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-LBD 的复性是通过两步的方式实现的, 首先是将变性样品稀释成含有尿素的缓冲液, 然后通过将样品 dialysing 在没有它的缓冲器上。使用此协议获得的折叠蛋白功能和 crystallisable18,23。但是, 所提出的方法有一定的局限性。众所周知, 当蛋白质变性和折叠存在尿素27,28,29时, carbamylation 的氨基酸经常发生。这是由于溶解尿素分解与时间和产生的氰酸30 , 反应与蛋白质氨基组形成稳定的 carbamylated 产品, 并在较小程度上与其他功能组31, 32. 在碱性 pH 和高温条件下, 尿素的分解速度加快。因此, 建议用超纯 (> 99%) 固体试剂制作新鲜尿素溶液, 并在低温下 (4 °c)30,33, 执行复用/透析步骤, 以减少氰酸酯的形成。此外, 选择含有主要胺的缓冲物, 如三、甘氨酸或碳酸氢铵, 对于复混混合是很重要的, 允许清除产生的氰酸29,33。其他选择是使用盐酸胍代替尿素, 因为盐酸胍没有被报告化学修饰蛋白。
除尿素外, 通常还需要一种还原剂 (巯基乙醇), 以溶解包涵体, 并通过保持其还原态中的半胱氨酸残留量来防止非本机内和分子间的二硫键形成26 ,34。Tlp3-LBD 有一个内二硫化物键, 并在本协议中, 10 毫米的被纳入纳入体内变性缓冲 (步骤 2.10), 以帮助不溶性蛋白的泥沙。然后将样品稀释成蛋白质复用缓冲液, 然后通过透析步骤逐步去除所有的德勤, 从而减少了100倍的浓度。重要的是要注意的是, 这一程序的应用, 在多硫键的蛋白质的复用可能需要优化氧化剂和还原剂的浓度 (如氧化和还原谷胱甘肽 (GSSH/GSH),GSSH/德勤, 半胱胺/cysteamineor 胱氨酸/半胱氨酸) 促进正确形成二硫化物桥梁34, 35.此外, 如果感兴趣的蛋白质具有未配对的半胱氨酸, 而不涉及二硫化物键形成, 则应将还原剂添加到所有纯化缓冲器中。从蛋白质的氨基酸序列预测潜在的二硫化物桥梁可以使用几个在硅片工具 (例如cys_rec: http://linux1.softberry.com/berry.phtml？topic=cys_rec & 组 = 程序 &子组 = propt)。
使用所提出的协议, 以纯化不同的蛋白质也可能需要优化的浓度咪唑在洗涤步骤4.4。如图 2B (车道标记 IB) 所示, 在本例中, 孤立的包含体主要是感兴趣的蛋白质。如果在 IB 样品的 SDS 页面凝胶上可见额外的有效带, 则需要增加步骤4.4 中的咪唑浓度以去除污染物, 但可能会降低蛋白质产量36,37。其他选择是应用咪唑浓度的线性梯度和池的洗脱分数含有的蛋白质的兴趣。
最后一步的纯化, 大小排除色谱, 提供了方法, 以同时估计的分子质量的洗脱粒子, 并得出的寡聚状态的蛋白质。然而, 通过大小排除色谱对分子质量的估计仅对球形分子是准确的, 而对于许多蛋白质38,39, 这种情况并非如此。通过将尺寸排除色谱耦合到多角度光散射 (SEC-加工)40或分析离心41, 可以确定高精度的蛋白质分子质量和寡聚状态。我们以前已经确认, Tlp3-LBD 是通过使用 SEC 加工分析23在解决方案中单体的, 此结果与其他 dCACHE LBDs42、43上的报告一致。
所提出的协议, 其原始或略有修改的形式, 已被用来再折叠餐巾和纯化的几个化学感受器 LBDs 从 dCACHE 系列 X 射线晶体学研究 17, 18, 19,23,42,44. 本程序一般可用于在可溶性和 crystallisable 形式下生产 LBDs 其他细菌 chemoreceptors 的周质的毫克量。在每个单独的情况下, 可能需要协议优化。除了上面讨论的要点外, 最佳 solubilisation 的包涵体和蛋白质复性可能需要使用洗涤剂 (例如SDS 或 n-乙酰基三甲基氯化铵), 添加剂 (如l-精氨酸) 和在单个复用步骤中调整其浓度和孵化时间26,34,45,46。此外, 复化步骤中的蛋白质浓度对复性产量有显著影响。通常应测试从 1 ng ml-1到10毫克 ml-1的浓度范围。对于 Tlp3-LBD, 复混组合中蛋白质的最佳浓度为0.2 毫克 mL-1。
极低的复用率通常是试验复用条件远未达到最佳的迹象。人们可以预期, 高产量与折叠蛋白质是一致的, 可以使用 CD 光谱学验证 (如本过程所述)。此外, crystallisability 蛋白的合成可以 sugguest 蛋白质的折叠状态。另外, 可以使用功能性化验 (如果可用) 来确认蛋白质是否折叠。例如, 在 Tlp3-LBD 的情况下, 折叠蛋白被证明保留其配体结合能力, 如等温量热法所示。23

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。

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			<td>497</td>
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			<td>MERK MILLIPORE</td>
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			<td>G-BIOSCIENCES</td>
			<td>A051-B</td>
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			<td>52332</td>
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			<td>694</td>
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			<td>Isopropyl &#946;-D-thiogalactoside (IPTG)</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>AST0487</td>
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			<td>G4251</td>
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			<td>Oxidized L-glutathione</td>
			<td>SIGMA-ALDRICH</td>
			<td>G4376</td>
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			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>SIGMA-ALDRICH</td>
			<td>&#160;7558-79-4</td>
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			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>SIGMA-ALDRICH</td>
			<td>10049-21-5</td>
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			<td>Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA)</td>
			<td>AMRESCO</td>
			<td>VWRC20302.260</td>
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			<td>Imidazole</td>
			<td>SIGMA-ALDRICH</td>
			<td>I2399</td>
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			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>BIOGB0232</td>
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			<td>SIGMA-ALDRICH</td>
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			<td>FALCON</td>
			<td>BDAA352070</td>
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			<td>LIVINGSTONE INTERNATIONAL PTY</td>
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			<td>Prepacked HiTrap Chelating HP column</td>
			<td>GE HEALTHCARE LIFE SCIENCES</td>
			<td>17-0408-01&#160;</td>
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			<td>EmulsiFlex-C5 high-pressure homogeniser</td>
			<td>AVESTIN</td>
			<td>EmulsiFlex&#8482; - C5</td>
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			<td>Peristaltic Pump P-1</td>
			<td>GE HEALTHCARE LIFE SCIENCES</td>
			<td>18-1110-91&#160;</td>
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			<td>Superdex 200 HiLoad 26/60 size-exclusion column&#160;</td>
			<td>GE HEALTHCARE LIFE SCIENCES</td>
			<td>28989336</td>
			<td></td>
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			<td>JASCO J-815 spectropolarimeter</td>
			<td>JASCO</td>
			<td>J-815</td>
			<td></td>
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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 Tlp3-LBD 每1升的细菌培养。
蛋白质洗脱从凝胶过滤柱作为一个单一的, 对称的峰值对应于220毫升的保留量 (图2A)。利用对数 (兆瓦) 与保留容积 (V保留(mL) = 549.3-73.9 x 对数 (兆瓦)) 的校准图计算分子量 (兆瓦) 在 EMBL 蛋白表达和纯化核心设施网站 (http://www.embl.de/pepcore/pepcore_services/protein_purification/色谱/hiload26-0_superdex200/指数 html), 产生了 29 kDa 的价值。这个值非常接近于从氨基酸序列 (28.7 kDa) 计算出来的, 这表明 Tlp3-LBD 是溶液中的单体。
为评价纯化过程, 采用 SDS 页分析法对不同步骤收集的样品进行评价。如图 2B所示, 包容体主要包含他的6Tlp3-LBD。少量的这种蛋白质也存在于诱导培养 (IPTG (+)) 和 uninduced 培养 (IPTG (-)) 的可溶性分数中, 显然是由于 T7 聚合酶的漏表达。他的6-Tlp3-LBD 迁移到聚丙烯酰胺凝胶上, 其表面的分子量为 28 kDa, 接近于从氨基酸序列 (31.8 kDa) 计算出来的值。他的6标记去除, 亲和层析和凝胶过滤步骤产生了高度纯净的蛋白质 (> 90% 电泳均匀性)。
为了证实这个过程获得的蛋白质是折叠的, 他的6-Tlp3-LBD 的二级结构被 CD 光谱学评价。使用 CDSSTR 估计从 CD 频谱 (图 3) 中α螺旋线和β片的含量给出了31% α和23% β的值。这些值与使用 Jpred3 服务器 (http://www.compbio.dundee.ac.uk/www-jpred/) (37% α和26% β) 的序列分析预测的结果相近, 表明从尿素变性包涵体中回收的蛋白质被折叠。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57092/57092fig1.jpg"> 图 1: 所介绍方法的示意图.重组他的6-Tlp3-LBD 是 expressd 在大肠杆菌后诱导与 IPTG (见第 1)。经过4小时的表达, 细菌细胞被收获和裂解 (见2节)。含有包含体 (ib) 的不溶性分数被洗涤, 用于去除膜和膜蛋白杂质, 然后将 IB 溶于含有高浓度尿素的缓冲液中 (见第2节)。他的6-Tlp3-LBD 是折叠稀释到缓冲 E 后, 彻底透析, 以逐步尿素去除 (3 节)。折叠他的6-Tlp3-LBD 然后被固定化的金属离子亲和层析纯化, 如第4节所述。使用 TEV 蛋白酶删除他的6标记 (第5节)。用凝胶过滤层析法 (6 节) 对未加标签的 Tlp3-LBD 进行浓缩和进一步纯化。为 SDS 页分析收集样本的点数用 * 表示。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57092/57092fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57092/57092fig2.jpg"> 图 2: 重组 Tlp3-LBD 的纯化。(a) Superdex 200 HiLoad 26/60 凝胶过滤柱上的未加标签 Tlp3-LBD 的尺寸排除色谱痕。蛋白洗脱, 保留体积为220毫升。(B)减少了15% 页考马斯蓝染色凝胶。M: 蛋白质分子量标记;IPTG (-): 在步骤1.3 中收集的 uninduced 控制样本;IPTG (+): IPTG 诱导后可溶性分数 (在步骤2.3 中收集);IB: 孤立的包容体 (步骤 2.13);折叠他的6-Tlp3-LBD: 从步骤4.6 折叠蛋白质样品;未加标签的 Tlp3-LBD: 在步骤5.13 中获得的蛋白质样本;凝胶过滤1和 2: 两个分数从蛋白质峰值洗脱从凝胶过滤柱 (步骤 6.6)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57092/57092fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57092/57092fig3.jpg"> 图 3: 纯化重组 Tlp3-LBD 的圆形二色谱谱。光谱记录在25°c 50 毫米磷酸磷酸钠 pH 7.4。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57092/57092fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

对于拟肽与他们抗利什曼原虫活性混合赖氨酸型/精氨酸型单体及评价合成的有效方法

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

科研

生物化学

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

滴定 ELISA 法测定受体配体相互作用的离解常数

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

龙苷受体 n 端域的晶体结构

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.