这项工作得到了波兰克拉科夫国家科学中心（NCN）第2016/23/D/NZ3/01314号资助。

1. 表达质粒的产生
<ol><li>使用拓扑预测工具检查目标蛋白质的膜拓扑结构。</li>
	<li>设计克隆策略，以便一旦融合蛋白插入高尔基体膜，较大和较小的片段将面对细胞质。如果，如这里所呈现的情况，目标蛋白质的N-和C-末端都是细胞质定向的，则以八种可能的方式标记蛋白质（见 图1B）。如果一种或两种感兴趣蛋白质的N-或C-末端是发光定向的，则将其从标记中排除。</li>
	<li>通过遵循标准克隆方案将感兴趣的基因亚克隆到适当的表达载体中（见 材料表）。 注意:建议测试所有可能的方向，因为某些标记选项可能由于距离不足、方向欠佳或空间限制而不起作用。</li>
</ol>2. 将表达质粒瞬时转染到细胞中
<ol><li>通过胰蛋白酶消化收获贴壁HEK293T细胞培养物，并将细胞重悬于专用的完整生长培养基中。将细胞（2 x 104 / 100μL /孔）接种到透明底部，白色侧96孔板上。调整孔的总数，以适应所有测试的组合和对照，包括重复。 注意:尝试仅使用板的内部60孔，以尽量减少热偏移并避免过夜蒸发。强烈建议使用聚-D-赖氨酸包被的板，例如 材料表中指示的板，否则细胞可能会在随后的洗涤步骤中分离。</li>
	<li>在标准条件（37°C，5%CO 2）下培养细胞过夜。</li>
	<li>第二天，用在1.1点获得的表达质粒的所需组合转染细胞。<ol>
<li>对于每种构建体，将无血清培养基中的表达质粒稀释至6.25ng / μL。</li>
		<li>以适当的脂质与DNA比例加入基于脂质的转染试剂，并根据制造商的说明进行孵育。</li>
		<li>将8μL脂质:DNA混合物加入指定的孔中。通过轻轻旋转混合板的内容物。这导致在50 ng /孔下转染两种表达结构。</li>
	</ol>
</li>
	<li>在标准条件（37°C，5%CO 2）中培养细胞20-24小时。 注意:将细胞培养更长的时间可能会导致更高水平的融合蛋白表达，这可能会促进片段之间的非特异性关联。</li>
</ol>3. 介质交换
<ol><li>第二天，在每个孔中用100μL无血清培养基替换调节培养基。确保细胞在培养基交换时没有脱落。 注意:此步骤应在加入呋马嗪工作溶液前2-3小时完成。血清戒断可最大限度地减少呋喃嗪自发光引起的背景。</li>
</ol>4. 呋马嗪工作液的制备
<ol><li>在测量之前，将1体积的呋喃嗪与19体积的稀释缓冲液（20倍稀释）混合。 注意:要制备的呋马嗪工作溶液的总体积取决于要分析的单个孔的数量（呋马嗪工作溶液以1:5的比例加入细胞培养基中，因此，向先前充满100μL无血清培养基的每个孔中加入25μL呋喃嗪工作溶液）。</li>
	<li>将呋马嗪工作溶液加入指定的孔（25μL/孔）。用手或使用轨道振荡器（例如，在300-500 rpm时15秒）轻轻混合板。</li>
</ol>5. 测量发光度
<ol><li>将板插入发光酶标仪。<ol>
<li>对于要在37°C下进行的实验，在指示的温度下平衡板10-15分钟。</li>
	</ol>
</li>
	<li>选择要分析的孔。</li>
	<li>读取集成时间为 0.3 s 时的发光。需要时，继续监测发光长达 2 小时。</li>
</ol>6. 数据分析
<ol><li>计算所有测试和对照组合的平均值和标准偏差。</li>
	<li>使用具有多重比较的单因素方差分析数据分析数据。</li>
	<li>通过将感兴趣的组合获得的平均发光除以为相应负对照获得的平均发光来计算折叠变化值。评估结果。 注:这里提出的数据分析方法假设，如果为测试组合获得的发光在统计上显着高于为相应控制组合获得的发光，并且同时，这两个值的比率超过10，则可以声称相互作用。</li>
</ol>

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作者没有什么可透露的。

在这里，我们提供了一个详细的方案，能够使用分裂荧光素酶互补测定法演示高尔基体驻留的III型膜蛋白（如NSTs）之间形成的异源复合物。所提出的数据分析和解释方法涉及将感兴趣的蛋白质组合获得的发光与为相应对照组合获得的发光相关联，其由与较大片段融合的目标蛋白质之一和与较小片段融合的细菌来源的控制蛋白组成。后者在哺乳动物细胞的细胞质中表达;因此，将其用作参考需要分析其相互作用的蛋白质的N和/或C末端位于高尔基体膜的细胞质侧。
该方案的关键步骤是质粒设计，接种要转染的细胞，转染本身，培养基交换，制备呋马嗪工作溶液并将其添加到细胞中。质粒设计应使两个荧光素酶片段都面向细胞质，否则对照组合将不起作用，并且将缺少参考RLU值。要转染的细胞应按照方案中的规定进行接种，否则转染效率可能次优。我们建议使用具有聚-L-赖氨酸涂层表面的多孔板，以便在交换培养基时支持细胞附着。最后，应新鲜制备呋喃嗪工作溶液并立即加入细胞中。添加后，应尽快执行读出。
为避免无结果，应始终包括阳性和阴性对照。应始终采用阳性对照，以便监测转染效率。生成和组合所有可能的融合蛋白非常重要，这些融合蛋白在拓扑上彼此相容以及与基于HaloTag的阴性对照相容。如果可能，应使用包含膜蛋白的阴性对照，该膜蛋白不与任何感兴趣的蛋白质相互作用。在这里，我们没有采用这种控制，因为它的发展仍在进行中。相反，我们通过共表达一种分析蛋白质的额外，未标记的副本来强制对感兴趣的复合物进行特定的反汇编。我们使用的表达载体携带相对较弱的单纯疱疹病毒 - 胸腺嘧啶激酶（HSV-TK）启动子，这确保了低表达水平，是获得特定结果的最佳选择。然而，在结果欠佳的情况下，使用基于CMV的载体或优化用于转染的质粒量可能是有益的。
所提出的方法虽然功能强大且方便，但存在一些局限性。首先，该方法不允许得出所鉴定的复合物是否对应于二聚体或高阶低聚物的结论。此外，无法监测相互作用蛋白质的亚细胞定位。然而，在扩展生物发光成像的基本方案时，这是可能的，尽管与基于荧光的方法相比，这些图像的空间分辨率会显着降低。
所提出的方法非常快速和有效（测量需要几分钟，数据来自数千个细胞）。数据处理和解释也相对简单。很少或根本不需要对基本协议进行优化。唯一需要的特定设备是光度计。分裂荧光素酶互补测定和BiFC的工作原理相同，即从其非功能性片段中重建功能蛋白。与基于荧光的方法相比，基于生物发光的方法的一般优势已在引言中列出。与BiFC相比，拆分荧光素酶补体测定的一个具体优点是前者是完全可逆的15。在BiFC中，一旦荧光蛋白被重组，它就不会解离回相应的非荧光片段17。相比之下，NanoLuc亚基的组装是可逆的，这为研究PPI的动力学创造了一个独特的机会。最后，所提出方法的特殊灵敏度允许假设这种方法应该适用于难以转染的细胞系。
这里提出的方案允许确定两种高尔基体驻留的III型膜蛋白是否相互作用。如前所述，该方法的基本设置可以与生物发光成像相结合，以确认目标PPI的亚细胞定位。这种分裂荧光素酶互补测定的可逆性允许实时研究PPI的动力学。来自呋喃嗪的发光信号持续约2小时。然而，用于分裂荧光素酶互补测定的底物也可用，以确保所得信号的持续时间大大延长（数小时至数天）。这种方法允许识别触发或阻止感兴趣的PPI的因素。其应用的一些最新例子包括对G蛋白与G蛋白偶联受体之间相互作用的研究18，蛋白质构象变化19，蛋白质泛素化20，细胞表面受体内化21以及鉴定调节PPI的因子22，23。因此，这种方法似乎是一种多功能工具，具有很高的潜力，甚至可以实现极具挑战性的实验目标。

本手稿描述了一种分步方案，以使用分裂荧光素酶互补测定的最新变体检查瞬时转染的人细胞中高尔基体驻留的III型膜蛋白之间是否存在异源相互作用。该程序已经针对核苷酸糖转运蛋白（NST）进行了最广泛的测试，但我们也能够获得其他高尔基体驻留的III型膜蛋白的阳性结果，其N和/或C-末端面向细胞质。
我们的研究小组探索了NST在大分子糖基化中的作用。NST是高尔基体和/或ER驻留的III型膜蛋白，其N端和C端朝向器官膜的细胞质侧1。NST被认为携带核苷酸激活的糖穿过细胞器膜，为其底物提供糖基转移酶。NST形成二聚体和/或更高的低聚物2，3，4，5，6，7，8，9，10。此外，还报道了不同NST之间的异源相互作用6，11。NST也被证明与功能相关的糖基化酶形成复合物12，13，14。我们寻求一种替代目前使用的技术，基于荧光寿命成像（FLIM）的FRET方法，用于研究NST和功能相关的高尔基体驻留蛋白的相互作用，因此我们决定测试分裂荧光素酶互补测定。它使我们能够确定NST和功能相关的糖基化酶之间的新相互作用9。
拆分荧光素酶补体测定的最新修改，NanoBiT，用于此处介绍的方案15。它依赖于从两个片段中重建荧光素酶（例如，NanoLuc） - 大的，称为大BiT或LgBiT，一种17.6 kDa蛋白，以及小的，仅由11个氨基酸组成，称为小BiT或SmBiT。感兴趣的两种蛋白质与互补片段融合，并在人类细胞系中瞬时表达。如果两种融合蛋白相互作用，则在添加细胞可渗透底物时原位产生发光。这两个片段已经过优化，因此它们与最小的亲和力结合，除非它们通过它们融合到的目标蛋白质之间的相互作用而聚集在一起。
一般来说，基于生物发光的方法比基于荧光的方法具有一些优势。生物发光信号具有更高的信噪比，因为与荧光素酶衍生的信号相比，背景发光可以忽略不计16。相比之下，基于荧光的方法通常受到由自发荧光现象引起的相对较高的背景的影响。此外，生物发光对分析细胞的危害小于荧光，因为在前一种情况下，不需要激发样品。由于这些原因，在体内研究PPI的生物发光方法优于常用的荧光方法，如Förster共振能量转移（FRET）或双分子荧光互补（BiFC）。
我们的方案依赖于将感兴趣的蛋白质组合获得的发光参考为对照组合获得的发光。后者包括一种被测试的蛋白质，它与较大的片段和对照蛋白（例如HaloTag）融合，与较小的片段融合。后者是一种细菌来源的蛋白质，预计不会与任何哺乳动物蛋白质相互作用。使用这种蛋白质作为对照会限制要分析的高尔基体驻留蛋白质对的拓扑结构。由于在哺乳动物细胞中，这种蛋白质是在细胞质中合成的，因此感兴趣的两种蛋白质都应该至少有一个细胞质尾巴。
这种方法对于PPI的初始筛选特别有用。当感兴趣的融合蛋白以不足以应用其他方法的水平表达时，它可能成为首选方法。同样，如果感兴趣的蛋白质以高水平表达，则分裂荧光素酶互补测定可能是最佳选择，但这会对其亚细胞定位产生不利影响或已知会迫使非特异性相互作用。由于较小的片段只有11个氨基酸，因此当使用较大的标签是不可能的时，可以应用分裂的荧光素酶互补测定。最后，它可以用来进一步确认使用其他技术获得的数据，如这里介绍的情况。

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adherent mammalian cell line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid</td>
			<td>Corning</td>
			<td>356651</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE HD Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax Discover Microplate Reader (or a different luminescence microplate reader)</td>
			<td>Promega</td>
			<td>GM3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth medium dedicated to the cell line used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT MCS Starter System</td>
			<td>Promega</td>
			<td>N2014</td>
			<td>This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.</td>
		</tr>
		<tr>
			<td>Nano-Glo Live Cell Assay System</td>
			<td>Promega</td>
			<td>N2011</td>
			<td>This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium, no phenol red</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Oribital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for data analysis (e.g. GraphPad Prism)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

该协议的目标是探索分裂荧光素酶互补的最新变体的适用性，以证明由核苷酸糖转运蛋白（NST）形成的异源复合物。这些ER和高尔基体驻留的多跨膜蛋白携带细胞质合成的核苷酸糖穿过细胞器膜，以提供介导糖基化及其底物的酶。NST以二聚体和/或更高的低聚物形式存在。不同 NST 之间的异源相互作用也有报道。为了验证该技术是否适合研究NST异色化现象，我们针对两种高尔基体驻留的NSTs的组合对其进行了测试，这两种NSTs先前已被证明可以通过其他几种方式关联。荧光素酶补体测定似乎特别适合研究高尔基体驻留膜蛋白之间的相互作用，因为它不需要高表达水平，这通常会引发蛋白质错位并增加假阳性的风险。

为了在这种方法中获得最可靠的数据，应测试所有可能的组合（参见 图1）。同时，应包括阳性和阴性对照。阳性对照应由已知相互作用的两种蛋白质组成，其中一种与较大的片段融合，另一种与较小的片段融合。理想情况下，阴性对照应由同样标记的两种非相互作用的III型膜蛋白组成。然而，建立这种对照可能具有挑战性，因为应使用几种替代方法彻底确认两种对照蛋白缺乏相互作用。因此，如果蛋白质的N-和/或C-末端（其相互作用有待确定）像这里介绍的情况一样细胞质暴露，则可以使用预计不会与任何人类蛋白质（例如HaloTag）相互作用的非人源胞质蛋白作为阴性对照。我们强烈建议通过共表达任何一种目标蛋白质的未标记变体并结合相应质粒的滴定，对使用这种类型的对照获得的结果进行额外的验证。如果两种蛋白质特异性相互作用，则其中任何一种缺乏荧光素酶片段的额外拷贝应导致发光的特定降低。
相对发光单元（RLU）值通常为正负组合，如表1所示。两种NST，SLC35A2和SLC35A3的结果被证明通过共免疫沉淀和FLIM-FRET6以及原位接近连接测定11相关联。SLC35A2和SLC35A3都是高尔基体驻留的III型膜蛋白，其N端和C端朝向细胞质（图1A）。因此，有八种可能的标记选项，并且所得的融合蛋白也可以以八种可能的方式设置在一起（图1B）。与所有测试和对照组合相对应的平均RLU值如图2A所示。阳性对照也包括在内。要将所选结果视为交互作用的指示，最初的要求是，为感兴趣的组合获得的RLU值在统计上显着高于为相应对照组合获得的RLU值。在图2A中可以看到三种这样的组合（LgBiT-SLC35A2 + SLC35A3-SmBiT，SmBiT-SLC35A2 + LgBiT-SLC35A3和SLC35A2-SmBiT + LgBiT-SLC35A3）。结果分析的下一步涉及通过将相应的RLU值除以为相应控件获得的RLU值来获得感兴趣组合的比率值。这种分析的结果如图2B所示。根据制造商的建议，10 到 1000 之间的比率高度指示了特定的相互作用。有两种组合符合这些标准（SmBiT-SLC35A2 + LgBiT-SLC35A3 和 SLC35A2-SmBiT + LgBiT-SLC35A3），并支持 SLC35A2 和 SLC35A3 相互作用的想法。然而，其他六种组合为阴性的事实并不意味着各自的融合蛋白之间根本没有相互作用，而是表明标记策略在这些情况下不是最佳的。此示例显示了测试所有可能的组合的重要性。
图2中提供的数据还通过HA标记的SLC35A2或SLC35A3与导致最高相对发光的组合（即SLC35A2-LgBiT + SmBiT-SLC35A3）的共表达得到证实。使用不同量的编码HA标记的NST变体的质粒进行共转染。这导致RLU值的剂量依赖性降低，具有统计学意义（图3）。通过HA标记的NST变体同时共表达SLC35A2-LgBiT和SmBiT-SLC35A3之间相互作用的特异性和剂量依赖性破坏如表2所示。
与本文介绍的方法相关的最常见问题是转染效率低。为了验证这是否是次优结果的原因，请在所有实验中包括阳性对照。我们用于获得此处提供的数据的阳性对照由两种相互作用的融合蛋白组成:与较小片段融合的cAPM依赖性蛋白激酶催化亚基α（PRKACA）和与较大片段融合的cAPM依赖性蛋白激酶II-α型调节亚基（PRKAR2A）。在我们手中，相应的RLU值达到了~6 x 105。这个数字的显着下降（2-3个数量级）可能表明所进行转染的效率欠佳。在这种情况下，我们建议检查培养细胞的健康状况，并确保接种适当数量的细胞进行转染。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61669/61669fig1v3.jpg"> 图1:膜拓扑结构和标记的示意图。 （A）SLC35A2和SLC35A3蛋白的膜拓扑结构。（B）使用分裂的荧光素酶互补测定片段标记SLC35A2和SLC35A3蛋白的可能性，并将所得的融合蛋白组合用于测定。 <a href="https://www.jove.com/files/ftp_upload/61669/61669fig1v3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61669/61669fig2v3.jpg"> 图2:在HEK293T细胞中对所选蛋白质组合和相应的阴性对照（处理数据）进行的分裂荧光素酶互补测定的结果。 （A）为所选蛋白质组合和相应的阴性对照获得的RLU值。负面，HaloTag标记有SmBiT;PRKAR2A，cAPM依赖性蛋白激酶II-α型调节亚基;PRKACA，cAPM依赖性蛋白激酶催化亚基α。使用单因素方差分析进行多重比较，并以三个技术重复的平均±标准差（SD）表示。 p < 0.1 *， p < 0.05 **， p < 0.01 ***.（B）通过将测试组合获得的平均发光（RLU样品）除以相应阴性对照获得的平均发光（RLU CONTROL）来计算的折叠变化。被视为指示交互作用的阈值设置为 10。 <a href="https://www.jove.com/files/ftp_upload/61669/61669fig2v3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61669/61669fig3v3.jpg"> 图3:通过同时共表达HA标记的NST变体，SLC35A2-LgBiT和SmBiT-SLC35A3之间相互作用的特异性和剂量依赖性破坏。HEK293T细胞转染编码SLC35A2-LgBiT和SmBiT-SLC35A3的质粒，此外，用空的pSelect质粒（模拟）或编码HA标记的SLC35A2（左图）和SLC35A3（右图）的pSelect质粒量增加。使用单因素方差分析进行多重比较，并以三个技术重复的平均±标准差（SD）表示。 p < 0.05 **， p < 0.001 ****。 <a href="https://www.jove.com/files/ftp_upload/61669/61669fig3v3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
表1:在HEK293T细胞中对所选蛋白质组合和相应的阴性对照进行的测定结果（原始数据）。 显示为所选蛋白质组合和相应的阴性对照获得的未处理的RLU值。负面，HaloTag标记有SmBiT;PRKAR2A，cAPM依赖性蛋白激酶II-α型调节亚基;PRKACA，cAPM依赖性蛋白激酶催化亚基α，TR，技术复制。 <a href="https://cloudflare.jove.com/files/ftp_upload/61669/JoVE_table_1.xlsx" target="_blank">请点击此处下载此表格。</a>
表2:通过同时共表达HA标记的NST变体（原始数据），SLC35A2-LgBiT和SmBiT-SLC35A3之间相互作用的特异性和剂量依赖性破坏。 图中显示了为所选蛋白质组合获得的未处理的RLU值。模拟，表达SLC35A2-LgBiT和SmBiT-SLC35A3的细胞与空pSelect载体共同转染;HA-SLC35A2，表达SLC35A2-LgBiT和SmBiT-SLC35A3的细胞与编码SLC35A2的pSelect质粒的指示量共同转染，编码SLC35A2在N末端标记HA表位;HA-SLC35A3，表达SLC35A2-LgBiT和SmBiT-SLC35A3的细胞与编码SLC35A3的pSelect质粒的指示量共同转染，编码SLC35A3在N末端标记HA表位;TR，技术复制。 <a href="https://cloudflare.jove.com/files/ftp_upload/61669/JoVE_table_2.xlsx" target="_blank">请点击此处下载此表格。</a>

Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

这里提出的方案旨在使用分裂荧光素酶互补测定的最新变体，证明高尔基体驻留的III型膜蛋白与活哺乳动物细胞中细胞质暴露的N-和/或C-末端之间异源相互作用的发生。

使用分裂荧光素酶互补测定法展示高尔基体驻留III型膜蛋白形成的异源复合物

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

demonstration-heterologous-complexes-formed-golgi-resident-type-iii

Universidad San Sebastian

Research

JoVE Journal

Biology

2.2K 次观看.  University of Wroclaw. 这里提出的方案旨在使用分裂荧光素酶互补测定的最新变体，证明高尔基体驻留的III型膜蛋白与活哺乳动物细胞中细胞质暴露的N-和/或C-末端之间异源相互作用的发生。

视频: Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...