分钟。在4°C下以20，000× g 离心30分钟。</li>
	<li>用0.45μm过滤器过滤上清液，并将其加载到柱体积（CV）为40mL的Q-琼脂糖柱上，使用蠕动泵以4mL/min的流速与2 CV平衡缓冲液（30 mM Tris-HCl，pH 8.0，10 mM EDTA，50 mM NaCl，5%甘油和10 mM 2-巯基乙醇）平衡。然后，将色谱柱连接到UV检测器，并继续用平衡缓冲液洗脱，直到280nm处的UV信号达到稳定的基线。</li>
	<li>以 3 mL/min 的流速以 50 mM 至 500 mM NaCl 的梯度洗脱 T7RNAP。收集 1 mL 级分，通过将每种馏分的 10 μL 与 2 x SDS 样品缓冲液混合，制备用于 SDS-PAGE 分析的样品。运行SDS-PAGE并用考马斯蓝R染色凝胶，T7RNAP应在大约90 kDa处显示为突出的条带以及许多其他杂质条带。</li>
	<li>含有T7RNAap的池级分，并使用具有12-14kDa MWCO的膜在透析缓冲液（10mMK2 HPO 4 / KH2PO4，pH 8.0，10mM NaCl，0.5mM EDTA，1mM DTT，5%（w / v）甘油）中透析12-16小时。</li>
	<li>第四天: 收集 T7RNAP 溶液并使用具有 12-14 MWCO 的过滤装置浓缩至终浓度为 3-10 mg/mL。T7RNAP可能在浓缩过程中开始沉淀。一旦样品出现浑浊，立即停止浓缩。加入甘油至终浓度为50%（w / v）并轻轻混合。速冻0.5-1mL等分试样并储存在-80°C。 工作T7RNAP等分试样可以储存在-20°C。 注意:应从 1 L 发酵中获得大约 20-40 mL 的 5 mg/mL 部分纯化的 T7RNAP，含 20，000-40，000 单位。为了测试每个T7RNAP批次的最佳量，应进行含有0.02mg / mL-0.1mg / mL制备的T7RNAP样品的绿色荧光蛋白（GFP）的CF表达反应。</li>
</ol>3. MSP1E3D1的表达和纯化
<ol><li>第一天:使用标准热冲击转化或电穿孔方案，用pET28（a）-MSP1E3D1载体31转化大肠杆菌BL21（DE3）星形细胞。在含有30μg/ mL卡那霉素的LB琼脂上划线或板转化细胞，并在37°C下孵育12-16小时。</li>
	<li>第二天: 接种200mL补充有0.5%（w / v）葡萄糖和30μg/ mL卡那霉素的LB培养基，从琼脂平板上挑选单个菌落，并在37°C下以180rpm孵育12-16小时。</li>
	<li>第三天: 在搅拌罐生物反应器中接种补充有 0.5% （w/v） 葡萄糖和 30 μg/mL 卡那霉素和 100 mL 预培养物的 10 L LB 培养基。在37°C，500rpm下进行发酵，每分钟曝气约3个生物反应器体积。在起泡过度的情况下，添加消泡剂。 注意:可以使用带挡板的摇瓶代替发酵罐。</li>
	<li>在OD 600为6.5-7.5时，加入IPTG至终浓度为1mM，并继续在37°C下孵育1小时。通过在4°C下以4，500× g 离心20分钟来收获细胞。 用200mL的0.9%（w / v）NaCl洗涤细胞沉淀，并在4°C下以8，000× g 再次离心10分钟。 弃去上清液并使用刮刀将细胞转移到 50 mL 管中。预期的湿颗粒重量为每升生物反应器培养物8-12g。在液氮中速冻细胞沉淀并储存在-80°C直至进一步使用。 注意:协议可以在此处暂停。</li>
	<li>第四天: 为了纯化，在室温水浴中解冻细胞沉淀。 将细胞悬浮在MSP-A缓冲液（40mM Tris-HCl，pH 8.0，300mM NaCl，1%（w / v）Triton X-100，每50mL细胞悬液1片c0mplete蛋白酶抑制剂混合物）中，以产生30-40%（w / v）细胞悬液。</li>
	<li>通过超声处理或使用法式压榨机破碎细胞，并在4°C下以30，000× g 离心30分钟。 将上清液转移到新管中并通过0.45μm过滤器过滤。使用蠕动泵将滤液涂在与5 CV的MSP-B缓冲液（40mM Tris-HCl，pH 8.0，300mM NaCl，1%（w / v）Triton X-100）平衡的Ni2 + 负载的次氮基三乙酸柱（CV > 15 mL）上。 注意:为了便于过滤，可以将上清液再超声处理一分钟以分解大的DNA片段。</li>
	<li>样品上样后，用 5 CV MSP-B、5 CV MSP-C（40 mM Tris-HCl，pH 8.9，300 mM 氯化钠，50 mM 胆酸）、5 CV MSP-D（40 mM Tris-HCl，pH 8.9，300 mM NaCl）、5 CV MSP-E（40 mM Tris-HCl，pH 8.0，300 mM NaCl）和 5 CV MSP-F（40 mM Tris-HCl，pH 8.0， 300 mM 氯化钠，50 mM 咪唑）。 注意:MSP-C缓冲液中的胆酸对于去除附着在MSP上的脂质很重要。胆酸在低pH值下不完全溶解。因此，在MSP-C和MSP-D中，pH值必须调节到8.9。用MSP-D缓冲液清洗色谱柱非常重要，因为较低的pH值可能会导致残留的胆酸沉淀并堵塞或损坏色谱柱。</li>
	<li>用MSP-G（40 mM Tris-HCl，pH 8.0，300 mM NaCl，300 mM咪唑）洗脱MSP。收集 1 mL 的馏分并监测 280 nm 处的紫外线吸收。收集并合并洗脱峰的馏分。</li>
	<li>将汇集的MSP级分转移到12-14kDa MWCO透析膜管中，并在4°C下对5L的MSP-H（40mM Tris-HCl，pH 8.0，300mM NaCl，10%（w / v）甘油）透析2-3小时。 更换为新鲜的5L MSP-H缓冲液，并在4°C下再透析12-16小时。</li>
	<li>第五天:将MSP溶液转移到离心管中，并在4°C下以18，000×g离心30分钟以除去沉淀物。将上清液转移到新管中，并在4°C下使用具有12-14kDa MWCO的超滤设备浓缩至200-800μM。 使用紫外可见分光度计测量设备测量浓度。使用消光系数27.31 M-1 cm-1和分子量31.96 kDa计算浓度。 注意:在生物反应器中的表达通常每L培养物产生15-30mg的MSP1E3D1。</li>
	<li>将浓缩的MSP溶液在液氮中减震冷冻等分试样并储存在-80°C。</li>
</ol>4. MSP1E3D1纳米盘的组装
<ol><li>第一天: 在 100 mM 胆酸钠中制备 1-2 mL 的 50 mM 脂质储备液（DMPG、DOPG、POPG DMPC、DOPC 或 POPC）。如果在同一天不使用，则储存在-20°C。 注意:脂质溶液必须澄清。如果溶液不澄清，胆酸钠浓度可以增加到150mM。</li>
	<li>将MSP1E3D1溶液与脂质溶液混合。加入终浓度为0.1%（w / v）的十二烷基磷酸胆碱（DPC）。组装反应可以调节至3 mL（表2）或12 mL的最终体积，对应于典型体积的透析盒（10 kDa MWCO）。在温和搅拌下在21°C孵育组装反应1小时。 注意:对于每种脂质，必须使用特定的MSP1E3D1:脂质比以确保均匀的纳米盘制备（表2）。对于新的脂质或脂质混合物，应通过中试实验和体积排阻色谱分析确定最佳比例14。</li>
	<li>用椎间盘形成（DF）缓冲液（10mM Tris-HCl，pH 8.0，100mM NaCl）预润湿透析盒的膜。用注射器将组装混合物转移到透析盒中。潜在的气泡可以通过使用注射器抽吸去除。</li>
	<li>第1-4天: 使用搅拌棒在室温下在室温下对3 x 5 L DF缓冲液透析10-20小时。然后将组装混合物从透析盒转移到具有10 kDa MWCO的离心过滤单元中，该单元与DF缓冲液平衡。在4°C下浓缩4，000× g 。 注意:浑浊的透析混合物可能表明MSP:脂质的化学计量比错误。沉淀物必须在4°C下以18，000× g 除去20分钟，然后才能浓缩样品。为避免样品浓缩过程中沉淀，请使用具有大死角的超滤装置。</li>
	<li>将组装好的纳米盘浓缩至至少300μM的浓度。 使用紫外可见光读数器测量浓度。考虑到一个纳米盘由两个MSP1E3D1分子形成，因此，MSP的浓度必须除以2才能确定正确的纳米盘量。 注意:所述设置（表2）将产生0.6-1 mL的300 μM纳米盘溶液。</li>
	<li>将浓缩的纳米盘制剂在4°C下以18，000× g 离心20分钟以除去沉淀物。然后，吸出上清液并在液氮中速冻50μL等分试样并储存在-80°C。 注意:建议使用体积排阻色谱法评估纳米盘形成的成功。样品应该是单分散的，并且应该只含有少量的聚集体。聚集体表明设置中的脂质量过高。作为参考，可以使用纯化的MSP1E3D1。如果纳米盘制备在参比MSP1E3D1峰的高度处显示峰，则所选脂质与MSP1E3D1的比率太低。</li>
</ol>5. 制备级 3 mL CECF 反应装置
<ol><li>第一天: 准备列出的所有必要的储备溶液（表3）。将储备储存在-20°C并在室温下解冻。确保在解冻后和移液前彻底混合所有原液。计算所有库存所需的体积并制定移液方案（表4）。高分子量化合物将仅添加到RM中。对于低分子量化合物，可以制备用于RM和FM的3 x预混液。 注意:由于混合过程中的体积损失，化合物混合物的最终体积可能小于计算值。这可以通过添加 2-5% 的过量体积来补偿体积损失来避免。</li>
	<li>在 100 mM 三乙酸酯（pH 8.0）中平衡 3 mL 透析盒的膜。确保去除多余的缓冲液。</li>
	<li>使用注射器将RM转移到透析盒中。用注射器抽吸去除RM室中过多的空气。将透析盒放入透析室。</li>
	<li>用 60 mL 的 FM 填充透析室。 将盖子放在室上并拧紧螺钉以固定它。将室在30°C以200rpm孵育12-16小时。 注意:确保在搅拌过程中腔室保持直立位置，否则会阻碍低分子化合物的交换。</li>
	<li>第二天: 使用注射器，从透析室吸出RM。将RM转移到新管中，并在4°C下以16，000× g 离心10分钟以除去沉淀物。将上清液转移到新鲜管中。现在可以进一步分析上清液中的蛋白质。 注意:在某些情况下，离心后会出现沉淀。这可以提供有关所分析的纳米盘或反应化合物的适用性的重要信息，以保持合成的MP可溶性。因此，建议通过使用SDS-PAGE、蛋白质印迹或通过目视评估沉淀大小来分析沉淀物中可能存在的目标量。</li>
</ol>6. 用于 Mg2+ 离子筛选的分析规模 60 μL CECF 反应装置
<ol><li>第一天: 混合相应 3x 预混液的所有组分，并将 60 μL 分配给 RM，将 825 μL 分配给 FM。 用 H2O 填充 FM，并通过涡旋混合 FM。将FM转移到24孔微孔板的3个孔中（表5）。 注意:由于可能无法访问定制的Mini-CECF容器，因此计算表 5 中的体积是在透析柱（10-14 kDa MWCO，RM:100 μL）中进行的反应，FM体积为1.7 mL，在96个深孔（2 mL）板中（图1）。</li>
	<li>用 10-14 kDa MWCO 将再生纤维素透析管切成约 25 x 20 mm 大小的碎片，并储存在含有 0.05% （w/v） 叠氮化钠的 H2O 中。 注意:叠氮化钠毒性很大。避免与皮肤或粘膜接触。不要使用金属容器，因为叠氮化钠会与金属反应形成爆炸性金属叠氮化物。</li>
	<li>在Mini-CECF容器组装之前，用组织从透析膜中去除过量的H2O。在每个容器上放置一个膜片，并用聚四氟乙烯环固定膜。</li>
	<li>将 Mini-CECF 容器转移到装有 825 μL FM 的 24 孔板的孔中。 避免 Mini-CECF 容器透析膜下方的气泡。</li>
	<li>如表5所示向RM中加入高分子组分，并通过上下移液进行混合。向反应容器中加入 60 μL。避免在转移粘性溶液时产生气泡。</li>
	<li>切割 2 张 8 x 10 cm 的密封热塑性薄膜片，并将它们包裹在 24 孔板上，反应容器在里面。这将减少反应混合物的蒸发。现在将细胞培养板的盖子放在板上并用胶带固定。将板在30°C和200rpm搅拌下孵育12-16小时。</li>
	<li>第二天: 通过在Mini-CECF容器上用移液管尖端刺穿透析膜并吸出RM来收获反应混合物。 将RM转移到新管中，并在4°C下以16，000× g 离心10分钟以除去沉淀物。将上清液转移到新鲜管中。现在可以进一步分析上清液中的蛋白质。 注意:在某些情况下，离心后会出现沉淀。这可以提供有关所分析的纳米盘或其他反应化合物的适用性的重要信息，以保持合成的MP可溶性。因此，建议通过SDS-PAGE、蛋白质印迹或颗粒大小的目视评估来分析沉淀物中可能存在的靶标量。</li>
</ol>

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作者没有什么可透露的。

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

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

<table><tbody><tr><td>1,2-dimyristoyl-sn-glycero-3-phospho-(1&#039;-rac-glycerol) (sodium salt) (DMPG)</td><td>Avanti Polar Lipids (USA)</td><td>840445P</td><td></td></tr><tr><td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td><td>Avanti Polar Lipids (USA)</td><td>850345C</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphocholine (sodium salt) (DOPC)</td><td>Avanti Polar Lipids (USA)</td><td>850375C</td><td></td></tr><tr><td>1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DOPG)</td><td>Avanti Polar Lipids (USA)</td><td>840475C</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td><td>Avanti Polar Lipids (USA)</td><td>850457C</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#039;-rac-glycerol) (sodium salt) (POPG)</td><td>Avanti Polar Lipids (USA)</td><td>840034C</td><td></td></tr><tr><td>2-Amino-2-(hydroxymethyl)-propan-1,3-diol (Tris)</td><td>Carl Roth (Germany)</td><td>4855</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Carl Roth (Germany)</td><td>4227</td><td></td></tr><tr><td>2-Propanol</td><td>Carl Roth (Germany)</td><td>9781</td><td></td></tr><tr><td>[&lt;sup&gt;3&lt;/sup&gt;H]-dihydroalprenolol Hydrochloride</td><td>American Radiolabeled Chemicals (USA)</td><td>ART0134</td><td></td></tr><tr><td>Acetyl phosphate lithium potassium salt (ACP)</td><td>Merck (Germany)</td><td>1409</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo;-triphosphate (ATP)</td><td>Sigma Aldrich (Germany)</td><td>A9251</td><td></td></tr><tr><td>Alprenolol hydrochloride</td><td>Merck (Germany)</td><td>A0360000</td><td></td></tr><tr><td>Anion exchange chromatography column material: Q-sepharose&amp;reg;</td><td>Sigma-Aldrich (Germany)</td><td>Q1126</td><td></td></tr><tr><td>Autoclave Type GE 446EC-1</td><td>Gettinge (Germany)</td><td></td><td></td></tr><tr><td>Bioreactor Type 884 124/1</td><td>B.Braun (Germany)</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall RC12BP+; Thermo Scientific (Germany); Sorvall RC-5C; Thermo Scientific (Germany); Mikro 22 R; Hettich (Germany)</td><td></td><td></td></tr><tr><td>Cholic acid</td><td>Carl Roth (Germany)</td><td>8137</td><td></td></tr><tr><td>Coomassie Brilliant Blue R250</td><td>Carl Roth (Germany)</td><td>3862</td><td></td></tr><tr><td>Culture flasks 500 ml baffled flasks, 2 l baffled flasks</td><td>Schott Duran (Germany)</td><td></td><td></td></tr><tr><td>Cytidine 5&#039;-triphosphate disodium salt</td><td>Sigma-Aldrich (Germany)</td><td>C1506</td><td></td></tr><tr><td>D-glucose monohydrate</td><td>Carl Roth (Germany)</td><td>6780</td><td></td></tr><tr><td>Di-potassiumhydrogen phosphate trihydrate</td><td>Carl Roth (Germany)</td><td>6878</td><td></td></tr><tr><td>Dialysis tubing Spectrum&lt;sup&gt;TM&lt;/sup&gt; Labs Spectra/Por&lt;sup&gt;TM&lt;/sup&gt; 12-14 kD MWCO Standard RC tubing</td><td>Fisher Scientific (Germany)</td><td>8700152</td><td></td></tr><tr><td>Dithiothreit</td><td>Carl Roth (Germany)</td><td>6908</td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth (Germany)</td><td>K928</td><td></td></tr><tr><td>Folinic acid calcium salt hydrate</td><td>Sigma-Aldrich (Germany)</td><td>47612</td><td></td></tr><tr><td>French pressure cell disruptor</td><td>SLM; Amico Instruments (USA)</td><td></td><td></td></tr><tr><td>Glycerol</td><td>Carl Roth (Germany)</td><td>3783</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate di-sodium salt (GTP)</td><td>Sigma-Aldrich (Germany)</td><td>G8877</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Carl Roth (Germany)</td><td>K025</td><td></td></tr><tr><td>IMAC column: HiTrap IMAC HP 5 mL</td><td>GE Life Sciences (Germany)</td><td>GE17-5248</td><td></td></tr><tr><td>Imidazole</td><td>Carl Roth (Germany)</td><td>3899</td><td></td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranosid (IPTG)</td><td>Carl Roth (Germany)</td><td>2316</td><td></td></tr><tr><td>Kanamycin</td><td>Carl Roth (Germany)</td><td>T832</td><td></td></tr><tr><td>L-Alanine</td><td>Carl Roth (Germany)</td><td>3076.1</td><td></td></tr><tr><td>L-Arginine</td><td>Carl Roth (Germany)</td><td>6908</td><td></td></tr><tr><td>L-Asparagine</td><td>Carl Roth (Germany)</td><td>HN23</td><td></td></tr><tr><td>L-Aspartic Acid</td><td>Carl Roth (Germany)</td><td>T202</td><td></td></tr><tr><td>L-Cysteine</td><td>Carl Roth (Germany)</td><td>T203</td><td></td></tr><tr><td>L-Glutamic Acid</td><td>Carl Roth (Germany)</td><td>3774</td><td></td></tr><tr><td>L-Glutamine</td><td>Carl Roth (Germany)</td><td>3772</td><td></td></tr><tr><td>L-Glycine</td><td>Carl Roth (Germany)</td><td>3187</td><td></td></tr><tr><td>L-Histidine</td><td>Carl Roth (Germany)</td><td>3852</td><td></td></tr><tr><td>L-Isoleucine</td><td>Carl Roth (Germany)</td><td>3922</td><td></td></tr><tr><td>L-Leucine</td><td>Carl Roth (Germany)</td><td>1699</td><td></td></tr><tr><td>L-Lysine</td><td>Carl Roth (Germany)</td><td>4207</td><td></td></tr><tr><td>L-Methionine</td><td>Carl Roth (Germany)</td><td>9359</td><td></td></tr><tr><td>L-Proline</td><td>Carl Roth (Germany)</td><td>1713</td><td></td></tr><tr><td>L-Phenylalanine</td><td>Carl Roth (Germany)</td><td>1709</td><td></td></tr><tr><td>L-Serine</td><td>Carl Roth (Germany)</td><td>4682</td><td></td></tr><tr><td>L-Threonine</td><td>Carl Roth (Germany)</td><td>1738</td><td></td></tr><tr><td>L-Tryptophane</td><td>Carl Roth (Germany)</td><td>7700</td><td></td></tr><tr><td>L-Tyrosine</td><td>Carl Roth (Germany)</td><td>T207</td><td></td></tr><tr><td>MD100 dialysis units</td><td>Scienova (Germany)</td><td>40077</td><td></td></tr><tr><td>&lt;em&gt;N-2&lt;/em&gt;-Hydroxyethylpiperazine-&lt;em&gt;N&#039;-2&lt;/em&gt;-ethansulfonic acid (HEPES)</td><td>Carl Roth (Germany)</td><td>6763</td><td></td></tr><tr><td>n-dodecylphosphocholine</td><td>Antrace (USA)</td><td>F308S</td><td></td></tr><tr><td>PAGE chamber: Mini-Protean Tetra Cell</td><td>Biorad (Germany)</td><td></td><td></td></tr><tr><td>PAGE gel casting system: Mini Protean Handcast systems</td><td>Biorad (Germany)</td><td></td><td></td></tr><tr><td>PAGE gel power supply: Power Pac 3000</td><td>Biorad (Germany)</td><td></td><td></td></tr><tr><td>Tryptone/peptone from caseine</td><td>Carl Roth (Germany)</td><td>6681</td><td></td></tr><tr><td>Peristaltic pump: ip-12</td><td>Ismatec (Germany)</td><td></td><td></td></tr><tr><td>Phosphoenol pyruvate monopotassium salt</td><td>Sigma Aldrich (Germany)</td><td>860077</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Carl Roth (Germany)</td><td>P018</td><td></td></tr><tr><td>Potassium acetate</td><td>Carl Roth (Germany)</td><td>4986</td><td></td></tr><tr><td>Potassium chloride</td><td>Carl Roth (Germany)</td><td>6781</td><td></td></tr><tr><td>Pyruvate Kinase</td><td>Roche (Germany)</td><td>10109045001</td><td></td></tr><tr><td>Scintillation counter: Hidex 300 SL</td><td>Hidex (Finland)</td><td></td><td></td></tr><tr><td>SDS pellets</td><td>Carl Roth (Germany)</td><td>8029</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich (Germany)</td><td>K305</td><td></td></tr><tr><td>Sodium chloride</td><td>Carl Roth (Germany)</td><td>P029</td><td></td></tr><tr><td>Spectrophotometer Nanodrop</td><td>Peqlab (Germany)</td><td></td><td></td></tr><tr><td>Spectrophotometer/fluorescence reader Spark&amp;reg;</td><td>Tecan (Switzerland)</td><td></td><td></td></tr><tr><td>tRNA (E. coli)</td><td>Roche (Germany)</td><td>10109550001</td><td></td></tr><tr><td>Ultra sonificator</td><td>Labsonic U, B. Braun (Germany)</td><td></td><td></td></tr><tr><td>Uridine 5&amp;rsquo;-triphosphate tri-sodium salt (UTP)</td><td>Sigma-Aldrich (Germany)</td><td>U6625</td><td></td></tr><tr><td>Y-30 antifoam</td><td>Sigma-Aldrich (Germany)</td><td>A6457</td><td></td></tr><tr><td>Yeast extract</td><td>Carl Roth (Germany)</td><td>2904</td><td></td></tr></tbody></table>

co-translational insertion of membrane proteins into preformed nanodiscs

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

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

Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

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

膜蛋白共翻译插入预成型纳米盘

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

co-translational-insertion-membrane-proteins-into-preformed

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

3.3K 次观看.  Goethe University Frankfurt. 共翻译插入到预先形成的纳米盘中，使得在定义的脂质环境中研究无细胞合成的膜蛋白成为可能，而无需与去垢剂接触。该协议描述了基本系统组分的制备以及提高表达效率和样品质量的关键参数。

视频: Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

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

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

培养细胞功能化纳米化对跨 golgi 网络的内分泌吸收和逆行迁移分析

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

含纳米片生物活性剂的配制与表征

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

生物学

脂质膜上的光控可逆蛋白质图案

Dynamic Light-Induced Protein Patterns at Model Membranes

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

作者聚焦:用于人工细胞系统中可逆模式的光可切换蛋白质募集

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

模型膜上的动态光诱导蛋白质模式

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell ...

生物工程

Protein Translocation Machinery on the ER Membrane

The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61&#945;, Sec61&#946;, and Sec61&#947;, and is the largest subunit of the translocon complex. In higher eukaryotes, such as mammals, the channel is capped in its resting state to prevent the leakage of ions like calcium into the cytosol.
Accessory protein complexes associated with the Sec61 channel
Sec62 and Sec63 are integral transmembrane proteins associated with the Sec61 complex. They are primarily involved in the functioning of the Sec61 channel&#39;s lateral gate and aid in the exit of the signal sequence and transmembrane domains into the lipid bilayer.
The translocon-associated protein or TRAP complex is a hetero-tetrameric constitutive subunit of the translocon complex, present behind the Sec61 channel. It assists in the binding of signal peptides having low hydrophobicity to the Sec61 channel. The oligosaccharyltransferase (OST) complex is another closely associated complex, which transfers pre-assembled glycans to select asparagine residues.
Some proteins, like the signal peptidase complex, associate with the translocon complex under specific conditions. For instance, the signal recognition particle receptor (SR) only interacts with the Sec61 channel to hand over the ribosome-nascent peptide complex during active translocation. Other proteins like lectin chaperones (calnexin and malectin) and sensor molecules (IRE1) associate with the translocon to correctly fold the incoming polypeptide or signal misfolded proteins.

ER 膜上的蛋白质转位机制

The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides ...

教育

JoVE Core

细胞生物学

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

关键术语与概念

Cotranslational Protein Translocation

Translocation of proteins across membranes is an ancient process that occurs even in bacteria and archaebacteria. In fact, the components of the translocation machinery are still conserved between prokaryotes and eukaryotes.
Sec61 channel partners for cotranslational translocation
During cotranslational translocation, the Sec61 channel partners with the signal recognition particle (SRP), the signal recognition particle receptor (SR), and the ribosomes to transport the nascent polypeptide chain into the lumen.
SRP mediates ribosome attachment to the Sec61 complex via the polypeptide exit tunnel of the large ribosomal subunit. The GTPase domains on both SRP and SR carry out GTP hydrolysis, which supports the transfer of the target polypeptide to the Sec61 channel. Subsequently, the ribosomes act as primary translocation motors and push the protein towards the ER lumen. Except for the GTPs spent during peptide chain elongation, no additional energy is expended for the protein to descend through the channel.&#160;
Accessory translocon complex proteins involved in cotranslational translocation
The translocating chain-associating membrane (TRAM) protein and translocon-associated protein (TRAP) complex are auxiliary components associated with the Sec61 channel during SRP-mediated translocation. The TRAP complex is a substrate-specific complex essential for secure anchoring of the signal sequences having low hydrophobicity. Its proteins directly interact with Sec61 and keep the channel in open confirmation to compensate for the weak interaction with the signal sequence.
On the other hand, TRAM is a membrane protein that acts as a chaperone. It assists in folding and membrane integration of cotranslationally translocating proteins.

膜蛋白共翻译插入预成型纳米盘

摘要

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含纳米片生物活性剂的配制与表征

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

模型膜上的动态光诱导蛋白质模式

模型膜上的动态光诱导蛋白质模式

模型膜上的动态光诱导蛋白质模式

ER 膜上的蛋白质转位机制

共翻译蛋白易位