我们感谢 Tlp3-LBD 的早期工作。Mayra. Machuca 感激 Departamento Admistrativo de Ciencia, Tecnología e Innovación COLCIENCIAS 为博士奖学金。

1. 他的6-Tlp3-LBD 在大肠杆菌中的表达
<ol><li>接种150毫升不育 Luria Bertani (LB) 汤, 含有50µg 毫升-1氨苄西林, BL21 密码子加 (DE3) RIPL 细胞, 用 pET151/D-TOPO 向量变换, 用于表达其6Tlp3-LBD (氨基酸残留 42-291), 并孵化它在37°c 在一个振动筛 (轨道直径, 25 毫米) 与 200 rpm 隔夜。</li>
	<li>准备六2升锥形烧瓶, 含800毫升不育 LB 汤和50µg 毫升-1氨苄西林。接种每瓶20毫升的隔夜文化从步骤1.1 获得。孵化他们在37°c 与连续震动在200转每分钟, 直到光学密度在600毫微米到达0.6。</li>
	<li>从其中一个烧瓶中取一个1毫升的整除 (uninduced 控制样品), 用台式离心机 (1.3万 x g, 4 °c, 10 分钟) 的颗粒, 并丢弃上清。将细菌颗粒贮存在-20 摄氏度, 用于后续的 SDS 页分析。</li>
	<li>通过将1毫米异丙基-β-d (IPTG) 添加到每瓶的培养中, 诱导蛋白表达。继续孵化的烧瓶在37°c 在 200 rpm 的一个额外的4小时。</li>
	<li>以 5000 x g 为15分钟, 在4摄氏度处离心, 并丢弃上清, 以此来收割细胞。 注意: 在这一点上, 该过程可以暂停, 将细胞颗粒放入一个-80 °c 冷藏库储存。</li>
</ol>2. 包涵体的分离和变性
<ol><li>将步骤1.5 中获得的所有细胞颗粒转移到250毫升烧杯中, 并添加100毫升的缓冲器 a (10 毫米三盐酸, pH 8.0, 150 毫米氯化钠).允许细胞完全解冻 (如果冻结), 并并用重悬颗粒。把样品放在冰上, 除非另有说明。</li>
	<li>通过高压 homogeniser 三倍的悬浮细胞溶解细胞并确保基因组 DNA 的完全剪切。</li>
	<li>离心裂解液在 1万 x g 15 分钟在4摄氏度。收集1毫升的上清 (可溶性分数) 样本, 并将其存储在-20 °c, 用于后续的 SDS 页分析。扔掉剩下的上清, 把小球放在冰上。 注: 这个小球是白色的颜色 (很容易区分从完整的细胞, 这是褐色的颜色的阴影), 并包含身体。</li>
	<li>并用重悬在20毫升的冰冷缓冲器 B (10 毫米三盐酸 pH 8.0, 0.2 毫米 phenylmethanesulfonyl 氟 (PMSF), 1% 海卫 X-100) 中, 包裹体颗粒彻底。这促进了膜和膜蛋白的 solubilisation。漩涡 1-2 分钟, 以帮助泥沙。</li>
	<li>离心样品在 1万 x g 15 分钟在4°c 和放弃上清。</li>
	<li>并用重悬在20毫升冰冷缓冲器 B 中彻底地将球团涡流 1-2 分钟. 确保颗粒破碎成小块。吸管样品向上和向下, 以帮助颗粒泥沙。 注: 必须彻底并用重悬颗粒, 以获得不含杂质的包裹体。</li>
	<li>重复步骤2.5。如果上清是多云或有色, 重复步骤2.6 和 2.5 (按顺序), 直到上清是明确和无色。</li>
	<li>并用重悬20毫升冰冷缓冲器 C (10 毫米三盐酸 pH 8.0, 0.2 毫米 PMSF) 的颗粒, 通过涡流管为 1-2 分钟。</li>
	<li>重复步骤2.5。</li>
	<li>加入25毫升冰冷变性缓冲 D (10 毫米三盐酸 pH 8.0, 8 米尿素, 10 毫米 dithiothreitol (德勤), 0.2 毫米 PMSF), 以溶解包裹体颗粒。并用重悬彻底由涡流 1-2 分钟, 或直到颗粒被分解成小块。</li>
	<li>采用轴向旋转的方法将悬浮液混合, 旋转速度为 30 rpm, 在4摄氏度时为 30-120 分钟。</li>
	<li>用离心法澄清变性蛋白溶液的 3万 x g, 4 °c 30 分钟, 将上清液放在冰上并丢弃颗粒。</li>
	<li>使用布拉德福德测定法测定蛋白质浓度。24为 SDS 凝胶分析整除, 并将其放置在-20 摄氏度。 注意: 在这一点上, 该过程可以暂停的 aliquoted 样品在液氮, 并保持在-80 摄氏度的存储。</li>
</ol>3. 蛋白质复用
<ol><li>在0.4 毫升烧杯中制备250毫升的缓冲 E (3 米尿素, 100 毫米三盐酸 pH 8.0, 20 米精氨酸盐酸盐, 2 毫米还原 l-谷胱甘肽, 500 毫米氧化 l-谷胱甘肽), 冷却缓冲液降至4摄氏度。</li>
	<li>添加60毫克变性蛋白混合物, 其中包含他的6-Tlp3-LBD 在步骤2.13 到250毫升的缓冲 E, 同时搅拌缓冲区在 500 rpm。在4°c 的情况下孵育复混混合物, 连续搅拌 500 rpm, 24-48 小时。此步骤中的最终蛋白质浓度为0.2 毫克 mL-1。</li>
	<li>在 8-10 l 桶中准备 7 l 的缓冲器 A, 并将其冷却到4摄氏度, 以准备下一步 (步骤 3.4), 该步骤将在 24-48 h 中进行 (请参见图 1中的流程图)。</li>
	<li>浸泡50厘米的透析油管28毫米充气直径, 与分子量切断在 12-14 kDa 成200毫升的10毫米乙二胺四乙酸钠盐 (EDTA-Na2) 和加热它在气体火焰, 直到沸腾。用200毫升的 ddH2O 冲洗透析膜。</li>
	<li>将透析膜的一端夹入透析导管闭合, 并将步骤3.2 中获得的250毫升复性混合物转移到透析管中。用另一夹钳关闭开口端。检查膜的完整性, 确保没有泄漏。</li>
	<li>将透析管放在透析桶中, 并在步骤3.3 中准备好缓冲液的预冷7升。放置一个磁力搅拌棒, 确保它不会接触透析油管, 而搅拌。</li>
	<li>透析样品在4°c 与继续搅动在500转每分钟和改变缓冲至少四次在 12 h 期间。最后一个缓冲区更改后, 将该示例留在透析过夜。 注: 在透析过程中, 尿素 (~ 3 米) 的过量会逐渐从变性蛋白溶液中去除, 从而使蛋白质再折叠餐巾。在透析结束时可以观察到重蛋白沉淀。</li>
	<li>从水桶中取出透析管, 将其内容转移到500毫升烧杯中。在冰上保留蛋白质溶液, 除非另有说明。</li>
	<li>将蛋白质溶液通过0.43 µm 孔径膜过滤成500毫升玻璃瓶, 以除去任何沉淀的蛋白质。</li>
</ol>4. 用固定金属离子亲和层析纯化其6标记蛋白
<ol><li>
电荷和平衡5毫升预螯合柱。
	<ol>
<li>将该列连接到蠕动泵, 确保连接油管中没有空气滞留。</li>
		<li>通过通过 25-50 毫升 ddH2O 来清洗该列。</li>
		<li>通过加载3毫升0.1 米 NiCl2然后通过 ddH2O 的 25 mL, 对该列进行充电。</li>
		<li>平衡柱与25毫升缓冲 F (装载缓冲, 20 毫米三 HCl pH 值 8.0, 500 毫米氯化钠, 20 毫米咪唑)。</li>
	</ol>
</li>
	<li>将步骤3.9 中获得的折叠蛋白样品加入2.5 毫升的1米三盐酸 pH 8.0, 25 毫升的5米氯化钠和2.5 毫升的2米咪唑库存溶液, 以此来调节缓冲 F 的组成。</li>
	<li>以5毫升/分或更少的速率将样品装载到柱上, 并丢弃流经 (未绑定的蛋白质)。</li>
	<li>用 50-100 毫升的缓冲 F 清洗柱体, 去除非特异的束缚蛋白, 并丢弃流经。</li>
	<li>洗脱他的 6-Tlp3-LBD 与25毫升的缓冲 G (洗脱缓冲器, 20 毫米三 HCl, 500 毫米氯化钠, 500 毫米咪唑) 和池的流动通过分数包含的蛋白质 (如使用布拉德福德试剂确定).</li>
	<li>使用布拉德福德测定法测定汇集样品中的蛋白质浓度。采取一个小整除为 SDS 页分析和地方在-20 °c。</li>
</ol>5. 他的6-标签去除使用 TEV 蛋白酶 (可选)
<ol><li>准备, 冷却到4摄氏度, 4 升的缓冲 H (TEV 裂解缓冲液, 10 毫米三盐酸 pH 值 8.0, 150 毫米氯化钠, 1% (v/v) 甘油, 2 毫米。</li>
	<li>切开大约 5-7 cm 透析油管 (直径 2-3 cm) 并且浸泡它在200毫升 ddH2O。</li>
	<li>将他的6标记的 TEV 蛋白酶添加到他在步骤4.5 中准备的6标记蛋白质中, 最终摩尔比 1 TEV: 8 蛋白。</li>
	<li>夹紧油管的一端与透析油管闭合, 并填充它与蛋白质/TEV 蛋白酶混合物。关闭另一端, 确保没有泄漏。</li>
	<li>将透析管放入缓冲 H 的预冷4升 (从步骤 5.1), 并在4摄氏度下孵化, 连续搅拌2小时. 改变缓冲和继续透析过夜, 以允许 TEV 介导的裂解反应完成。</li>
	<li>同时, 准备和冷却 (4 °c) 4 升的缓冲 I (10 毫米三盐酸 pH 值 8.0, 150 毫米氯化钠, 1% (v/v) 甘油) 准备第二天。</li>
	<li>在隔夜透析以后, 交换缓冲到缓冲我在步骤5.6 和透析准备了进一步 1-2 h 在4°c。</li>
	<li>从透析桶中取出管子, 松开管的一端, 将蛋白质溶液转移到50毫升的猎鹰管上。过滤解决方案通过0.43 µm 孔径注射器过滤器, 并保持在冰上, 除非另有说明。</li>
	<li>测量样品的体积并调整三个, 氯化钠和咪唑浓度对缓冲 F 的组成 (例如在缓冲器 J 中的25毫升蛋白质溶液中添加0.25 毫升1米三盐酸盐, 1.75 毫升氯化钠, 5 毫升的0.25 米咪唑)。</li>
	<li>按照步骤4.1 中的描述, 准备一个5毫升 HiTrap 螯合 HP 柱。</li>
	<li>将样品装载到柱上 (流速: 5 毫升/分) 并收集流经, 其中含有未加标签的 Tlp3-LBD (残滓 42-291)。Uncleaved 的蛋白质, 他的6标记和他的6-TEV 由列保留。</li>
	<li>用5毫升缓冲 F (加载缓冲器) 冲洗柱, 使所有未加标签的蛋白质都能流经并收集洗脱液。</li>
	<li>在步骤5.11 和5.12 中获得的洗脱液, 并测量蛋白质浓度。采取一个小整除, 并存储在-20 °c 的 SDS 页分析。</li>
</ol>6. Tlp3-LBD 的尺寸排除层析 (凝胶过滤)
<ol><li>准备 1 L 的缓冲 a (10 毫米三盐酸 pH 值 8.0, 150 毫米氯化钠) 和过滤它使用0.43 µm 膜。</li>
	<li>平衡26/60 大小排除列, 缓冲区 a 的流速为4毫升/分钟。</li>
	<li>将步骤5.13 中获得的蛋白质样品浓缩为 3-4 毫升的体积, 使用15毫升离心选矿机, 10 kDa 切断 (4 °c, 4000 x g)。</li>
	<li>通过离心 1.3万 x g, 4 摄氏度, 以30分钟的方式澄清蛋白质溶液, 去除任何沉淀的蛋白质。</li>
	<li>将样品装入预平衡26/60 凝胶过滤柱上。以4毫升/分的流速在缓冲器 a 上进行色谱检测. 监测紫外线痕迹。Tlp3-LBD elutes 210-220 毫升的保留体积。池的峰值分数。</li>
	<li>测量蛋白质浓度。用一个小整除进行 SDS 页分析。</li>
</ol>7. 在蛋白质纯化各个阶段收集的样品的 SDS 页分析
<ol><li>准备 1 L 的 SDS 页运行缓冲 (缓冲 J), 包含25毫米三, 250 毫米甘氨酸 pH 值 8.3, 和 0.1% (w/v) 月桂钠硫酸盐 (SDS)。</li>
	<li>准备10毫升的 5x SDS 页加载缓冲 (缓冲 K), 其中包含0.25 米三盐酸 pH 6.8, 10% (瓦特/v) SDS, 50% (v/v) 甘油, 1 毫米, 和0.05% 溴酚蓝色。</li>
	<li>允许在纯化过程的不同步骤中收集的整除数 (步骤1.3、2.3、2.13、4.6、5.13、6.6) 解冻。</li>
	<li>对于每个样本, 将对应于总蛋白的15µg 的体积与2µL 的 5x SDS 页加载缓冲区混合, 并使用 ddH2O 将卷调整为10µL。</li>
	<li>将样品加热95摄氏度, 5-10 分钟, 在三十年代将它们旋转 1万 x g, 然后将样品转移到干净的管子中。</li>
	<li>用15% 聚丙烯酰胺分离凝胶 (5 毫升: 2.5 毫升 30% (w/v) 双丙烯酰胺, 制备一个 SDS 页凝胶, 1.2 毫升1.5 米三盐酸盐 pH 8.8, 1.2 毫升 ddH2O, 50 µL 10% (w/v) SDS, 50 µL 20% (w/v) 过硫酸铵 (APS), 3 µL tetramethylethylenediamine (TEMED)和5% 聚丙烯酰胺堆积凝胶 (2 毫升: 340 µL 30% (w/v) 双丙烯酰胺, 250 µL 1 米三 pH 6.8, 1.37 毫升 ddH2O, 20 µL 10% (w/v) SDS, 20 µL 20% (w/v) ap, 2 µL TEMED)。</li>
	<li>按照制造商的建议, 组装电泳设备并填写 1x SDS 页面运行缓冲区。</li>
	<li>载入步骤7.5 中制备的蛋白质分子量标记和样品。在 25 mA 的恒定电流下进行电泳。</li>
	<li>将凝胶放入容器中, 用 ddH2将其冲洗干净. 添加150毫升的考马斯蓝染色溶液, 含 25% (v/v) 异丙醇, 10% (v/v) 醋酸和 0.03% (瓦特/v) 灿烂的蓝色 R-250。在室温下将容器放置在水平旋转平台上30分钟。</li>
	<li>用 ddH2O 将凝胶冲洗, 放入含有 5% (v/v) 甲醇和 7% (v/v) 乙酸的脱色溶液中。Destain 同时使用水平旋转平台进行1小时的混合。</li>
	<li>图像的凝胶和分析。</li>
</ol>8. 循环二色谱 (CD) 折叠纯蛋白二次结构的光谱分析
<ol><li>将步骤6.6 中获得的 0.5-1 毫克折叠蛋白转移到透析管中, 并将其放置在含有 5 l 的缓冲 l (50 毫米磷酸钠 pH 值 7.4) 的透析桶中, 预换热器到4摄氏度。透析样品在4°c 与连续搅动和执行至少 3-4 缓冲变动在 2-4 h 之前在离开样品到透析过夜。</li>
	<li>使用 10 kDa 离心浓缩器将透析蛋白溶液浓缩为0.06 毫克 mL-1 。</li>
	<li>通过离心在 1.3万 x g, 4 °c 为30分钟澄清溶液。</li>
	<li>使用具有 20 nm 最小-1扫描速率的 spectropolarimeter, 将样本的远紫外 CD 光谱记录在波长范围为 200-260 nm 的25摄氏度。使用透析缓冲区作为空白控件。重复记录, 并生成平均频谱。</li>
	<li>通过使用 CDSSTR 程序从 DichroWeb 服务器25 (http://dichroweb.cryst.bbk.ac.uk) deconvoluting CD 频谱, 计算辅助结构内容。</li>
</ol>

作者声明没有竞争的财政利益。

本文介绍了细菌化学感受器 Tlp3 周质 LBD 中包涵体的表达和复性的简便方法。纯蛋白的制备涉及在大肠杆菌中对 pET 质粒编码基因的过度表达, 包括体的纯化和 solubilisation, 变性蛋白的复性及其纯化的连续亲和性和尺寸排除色谱步骤。尿素促进变性和稀释/透析介导的复性是该议定书的关键步骤, 最优化往往需要确保适当复存放在包含体的蛋白质26。
Tlp3-LB...

趋化和运动已被证明在空肠弯曲菌发病机制中发挥重要作用, 通过促进细菌的殖民和入侵主机1,2,3。随着化学信号的引导, 趋化使细菌朝着最佳的生长环境迈进。这个过程包括通过一组称为 chemoreceptors 的蛋白质 (Tlps) 识别细胞内和环境化学线索。大多数 chemoreceptors 是膜嵌入蛋白与 extracytoplasmic 配体结合域 (LBD), 跨膜领域和细胞质信令域, 后者与胞浆信号蛋白相互作用, 传输讯号到鞭毛马达4,5,6,7。
十一种不同的 chemoreceptors 已在C.菌基因组4,8中确定。到目前为止, 只有其中一些 chemoreceptors 的特点和配体特异性的 Tlp19, Tlp310,11, Tlp411, Tlp712, Tlp1113是已知的。通过生产折叠和高度纯净的重组化学感受器LBDs14、, 可以极大地促进该物种中剩余 chemoreceptors 的天然配体的鉴定, 以及其他细菌中的许多 chemoreceptors.15, 16. 然而, 在大肠杆菌中 heterologously 表达周质 LBDs 细菌 chemoreceptors 的尝试往往导致将其定向到包含机构 17, 18, 19.然而, 这种现象可以方便地分离和恢复现有的蛋白质。本文以周质化学感受器 Tlp3 的 LBD 为例, 提出了一种从包涵体中提取蛋白质、复性和纯化的方法. 之所以选择此示例, 是因为 Tlp3-LBD 属于 dCACHE 家族20 , 这些传感域大量发现在双组分组氨酸激酶和 chemoreceptors 中, 原核生物20, 21,22,23。
在这种方法中, 基于 pET151/D-TOPO 向量的表达式构造被设计为将 N 终端合并为其6标记, 后跟一个烟草蚀刻病毒 (TEV) 蛋白酶解切站点, 用于随后的标记删除19。该协议描述了蛋白在大肠杆菌中的过度表达, 分离和尿素介导的 solubilisation, 以及尿素耗竭的蛋白质复用。在复性后, 用亲和层析法纯化样品, 采用选择性标记去除和粒度排斥层析。用循环二色谱光谱法确定纯化蛋白的折叠状态。这是以前开发的方法的修改版本, 用于恢复和纯化不同化学感受器、幽门螺杆菌TlpC19 的 LBD。此过程在图 1中总结, 产生 10-20 毫克的纯, 未标记的 Tlp3-LBD 每 1 L 细菌培养, 蛋白质纯度 > 90% 按 SDS 页估计。

<table><tbody><tr><td>Tris base</td><td>AMRESCO</td><td>497</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>MERK MILLIPORE</td><td>1064041000</td><td></td></tr><tr><td>Ampicillin</td><td>G-BIOSCIENCES</td><td>A051-B</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MERCK</td><td>52332</td><td></td></tr><tr><td>Triton x-100</td><td>AMRESCO</td><td>694</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-thiogalactoside (IPTG)</td><td>ASTRAL SCIENTIFIC PTY LTD</td><td>AST0487</td><td></td></tr><tr><td>Urea</td><td>AMRESCO</td><td>VWRC0568</td><td></td></tr><tr><td>Dithiothreitol</td><td>ASTRAL SCIENTIFIC PTY LTD</td><td>C-1029</td><td></td></tr><tr><td>L-arginine monohydrochloride</td><td>SIGMA-ALDRICH</td><td>A5131</td><td></td></tr><tr><td>Reduced L-glutathione</td><td>SIGMA-ALDRICH</td><td>G4251</td><td></td></tr><tr><td>Oxidized L-glutathione</td><td>SIGMA-ALDRICH</td><td>G4376</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>SIGMA-ALDRICH</td><td>7558-79-4</td><td></td></tr><tr><td>Sodium phosphate monobasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>SIGMA-ALDRICH</td><td>10049-21-5</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA)</td><td>AMRESCO</td><td>VWRC20302.260</td><td></td></tr><tr><td>Imidazole</td><td>SIGMA-ALDRICH</td><td>I2399</td><td></td></tr><tr><td>Glycerol</td><td>ASTRAL SCIENTIFIC PTY LTD</td><td>BIOGB0232</td><td></td></tr><tr><td>Nickel chloride (NiCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>SIGMA-ALDRICH</td><td>339350</td><td></td></tr><tr><td>Glycine</td><td>AMRESCO</td><td>VWRC0167</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>SIGMA-ALDRICH</td><td>L4509</td><td></td></tr><tr><td>Unstained Protein Ladder, Broad Range (10-250 kDa)</td><td>NEW ENGLAND BIOLABS</td><td>P7703</td><td></td></tr><tr><td>Amicon Ultracel centrifugal concentrator (Millipore)</td><td>MERCK</td><td>UFC901096</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>FALCON</td><td>BDAA352070</td><td></td></tr><tr><td>Dialysis tubing</td><td>LIVINGSTONE INTERNATIONAL PTY</td><td>Dialysis</td><td></td></tr><tr><td>Snakeskin dialysis tubing</td><td>THERMO SCIENTIFIC&amp;trade;</td><td>68100</td><td></td></tr><tr><td>Prepacked HiTrap Chelating HP column</td><td>GE HEALTHCARE LIFE SCIENCES</td><td>17-0408-01</td><td></td></tr><tr><td>EmulsiFlex-C5 high-pressure homogeniser</td><td>AVESTIN</td><td>EmulsiFlex&amp;trade; - C5</td><td></td></tr><tr><td>Peristaltic Pump P-1</td><td>GE HEALTHCARE LIFE SCIENCES</td><td>18-1110-91</td><td></td></tr><tr><td>Superdex 200 HiLoad 26/60 size-exclusion column&amp;nbsp;</td><td>GE HEALTHCARE LIFE SCIENCES</td><td>28989336</td><td></td></tr><tr><td>JASCO J-815 spectropolarimeter</td><td>JASCO</td><td>J-815</td><td></td></tr></tbody></table>

method for efficient refolding and purification of chemoreceptor ligand binding domain

chemoreceptors 和结构研究的天然配体的鉴定, 旨在澄清配位特异性的分子基础, 可大大促进了毫克量的纯, 折叠配体结合域的生产。试图 heterologously 表达周质配体结合域的细菌 chemoreceptors 在大肠杆菌 (大肠杆菌) 往往导致他们的目标纳入纳入机构. 本文采用空肠弯曲菌 (周质) 化学感受器 Tlp3 的 dCACHE 配体结合域为例, 提出了从包涵体中提取蛋白质、复性和纯化的方法.该方法涉及的兴趣蛋白的表达与裂解他的6标签, 分离和尿素介导 solubilisation 的包涵体, 蛋白质复用尿素耗竭, 并通过亲和层析纯化,其次是标签去除和大小排除色谱。循环二色谱光谱学用于确认纯蛋白的折叠状态。已经证明, 该协议一般是有用的生产毫克量的 dCACHE 周质配体结合域的其他细菌 chemoreceptors 在可溶性和 crystallisable 的形式。

重组表达他的6-Tlp3-LBD 在大肠杆菌导致蛋白质沉积在包涵体。在步骤2.13 中计算出的细菌培养 1 L 的表达率是其6-Tlp3-LBD 在包涵体中的大约100毫克。这里描述的蛋白质隔离过程和图 1中所说明的, 是通过亲和层析、标记去除和大小排除色谱的方法, 包括包含体的 solubilisation、蛋白质的复用和纯化。, 并产生 10-20 毫克纯, 未加标签的 T...

Watch this Scientific Journal Video about Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain at JoVE.com

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

提出了一种 dCACHE 周质配体结合域的复用过程, 该方法由包涵体化学感受器 Tlp3 和纯化, 产生毫克量的蛋白质.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

method-for-efficient-refolding-purification-chemoreceptor-ligand

Research

JoVE Journal

Biochemistry

17.7K 次观看.  Monash Biomedicine Discovery Institute. 提出了一种 dCACHE 周质配体结合域的复用过程, 该方法由包涵体化学感受器 Tlp3 和纯化, 产生毫克量的蛋白质. 

视频: Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

科研

生物化学

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

遗传学

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

生物工程

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPAR&#947;) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite ...

环境科学

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

使用单分子FRET可视化膜受体的构象动力学

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

免疫与感染

<meta charset="utf8"></head><body>预测蛋白质-蛋白质结合界面的氨基酸偏好

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

<meta charset="utf8"></head><body>作者聚焦:一种破译多特异性蛋白质-蛋白质相互作用中氨基酸偏好的计算方法

We describe a methodology based on sequence diversification to estimate the amino acid preferences of multispecific binding sites in protein-protein interactions (PPIs). In this strategy, thousands of potential peptide ligands are generated and screened in silico, thus overcoming some limitations of available experimental methods.

化学感受器配体结合域的高效复用与纯化方法

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此视频中的章节

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

测量TCR-的pMHC结合

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

使用单分子FRET可视化膜受体的构象动力学

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

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参与蛋白质-蛋白质相互作用的潜在多特异性肽结合结构域的氨基酸偏好的计算预测