这项工作得到了生物 ID 中心和上海交通大学交叉学科研究基金 (YG2016QN66)、中国国家自然科学基金 (31271217) 和中国国家基础研究项目 (2014CB910304) 的支持。

注: 连续表达标记标记的 hEAG1 通道蛋白的 HEK-293T 细胞系由转染 pCDH 慢病毒载体质粒组成, 其中含有 hEAG1 的 DNA 序列, 在远端 C 终点有一条旗, 随后为 HEK-293T 细胞。嘌呤霉素的选择, 如前面所描述的15。
1. HEK-293T 细胞标记标记 hEAG1 通道蛋白的亲和纯化
<ol><li>解冻细胞稳定地表达 hEAG1 渠道从液氮入温暖的水 (37 °c) 快速地。将细胞 (大约 5 x 106冷冻细胞) 种子放在10厘米的盘子里。在 Dulbecco 的改良鹰 (DMEM) 培养基中, 在一夜之间生长细胞, 辅以8毫升10% 胎牛血清 (血清), 100 单位/毫升青霉素, 100 微克/毫升链霉素, 4 毫米 l-谷氨酰胺, 第二天改变培养基。</li>
	<li>用荧光显微镜检查这些 HEK-293T 细胞的 GFP 荧光, 以确保细胞在培养系统中稳定表达 hEAG1 通道的比例很高。在室温和 thenadd 2 毫升的含血清培养液中, 以1毫升0.25% 胰蛋白酶的生长细胞以指数胰蛋白酶处理终止消化。将 400 ul 的细胞悬浮液转移到15厘米的盘子中, 含有15毫升的完全 DMEM 培养基。总共准备四15厘米的菜肴。</li>
	<li>
当细胞达到大约90% 融合时, 在2–3天的培养后收获这些细胞。
	<ol>
<li>从细胞中取出生长培养基, 用4毫升磷酸盐缓冲盐水 (1x PBS, pH = 7.4) 清洗两次。</li>
		<li>洗涤后丢弃 PBS。</li>
		<li>使用细胞刮刮, 并将刮伤细胞与1毫升吸管转移到15毫升管中, 将细胞分成2毫升 1x PBS。</li>
		<li>离心机细胞悬浮5分钟在 420 x g 在4°c。</li>
		<li>醒酒并丢弃上清。</li>
		<li>并用重悬细胞颗粒在4毫升的溶解缓冲 (10 毫米 HEPES, 1.5 毫米氯化镁2, 10 毫米氯化钠, 1% NP-40, pH 7.0, 包含完整的蛋白酶抑制剂) 为30分钟在冰上, 和漩涡彻底裂解每10分钟。</li>
		<li>离心细胞裂解液为10分钟在 1.2万 x g 在4摄氏度。</li>
		<li>将上清液转移至5毫升管, 并将其放在冰上立即使用。</li>
	</ol>
</li>
	<li>
准备抗旗 M2 亲和树脂。
	<ol>
<li>通过温和倒置, 彻底悬浮树脂 (在贮存缓冲器中提供50% 悬浮液), 以确保该瓶的反旗 M2 亲和凝胶是一个均匀悬浮的凝胶珠。</li>
		<li>立即将 400 ul 的悬浮液转移到冷却的1.5 毫升管上。</li>
		<li>离心机悬浮在三十年代在 8, 000 x g 在4°c 并且仔细地放弃上清清洗商店缓冲。</li>
		<li>将 500 ul 1x PBS 添加到树脂上, 用1毫升吸管将颗粒悬浮。然后离心悬浮三十年代在 8, 000 x g 在4°c 并且仔细地放弃上清 PBS。重复这些洗涤步骤以清除存储的缓冲区。</li>
	</ol>
</li>
	<li>添加 500 ul 蛋白提取液在步骤1.3.8 到树脂颗粒悬浮颗粒和转移悬浮到一个新的冷却5毫升管。再次重复这一步骤, 以确保没有凝胶珠离开。将左蛋白提取物添加到混合物中。</li>
	<li>用 8 rpm 在4摄氏度的振动筛上一夜之间孵化混合物, 捕捉旗融合蛋白。</li>
	<li>离心后的混合物12小时孵化10分钟在 1, 000 x g 在4°c。</li>
	<li>丢弃上清液, 用 1x PBS 的 500 ul 来清洗颗粒三次。把它放在冰上立即使用。</li>
	<li>
用3x 旗肽洗脱旗 hEAG1 蛋白。
	<ol>
<li>准备3X 标志洗脱液。溶解3X 旗肽在 500 ul 存货溶液 (0.5 M 三 HCl, 1 m 氯化钠, pH = 7.5) 在浓度8微克/ul。</li>
		<li>添加 10 ul 3x 旗洗脱液到 390 ul 的 PBS 是一个 200 ng/ul 最终浓度解决方案。</li>
		<li>添加 400 ul 的3x 旗洗脱液的凝胶珠准备在步骤1.8。</li>
		<li>在 8 rpm 的振动筛上孵化样品2小时4摄氏度。</li>
		<li>离心树脂三十年代在 8, 000 x g。</li>
		<li>将上清液转移到新鲜的1.5 毫升管, 并将其存储在4摄氏度, 以便立即使用。</li>
	</ol>
</li>
</ol>2. 蛋白质检测试剂盒和西方印迹对纯化旗融合 hEAG1 的浓度测定和确认
<ol><li>根据生产说明书确定纯化蛋白的浓度。</li>
	<li>使用 30 ul 样品进行西方分析, 以确认使用反旗抗体作为前述的15, 可以纯化出感兴趣的蛋白质。</li>
</ol>3. 用生物素对纯化通道蛋白进行 BLI 测定的标记
<ol><li>在 PBS 中准备5毫克/毫升生物素库存溶液。对于每个测试 (两个生物传感器), 添加一个3倍摩尔生物素超过20微克纯化蛋白, 以达到更好的 n-终端 biotinylation 纯化蛋白在 PBS。</li>
	<li>在黑暗的冰上孵化样品至少30分钟。</li>
	<li>准备样品稀释 (SD) 缓冲: PBS 与0.02% 吐温20和0.1% 牛血清白蛋白 (BSA, pH 值 7.4)。</li>
	<li>进行超滤, 将纯化通道蛋白的缓冲液转化为 SD 缓冲器。去除未绑定生物素, 使用超滤装置, 分子质量截止 30 kDa, 添加 SD 缓冲器和离心的样品在 1.2万 x g, 10 分钟在4摄氏度。</li>
	<li>从超滤装置的离心管中取出 ultrafiltrate, 将 200 ul SD 缓冲器添加到过滤器中, 离心 1.2万 x g 的样品, 在 4°c 10 分钟。重复此操作至少三次。</li>
	<li>为了收集缓冲交换样品, 反向插入过滤器到1.5 毫升管和离心他们在 2000 x g, 5 分钟在4°c。把样品放在冰上立即使用。</li>
</ol>4. 用于化验的 PIP2解决方案的准备
<ol><li>在冰上用 sonicating 30 分钟的方法准备在去离子 H2O 上的 PIP2 (1 毫米) 的库存解决方案, 如前所述17。在-20 摄氏度的玻璃瓶中贮存溶液, 并在试验前将其稀释至最终浓度, 然后用剧烈的涡流。</li>
</ol>5. BLI 化验
<ol><li>打开设备并检查 "仪器状态" 窗口, 确认机器处于 "就绪" 状态, 至少在 BLI 研究前30分钟 prewarm 设备。</li>
	<li>在打开数据采集软件之前, 一定要先关闭仪器的门, 然后在实验向导中选择 "新的动力学实验"。</li>
	<li>定义在96井板上使用的水井, 通过右键单击选择缓冲区、负载和样本。对于样品井, 生物素化旗融合 hEAG1 蛋白的浓度单位应输入摩尔 (10 微克, 0.09 微米)。</li>
	<li>定义检测步骤, 包括基线、载荷、关联和离解。选择一个检测步骤, 双击相应的列。为控制传感器设置了检测定义的副本。将 rpm 设置为1000。分别选择1分钟的基线步长和5–10最小值进行加载、关联和分离。在室温下进行测试 (约24摄氏度)。 注意: 有两个主要程序: 将生物素化通道蛋白加载到传感器中, 并测定与小分子化合物的相互作用。他们可以连续地进行 (基线, 装载, 基线, 协会并且离解)。另外, 它们可以单独进行, 以避免在第一个加载部件不成功时浪费测试化合物。</li>
	<li>单击包含传感器的列, 然后单击 "填充" 以指示传感器托盘中传感器的位置。</li>
	<li>查看所有计划好的步骤, 检查错误并返回以更正它们。</li>
	<li>设置数据文件的位置, 然后单击 "转到" 以开始检测。 注: 在 SD 缓冲器中, 传感器需要至少10分钟的 prewetted, 如果这一步骤已经完成, 则应跳过 "延迟实验启动" 设置。如果没有, 在预湿传感器之前设置一个600s 延迟。</li>
	<li>将一个黑色的96井板放在托盘底部, 将盘子的 A1 角插入托盘上的凹槽上, 以将盘子装上座椅。为使油井加载传感器, 每井 200 ul 的检测缓冲器, 在96井板 B 排 a 和2井中加入2井。</li>
	<li>准备另一条黑色96井板作为样品板, 并填写在 B 行 200 ul SD 缓冲区的水井作为控制或生物素化 hEAG1 蛋白溶液 (10 微克) 在排 A 在编程期间分配的步骤5.3。 注意: 避免引入气泡。</li>
	<li>打开仪器的门, 分别将传感器托盘和样品板插入左、右板架。检查传感器托盘和样品板是否正确定位的基础上左板持有人的形状和 "A1" 标志在右上角的右板持有人。关上门, 开始化验。</li>
</ol>6. 数据分析
<ol><li>打开数据分析软件并加载包含检测数据的文件夹。点击 "处理" 进入加工菜单界面, 我们可以看到色彩鲜艳的原始动力学曲线。</li>
	<li>在Step1: "数据选择" 下, 单击 "传感器选择"。在 "传感器托盘 #1" 上, 单击仅使用 SD 缓冲区润湿的传感器井, 右键单击可将传感器类型改为 "参考传感器"。在 "样本板图" 中, 指定所有非特定的结合井, 右键单击 "将井型" 改为 "参考井"。</li>
	<li>在步骤2的 "减法" 和点 "双参照" 之前的方框中勾选。</li>
	<li>在步骤 3: "对齐 Y 轴", 选择 "基线" 作为对齐步骤。对于 "时间范围", 输入该基线的最后十年代(即从: 0.1 到: 59.8)。</li>
	<li>在步骤 4: "跨步更正" 中, 选择 "对齐基线", 以最小化关联和离解步骤之间的信号转移。</li>
	<li>在步骤 5: "进程" 中, 在大多数情况下选择 Savitzky Golay 筛选功能, 然后继续 "进程数据"。</li>
	<li>使用步骤7中的其他软件保存原始数据以进行进一步的数据分析: "保存结果"。</li>
	<li>
点击 "分析 |曲线拟合 "。
	<ol>
<li>对于 "步骤分析", 选择 "关联 |离解 "。对于 "模型", 选择1:1。 注: 我们选择1:1 模型, 因为它适合我们的原始数据, 并避免了超过1:2 或2:1 模型, 由于他们的高自由度的可能性。但其他选择在这里是可利用的, 并且可以适当地被选择为其他拟合分析为不同的结合模型对物。</li>
		<li>对于 "拟合", 请选择 "全局 (完全)"。对于 "分组通过", 选择 "颜色"。选择 "Rmax 未链接的传感器", 以允许独立拟合最大信号响应 (Rmax)。</li>
		<li>单击 "适合曲线" 开始非线性回归分析。研究中采用了小山方程和单指数函数。</li>
		<li>单击 "数据导出 |保存报告 "以保存拟合结果。或单击 "数据导出 |导出拟合结果 "以保存原始数据, 以便与其他软件进行进一步的绘图和数据分析。</li>
	</ol>
</li>
</ol>

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作者声明他们没有竞争的财政利益。

膜离子通道已被证实为治疗各种人类疾病 (包括心血管和神经系统疾病) 的13% 以上的主要治疗靶点, 如18。膜片钳记录是测量小分子离子通道功能的黄金标准, 已广泛应用于离子通道配体的筛选。然而, 这种电生理方法无法证明小分子是否直接与通道绑定, 或者不是19, 因为小分子可以对与通道相互作用的其他蛋白质或细胞间通路采取行动。与广泛使用的放射性配体结合试验和其他常用的无标签生物传感器方法相比, BLI 在相对简单的排列、不受限制的关联相位、高贯穿、检测设计方面具有显著优势。体内系统和少量的固定化蛋白 (几微克) 的需求, 它可以提供详细的洞察力数据5,6,20。
为了最大限度地模拟体内的情况, 我们从哺乳动物的稳定表达系统中纯化了功能 hEAG1 通道蛋白。提出了一种简便有效的抗原离子通道蛋白的纯化和鉴定方法。成功纯化膜蛋白的一些重要提示是: 1) 根据哺乳动物表达系统中的兴趣蛋白表达丰度, 可以缩小或放大纯化过程;2) 我们在 hEAG1 通道的 C 总站上熔化了一个标志标记, 以利用商业套件和净化协议, 促进用反旗亲和珠纯化此蛋白。电生理测量表明, 融合标志对该通道15的功能没有影响;3) 将抗旗珠和蛋白提取物的混合孵化过夜, 轻轻摇动, 有利于捕捉旗融合蛋白;4) 采用亲和纯化方法, 可以从四15厘米的菜肴中获得足够的 hEAG1 离子通道蛋白, 融合90% 细胞, 用于一次化验。
采用过量生物素 (3-10 倍) 生物素化纯化 hEAG1 通道蛋白, 用 SD 缓冲器交换溶液缓冲器进行进一步的 BLI 试验。过量生物素可以最大程度地改变膜通道蛋白, 以提高传感器表面的涂层效率, 而含有低浓度 BSA 和吐温20的 SD 检测缓冲器可以将非特异性绑定9降至最低.尽管进行了这些关键操作, 但非理想的相互作用仍然存在, 特别是当进行具有高浓度小分子的约束研究时, 它们可能不专门绑定到 SA 传感器的表面, 从而导致假阳互 , 如图 4A所示的菁曲线。因此, 通过将传感器与小分子、生物素化 hEAG1 蛋白与小分子溶液之间的相互作用减去非特定的约束来获得可靠的结果, 还需要一个双参考减法协议, 并带有小分子溶液的传感器。这双参考减法操作需要四生物传感器一次化验。两种生物传感器将被涂上生物素化膜蛋白, 并进行与小分子及其缓冲剂的相互作用试验。另外两个传感器将与 SD 缓冲器一起润湿, 并与小分子及其缓冲器相互作用。只有膜蛋白固定化生物传感器与小分子的相互作用, 才显示出正信号, 其余三种交互信号作为控制 (图 2) 工作。通过使用此过程, 如图 3和图 4所示, 我们的结果清楚地显示了 hEAG1 通道蛋白和 PIP2之间的强大交互, 并显示了浓度依赖性剖面。必须指出的是, 我们的测量有几个限制。例如, 还有一些其他的膜脂可以与纯化的通道蛋白结合。此外, 还不清楚, 锚定纯化蛋白是否保持其原始构象和活性。当它们与蛋白质相互作用时, 很难测量小分子脂的真正构型。虽然详细的过程是未知的, 我们的研究表明, BLI 的结合动力学是一致的, 通过电生理记录, 这使得新的 BLI 应用于离子通道蛋白-小分子相互作用的补充方式。
总之, 我们的研究证实, 它是一个可靠的策略, 通过纯化的功能膜蛋白从哺乳动物表达系统, 以检测与其配体的直接相互作用。BLI 在膜蛋白-小分子相互作用中的成功应用将促进离子通道药物发现过程中的小分子筛查和机制探索。

靶向细胞表面可接触的离子通道蛋白质与小分子提供了巨大的潜力, 配体筛选和生物药物发现1,2,3。因此, 研究离子通道与小分子之间的相互作用及其相应的作用是需要适当的工具。在离子通道功能检测中, 膜片钳记录已被证明是一种独特而不可替代的技术。然而, 确定小分子是否直接靶向离子通道需要其他技术。传统上, 采用放射性配体结合试验观察小分子与靶离子通道蛋白结合的动力学。然而, 由于其对放射性标记和检测的要求, 这种技术的使用受到限制。此外, 在研究中对小配体进行标记的先决条件是防止其在许多类型的离子通道中使用, 而不需要已知的特异配体。一些无标签的技术, 如核磁共振光谱学, x 射线衍射, 微型 thermophoresis (MST)4和表面等离子共振 (SPR) 已被用来测量蛋白质-小分子相互作用。但是这些类型的化验通常无法提供足够的信息, 因为很难获得全长蛋白质, 低分辨率的动力学, 低吞吐量和高成本的5。与这些技术相比, 生物层干涉法 (BLI) 正在成为一种新的无标签方法, 以克服这些缺陷, 通过固定少量蛋白质样品在表面上的蛋白质-小分子相互作用生物传感器和测量光学变化信号6,7。BLI 技术作为一种有前途的生物传感器平台, 已经进行了观察小分子与天然水溶性蛋白质 (如人单克隆抗体 CR80208 ) 的相互作用, 并报告了详细的化验程序。上一篇文章9。虽然离子通道蛋白在新的治疗靶点发现中的关键作用已经得到了确认, 但是基于 BLI 的离子通道蛋白-小分子相互作用的研究还没有被描述。
人类的以太hEAG1) 在各种类型的癌细胞和中枢神经系统中表达, 这使得该通道成为许多癌症和神经疾病的潜在治疗目标10,11, 12,13,14。我们实验室的电生理研究证实了磷酸肌醇4、5-bisphosphate (PIP2) 对 hEAG1通道15 的抑制作用。根据我们的结果, 测试 PIP2与 hEAG1 的直接交互使用 BLI 技术可以作为其他类型的离子通道蛋白-小分子化合物相互作用的模型, 特别是那些缺乏特定配体的通道。根据 BLI 化验的指示, 我们准备生物素化 hEAG1 蛋白, 并将它们固定在链亲和素 (SA) 生物传感器的表面上, 然后将它们与 PIP 2 解决方案相互作用, 观察它们之间的直接绑定。蛋白质和脂质。在将 PIP2附着到 hEAG1 蛋白涂层表面后, 表面层的厚度增加, 这直接关系到光谱的变化, 可以实时地测量16。由于关联步长的正移和离解步的负移位, 可以确定约束动力学。根据这一原理, 利用亲和纯化方法对 HEK-239T 稳定表达系统中的功能 hEAG1 离子通道蛋白进行纯化, 以维持体外功能状态, 然后测定其结合的动力学不同浓度 PIP2, 并产生了 semblable 的动力学数据, 如观察到的电生理测量15。BLI 和电生理测量结果之间的密切对应, 首次证明了 BLI 作为离子通道膜蛋白-小分子相互作用的适当分析工具的适用性。

<table><tbody><tr><td>DMEM/High Glucose Medium</td><td>HyClone</td><td>SH30243.01</td><td></td></tr><tr><td>Phosphate Buffered Saline (1x)</td><td>HyClone</td><td>SH30256.01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10270</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>1697550</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Corning</td><td>430599</td><td>150 mm X 25 mm</td></tr><tr><td>Nonidet P-40 Substitute</td><td>Amresco</td><td>E109</td><td></td></tr><tr><td>Sodium chloride</td><td>BBI Life Sciences</td><td>A610476</td><td></td></tr><tr><td>Potassium chloride</td><td>BBI Life Sciences</td><td>A610440</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>BBI Life Sciences</td><td>A600332</td><td></td></tr><tr><td>Polyoxyethylene-20-Sorbitan Monolaurate</td><td>BBI Life Sciences</td><td>A600560</td><td></td></tr><tr><td>ANTI-FLAG M2 Affinity Gel</td><td>Sigma</td><td>A2220</td><td></td></tr><tr><td>3x FLAG peptide</td><td>Sigma</td><td>F4799</td><td></td></tr><tr><td>Octet-RED96</td><td>Pall/Fort&amp;eacute;Bio</td><td>30-5048</td><td></td></tr><tr><td>Data Acquisition software</td><td>Pall/Fort&amp;eacute;Bio</td><td>Version 7.1</td><td></td></tr><tr><td>Data Analysis software</td><td>Pall/Fort&amp;eacute;Bio</td><td>Version 7.1</td><td></td></tr><tr><td>Biosensor/Streptavidin</td><td>Pall/Fort&amp;eacute;Bio</td><td>18-5019</td><td></td></tr><tr><td>Microtiter plate</td><td>Greiner Bio-one</td><td>655209</td><td></td></tr><tr><td>Sulfo-NHS-LC-LC-Biotin</td><td>ThermoFisher</td><td>21338</td><td></td></tr><tr><td>Centrifugal Machine</td><td>ThermoFisher</td><td>75004250</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder</td><td>ThermoScientific</td><td>318120</td><td></td></tr><tr><td>Ultrafiltration device</td><td>MILLIPORE</td><td>UFC503008</td><td>NMWL of 30 kDa</td></tr><tr><td>phosphatidylinositol 4, 5-bisphosphate (PIP&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>P9763</td><td></td></tr><tr><td>Monoclonal ANTI-FLAG M2 antibody</td><td>Sigma</td><td>F1804</td><td>1:2000 dilution</td></tr><tr><td>goat anti-mouse HRP-conjugated secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc-2005</td><td>1:5000 dilution</td></tr><tr><td>Enhanced BCA Protein Assay Kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>Protease Inhibitor Cocktail Tablets</td><td>Roche</td><td>04693159001</td><td></td></tr><tr><td>Amersham Imager 600 Imaging System</td><td>GE Healthcare Bio-Sciences</td><td></td><td></td></tr><tr><td>Western blot system</td><td>BIO-RAD</td><td></td><td></td></tr></tbody></table>

capturing the interaction kinetics of an ion channel protein with small molecules by the bio-layer interferometry assay

生物层干涉法 (BLI) 是测定蛋白质和蛋白质-小分子相互作用的重要工具。在这里, 我们首先描述了这个新的无标签技术的应用研究人 EAG1 (hEAG1) 通道蛋白与小分子 PIP 的相互作用2。hEAG1 通道被认为是潜在的治疗靶点, 因为它在癌症中的异常过度表达, 以及某些类型的神经系统疾病所涉及的一些增益功能突变。我们从哺乳动物稳定表达系统中纯化 hEAG1 通道蛋白, 并用 BLI 测量与 PIP2的交互作用。hEAG1 蛋白与 PIP 的结合动力学的成功测量2表明 BLI 法是一种潜在的高通量方法, 用于离子通道药理学中新的小分子配体筛选。

我们从 HEK-293T 细胞稳定抗原 hEAG1 中纯化了旗融合 hEAG1 通道蛋白。采用膜片钳法对该融合蛋白的功能进行了验证, 纯化蛋白的质量和特异性由西方印迹 (图 1) 确定。纯化通道蛋白生物素化使用实时 BLI 检测与脂质 (PIP2) 进行交互试验。BLI 绑定检测配置显示在图 2中。hEAG1 和 PIP2之间的典型绑定曲线显示在图 3中。在这种情况下, 3 微米 PIP2在 PBS 缓冲区中被分解 (PIP2的配置显示在辅助图 1中), 并且使用双引用减法协议分析信号以减去非特定绑定 (传感器和 PIP2之间的绑定, 背景 (生物素化 hEAG1 蛋白和 pbs 之间的交互) 和信号漂移 (传感器和 pbs 之间的绑定) 由传感器的可变性引起。绑定跟踪是全局匹配的, 并显示了良好的拟合覆盖 (辅助图 2)。另外, 我们通过在不同浓度的 pip2上孵化蛋白质, 来测量 pip2与纯化的 hEAG1 通道复合体的结合动力学。经过分析, 我们得到一个0.35 ±0.04 微米的离解常数 (Kd) 值, 它类似于从电生理测量15获得的 IC50值。结果表明, BLI 测定法适用于离子通道膜蛋白和脂质相互作用分析。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56846/56846fig1.jpg"> 图 1: 分别通过 GFP 成像、补丁钳和 HEK-293T 细胞对重组 hEAG1 蛋白的表达、功能和特异性进行鉴定.(A) 通过对 hEAG1-pCDH 慢病毒北疆系统进行转染后的单克隆嘌呤霉素抗性选择成功地建立了稳定表达 hEAG1 HEK-293T 系统, 在几乎所有细胞中 GFP 表达都证明了这一点。(B) 脉冲协议 (顶部) 和叠加电流跟踪, 从一个具有代表性的全细胞补丁-钳位记录从 hEAG1 通道在一个稳定的细胞中。电流是由-80 mv 的持有电压到 70 mv 的去极化型电压引起的, 其步骤为 10 mv, 后跟复极化到-80 mv。这些单元格在正常的 K+信道记录解决方案中进行孵化, 如前所述15。电压依赖性外向钾电流表明, 功能 hEAG1 通道是高度表达的 HEK293T 细胞。(C) 从纯化蛋白样品中提取的 hEAG1 通道蛋白的印迹。反旗抗体识别一个单一的蛋白质带的〜 110 kDa, 显示一个全长的标志标记 hEAG1 通道表达。此图 1C已从汉族et . 中修改。15.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56846/56846fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56846/56846fig2.jpg"> 图 2: 显示 BLI 绑定检测协议的示意图.四传感器并行使用生物素化通道蛋白或其溶解 SD 缓冲器加载通道蛋白质和参考。之后, 这四传感器转移到检测阶段, 以检测与磷脂或其溶液缓冲剂的关联和解除。通道蛋白、磷脂和缓冲液的位置颜色如所示。水平红色虚线表示 BLI 研究的两个主要步骤: 加载阶段和交互检测阶段。此图 2已从汉族et . 中修改。15.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56846/56846fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56846/56846fig3.jpg"> 图 3: 在典型的 BLI 研究中显示原始数据和已处理数据的屏幕捕获.(A) 典型的加载和平衡曲线, 显示平衡步骤 (六十年代) 与 SD 缓冲 (基线), 加载步骤与 hEAG1 蛋白 (加载) 和参考曲线平衡和负载 hEAG1 蛋白 (加载), 同时测量两个单独的传感器提示。(B) 垂直红线表示在检测操作期间将传感器从脂质溶液转移到缓冲液。(C) 在处理双参考减法后, 在关联和分离阶段的原始光信号减去非特定的绑定信号。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56846/56846fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56846/56846fig4.jpg"> 图 4: BLI 检测结果显示 hEAG1 通道蛋白与 PIP2直接交互.(A) 屏幕捕获, 显示以浓度依赖性方式 (0.03–3微米) 的 PIP2的 hEAG1 蛋白绑定的原始数据。PIP2的累计浓度表示对应于生物传感器的数据跟踪。(B) 原始数据处理, 表明在一个代表性的分析中, 不同浓度的 PIP2的光学干扰的变化。(C) 曲线适合于从不同 PIP 的光干涉信号的峰值值中获得的小山方程, 用于测定相互作用的平衡离解常数 (K d)在 hEAG1 通道蛋白和 PIP2 (n = 3) 之间。图 4B和4C已从汉等. 修改。15.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56846/56846fig4large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
补充图 1: 长链磷酸肌醇4、5-bisphosphate (PIP2) 的配置。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56846/Supplementary_Figure_1.tif">请单击此处下载此图.</a>
补充图 2: 从图3的 BLI 分析中得到的拟合叠加的屏幕捕获。数据被处理并且被装备并且只显示了协会和离解阶段。处理后的数据曲线为蓝色, 非线性拟合曲线为红色。适合的优点: R2 = 0.984789, X2 = 0.019109。最大绑定参数 (Rmax) = 0.2875 nm (0.0006).<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56846/Supplementary_Figure_2.tif">请单击此处下载此图.</a>

Watch this Scientific Journal Video about Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay at JoVE.com

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

该协议描述了纯化 hEAG1 离子通道蛋白与小分子脂配体磷酸肌醇4、5-bisphosphate (PIP2) 的相互作用。实验表明, BLI 可能是一种新的小分子离子通道配体筛选的潜在方法。

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

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Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.2K 次观看.  Shanghai Jiao Tong University School of Medicine. 该协议描述了纯化 hEAG1 离子通道蛋白与小分子脂配体磷酸肌醇4、5-bisphosphate (PIP2) 的相互作用。实验表明, BLI 可能是一种新的小分子离子通道配体筛选的潜在方法。

视频: Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

生物层干涉测量的蛋白质 - 蛋白质相互作用和变构配体反应动力学的影响

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

化学

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

开放补丁夹具移液器测量未搅拌边界层中的离子浓度: 流体流动对离子通道控制的启示

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

<meta charset="utf8"></head><body>使用无标记方法定量分析 RPA-ssDNA 结合动力学

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

<meta charset="utf8"></head><body>使用基于链霉亲和素的生物膜干涉测量法分析 DNA-蛋白质相互作用

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

使用基于链霉亲和素的生物膜干涉测量法分析 DNA-蛋白质相互作用

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

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方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

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用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

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