Name: a guide to production, crystallization, and structure determination of human ikk1/&#945;
Uploaded: 2018-11-02T13:00:00
Description: Watch this Scientific Journal Video about A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃŽÂ± at JoVE.com

我们感谢位于高级光子源、lemont、il 的 beamlines 19id、24id 和13id 的工作人员在收集各种晶体的数据时提供的支持。我们感谢 salk 研究所 dmitry lyumkis 在 em 地图模型构建的早期阶段为我们获取了低分辨率的低温-em 图, 该模型被用来构建最初的 ikk1 分子替换搜索模型。导致这些结果的研究得到了国家卫生研究院向 gg 提供的 ai064326、ca141722 和 gm071862 赠款的资助。sp 目前是 wellcome 信托 dbt 印度中级研究员。

1. 适用于 ikkniα大规模表达的重组病毒的制备
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p1 病毒制备32<ol>
<li>第1天: 6 井板的每口井中的板 sf9 细胞 (~ 6x105) (通道号小于 10) 在每口井中的6孔板和孵育。当通道细胞达到密度为2至 3 x 10 6 细胞时 , 每毫升悬浮在悬浮状态, 在密度约为 6 x 105 的情况下稀释到新鲜介质中。</li>
		<li>第2天: 在没有抗生素和血清的情况下, 在100μl 的 Sf9 iii ?...

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两种相关 ikk 蛋白的生产、结晶及结构解 我们着手确定 ikk半α的 x 射线晶体结构, 并认为这将是一个相对简单的练习, 考虑到我们在 ikk2/ββ蛋白生产、结晶和结构测定方面的经验。然而, 我们感到非常惊讶的是, 这两种相关的蛋白质在结晶的易用性方面表现得非常不同。尽管几个知名实验室做出了努力, ikk1 结构的确定还是用了近20年的时间。这在很大程度上是由于几个关键瓶颈造成?...

从炎症和免疫到生存和死亡, 不同的细胞功能都需要 nf-b 家族的转录活性。这些活动在细胞中得到严格控制, 失去调节导致各种病理条件, 包括自身免疫性疾病和癌症1、2、3.在没有刺激的情况下, n f-b 的活性被 b (-b) 蛋白4的抑制剂所抑制。在 b 蛋白上特定的尔斯残基的磷酸化标志着它们的泛化和随后的蛋白酶体降解或选择性处理5。两个高度同源的 ser/thr 激酶, ikk2/ββ和 ikkneα, 通过执行这些磷酸化事件6,7作为 nf-b 活动的中央调节器。
配体和受体之间的相互作用通过一系列介质传输信号, 从而激活 nf-b 因子。nf-b 信号过程大致可分为两种不同的途径--规范和非规范 (替代)8。ikk2/β的活性主要调节对炎症和先天免疫反应至关重要的规范途径的nf-b 信号9。这种途径的一个显著特点是 ik2/β10在一个迄今没有生化特征的 ikk 复合体中快速而短暂地激活--假定由 ikk1 和 ikk2 以及一个调节组件--nemo (nf-b 调制器) 组成)11,12,13。在 ikk 复合体的两个催化 ikk 亚基之间, ikk2 主要负责14特定残基的原型 b (α,-β, 和-γ) 的特定残基的磷酸化, 并与 nf-b 结合, 并且非典型的 b 蛋白 nf-b送往 105, 这是一种nf-b p50 亚基5的前体.磷酸化诱导的泛化和蛋白酶体降解的 i b (或 p105 的处理) 导致释放和激活一组特定的 nf-b 二聚体 15.由于 ikk2 功能失调而引起的异常 nf-b 活性已在许多癌症以及 2, 3,16例自身免疫性疾病中观察到。
与 ikk2/βl 相比, ikk这家α的活性调节非规范途径的 nf-gitemb 信号, 这对发育和免疫是必不可少的。ikk1 磷酸化了其 c 端段上 nf-b2/p100 的特定残留物, 从而导致其加工和 p52 的生成。转录活性 p52:RelB 异质化物的形成引发了对发育信号7、17、18、19、20的缓慢而持续的反应。有趣的是, 中心 nf-b 因子 p52 的生成在很大程度上依赖于另一个因子, nf-b 诱导激酶 (nik)21,22, 但不是 ikk2 或 nemo。在静息细胞中, 由于其持续依赖蛋白酶体的降解23,24, 25,nik的水平仍然很低。在 "非规范" 配体刺激细胞时, 在某些恶性细胞中, nik 变得稳定, 可以招募和激活 ikkgeα. nik 和 ikk1 的激酶活性对于将 p100 有效地处理为 p1007 是必不可少的。ikk1 和 nik 磷化三种丝氨酸 (ser866、870和 872) 的 nf-b2/p100 在其 c-终端段上导致其处理和 p52 的生成。非规范途径的异常激活已涉及到许多恶性肿瘤, 包括多发性骨髓瘤26,27,28。
一些高效和特定的 ikk2/β抑制剂是已知的, 虽然到目前为止没有一个已被证明是一种有效的药物。相反, ikk半特异性抑制剂是稀疏的。这在一定程度上可能是由于我们缺乏关于 ikkniα的结构和生化信息, 这限制了我们对 ikk1 在细胞中激活 nf-b 的机械基础的理解, 以及合理的药物设计。ikk2/β的 x 射线结构为我们提供了关于 ikk2/β29激活机理的见解;然而, 这些结构不能揭示如何不同的上游刺激触发激活 ikk% α或 ikk2/β来调节不同的集合 nf-b 活动30,31。为了了解 ikknα独特信号功能的机制基础, 并建立了一个合理药物设计的平台, 我们重点确定了 ikk半α的结构。

<table>
	<tbody>
		<tr>
			<td>Cellfectin/Cellfectin II</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362100</td>
			<td>Cellfectin is now discontinued, replaced by Cellfectin II</td>
		</tr>
		<tr>
			<td>Sf900 III Insect cell medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12658-027</td>
			<td></td>
		</tr>
		<tr>
			<td>SF9 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>12659017</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IKK1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-56704</td>
			<td>Previously sold by Imgenex</td>
		</tr>
		<tr>
			<td>anti-PentaHis antibody</td>
			<td>Qiagen</td>
			<td>34460</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Nitrocellulose can also be used</td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay reagent</td>
			<td>BioRad</td>
			<td>500001</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 column</td>
			<td>GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon concentrator</td>
			<td>Millipore</td>
			<td>UFC801008, UFC803008, UFC201024, UFC203024</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound A</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calbiochem IKK-inhibitor XII</td>
			<td>Calbiochem</td>
			<td>401491</td>
			<td></td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>SIGMA</td>
			<td>S4400</td>
			<td></td>
		</tr>
		<tr>
			<td>MLN120B</td>
			<td>Millenium</td>
			<td>Gift item</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPPNP</td>
			<td>SIGMA</td>
			<td>A2647</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA</td>
			<td>51227, 42867, 31404,</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>Alfa Aesar</td>
			<td>J62101</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>SIGMA</td>
			<td>93593, 81210, 88276, 95904, 81255, 89510, 92897, 81285, 95172</td>
			<td>Some of them are new Cat # on SIGMA catalogue. What we had was originally from Fluka that had different Cat #.</td>
		</tr>
		<tr>
			<td>Crystallization Screens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Screen I and II (Crystal Screen HT)</td>
			<td>Hampton Research</td>
			<td>HR2-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Index HT</td>
			<td>Hampton Research</td>
			<td>HR2-134</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG/Ion and PEG/Ion2 (PEG/Ion HT)</td>
			<td>Hampton Research</td>
			<td>HR2-139</td>
			<td></td>
		</tr>
		<tr>
			<td>PEGRX 1 and PEGRx 2 (PEGRx HT)</td>
			<td>Hampton Research</td>
			<td>HR2-086</td>
			<td></td>
		</tr>
		<tr>
			<td>SaltRx 1 and SaltRx 2 (SaltRx HT)</td>
			<td>Hampton Research</td>
			<td>HR2-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal mounts</td>
			<td>Hampton Research</td>
			<td>HR8-188, 190, 192, 194</td>
			<td></td>
		</tr>
		<tr>
			<td>Synchrotron</td>
			<td>The Advanced Photon Source (APS) at the U.S. Department of Energy&#8217;s Argonne National Laboratory</td>
			<td>Beamline 19 ID</td>
			<td>The Advanced Photon Source (APS) at the U.S. Department of Energy&#8217;s Argonne National Laboratory provides ultra-bright, high-energy storage ring-generated X-ray beams for research in almost all scientific disciplines.</td>
		</tr>
	</tbody>
</table>

a guide to production, crystallization, and structure determination of human ikk1/&#945;

一类细胞外刺激需要激活 ikkenα, 以诱导 nf-b 亚基 (p52) 通过处理其前体 p52 来产生。p52 的功能是作为一个同相或异质体与另一个 nf-b 亚基, reb。这些二聚体反过来调节与炎症、细胞存活和细胞周期有关的数百种基因的表达。ikktuα主要与 ikk2/β和 nemo 作为三元复合体保持联系。然而, 它的一小部分也被观察为低分子量复合物。不知道 p100 处理活动是否由在较大或较小的复杂池中激活 ikkenα触发。在几种癌症和炎症性疾病中检测到了 ikk% α的组织活性。为了了解 ikknα的激活机制, 并使其作为药物靶标的使用, 我们在不同的宿主系统中表达了重组的 ikknα, 如大肠杆菌、昆虫和哺乳动物细胞。我们成功地在感染杆状病毒的昆虫细胞中表达了可溶性 ikk定α, 获得了毫克数量的高纯度蛋白质, 在抑制剂存在的情况下将其结晶, 并确定其 x 射线晶体结构。在这里, 我们描述了生产重组蛋白的详细步骤, 它的结晶, 以及它的 x 射线晶体结构的测定。

ikknenα不同结构的克隆与表达 全长人类 ikk% α被克隆到杆状病毒表达载体 pfastbhta 在其 ecri 和 noi 限制站点内, 以获得一个 n-端六-组氨酸标记 ikk1。该标签可通过 tev 蛋白酶消化去除。由于全长 ikk% α两端都含有灵活的区域, 而灵活的区域通常使蛋白质难以结晶, 因此我们在 pfastbachta 向量的上述站点中克隆了 ikk% α的各种截断片段。利用野生类型 (wt) 和 s176e180e (e...

Watch this Scientific Journal Video about A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃŽÂ± at JoVE.com

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

b 激酶 1/α (ikkniα-chuk) 是一种 ser/thr 蛋白激酶, 主要通过激活 nf-b 转录因子参与无数的细胞活动。在这里, 我们描述了生产和晶体结构测定这种蛋白质所需的主要步骤。

人的 ikkknα的生产、结晶和结构测定指南

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

本程序的目标是为人类 IKK1-alpha 的生产、结晶和结构测定提供指导，以了解其信号功能的机械基础，并建立合理药物设计平台。这种方法应该帮助研究人员确定 IKK1 的结构，它将为免疫信号领域的关键查询提供答案。该技术描述了获得 IKK 蛋白晶体的简化路径，这些晶体对结晶相当难耐，在克服确定结构的瓶颈方面提供了各种关键信息。一般来说，新方法的个体将难以为之，因为产生千毫克的可溶性、表现良好的蛋白质，需要它在昆虫细胞中表达，而结晶只能在极窄的条件下进行。用于确定 IKK1 结构的分子替换程序也可能非常棘手，特别是由于晶体的偏转性能较差，在缺乏适当搜索模型的情况下，不在包含许多 IKK1 分子的不对称单元的位点。演示程序将是凯尔舒马特和桑贾拉霍奇基斯，学生从我的实验室。第一天，在Sf903昆虫细胞培养的两毫升中，在六井板的每个井中，在27摄氏度下孵育。当细胞达到每毫升悬浮细胞的2到3倍的密度，通过稀释到新鲜介质，其密度约为6倍10至5。第二天，在SF-903或格雷斯昆虫介质的100微升中稀释8微升Sf9转染试剂。然后短暂地涡旋混合物。在单独的管中，用同一介质的100微升稀释1微克的套件纯化重组百花。将稀释的DNA和转染试剂结合。在室温下孵育15至30分钟后，将介质从井中取出。然后加入一毫升新鲜介质。接下来，将稀释的DNA转染试剂混合物滴入细胞，调整滴体大小，使粘附细胞不脱。六小时后，在细胞顶部加入1.5毫升的新鲜介质。在27摄氏度下孵育细胞，每12小时定期检查一次井中是否有病毒感染的迹象。第五天，在感染后60至72小时取出含有细胞的介质。将介质转移到管中后，在500倍g和4摄氏度下离心5分钟。完成后，保存上一点，这是P1病毒堆栈。在第一天，将先前准备的 His-IKK1 细胞颗粒重新在 40 毫升的解解缓冲液中重新暂停。将细胞悬浮液放在冰上，以60%至70%的占空比和5到10个30秒持续时间的脉冲，以超过1分钟的间隔，通过声波对细胞进行悬浮。在4摄氏度下，在大于或等于28，000g的离心下澄清酸盐，45分钟。在制备镍-NTA阿加罗斯树脂后，根据文本协议，使用20毫升的洗脱缓冲液在重力流下洗脱他标记的 IKK1α 蛋白。收集一到1.5毫升的分数。将含有蛋白质的馏分结合后，在4摄氏度下将样品与TEV蛋白酶一起消化过夜。第二天早上，在27摄氏度的亚硫酸镁、β-甘油磷酸盐、氟化钠和正极酯钠的温度下，用ATP一毫升孵育蛋白质一小时。孵育后，通过0.45微米过滤器过滤蛋白质溶液。然后将大约 6 毫升的样品装到 120 毫升的制备尺寸排除柱上，该列连接到自动液相色谱系统。平衡后，以每分钟一毫升的流速运行大小排除色谱，并收集两毫升的分数。在运行过程中，监测洗脱 280 和 254 纳米。拉纯馏分后，按照制造商的指示，浓缩在30千元分子量截止离心浓缩器中。然后分配25微升的浓缩蛋白等分，并在液氮中闪冻。使用机器人将结晶试剂的 80 到 100 微升移液器放入 96 井板的储液罐中。使用结晶机器人，分配和混合0.2至0.25微升的 IKK1 及其抑制剂复合物，其储液罐溶液体积相同。设置跌落后，立即用光学透明薄膜密封每个板，以避免蒸发。在18摄氏度下孵育一个板，在寒冷的房间里孵育一个4摄氏度的盘子。使用带偏振器的立体显微镜，每天检查每滴晶体的外观，前七天，然后以更长的间隔。在准备 IKK1 及其抑制剂复合物后，将井溶液转移到 24 井板的每个井中。将 1 至 1.5 微升的井溶液放在干净的玻璃盖玻片上。将同等体积的 IKK1 及其抑制剂复合物添加到玻璃盖玻片中，通过上下移液三到四次轻轻混合。在板的每个井的环上涂抹润滑脂后，将玻璃盖玻片翻过来，用钳子放在相应的井上。然后按压玻璃盖玻片，密封井。在24井板中设置所有滴水后，在黑暗中在寒冷的房间里孵育。偶尔检查显微镜下的滴，晶体的外观和生长。一旦晶体生长完成，轻轻取下含有晶体的玻璃盖玻片，并将其放在固体表面上，晶体滴朝上。轻轻添加 10 微升低温 A 溶液，通过移液轻轻混合，使晶体不接触。使用显微镜慢慢去除晶体中的一些液体，并保持约五到八微升溶液。轻轻将 2.5 至 4 微升的低温 B 溶液添加到落液上，然后通过移液轻轻混合。用小 Petrie 盘盖住玻璃盖玻片，以避免直接气流在晶体上。等待五分钟后，在适当的基座上，从掉落的晶体中仔细挑选一个晶体。在液氮中闪冻结晶体。然后将含有低温环的晶体存放在浸在德瓦瓶中的液氮中，直到同步加速器上准备进行 X 射线衍射。经过对几种不同IKK1变种的广泛试验，晶体通过一个截断构造获得，只有在IKK抑制剂XII存在的情况下才显示合适的X射线特性。与已知的 IKK2 模型相比，低分辨率 X 射线数据与弱强度、非对称单元中的大量 IKK1 分子和 IKK1 单体-二元模型的构象变化相结合，使得难以获得分子替代溶液 IKK1 并确定其结构。不同的 IKK2 结构在其二元中指示不同的单体间向方向，因此，在两个不同的二元模型中，P578 的α碳之间的距离在 39 到 61 个焦虑之间变化。由于低分辨率的低温显微镜图和基于高分辨率 IKK2 结构生成的 IKK1 域的高精度模型，因此有可能获得有用的搜索模型。初始模型显示激酶域相对于 IKK2 单体旋转 24 度的方向和 58 个焦虑的 N 端子开口方向。使用其中一个以 52- angstrom 开口作为模型的二元，在非对称单元中找到了六个二元。一旦掌握，这项技术可以在几个月内执行，如果它执行得当。在尝试此程序时，重要的是要记住，获得可溶性、活性和良好表现的蛋白质是第一个关键步骤。看完这段视频后，您应该对需要详细遵循的所有步骤有一个良好的一般理解，以便成功地结晶和确定 IKK1 蛋白的结构。学习这个程序，仍然有其他EKK家族蛋白质的结构，也可以确定，以回答有关激酶-米托源信号的其他问题。

Research

JoVE Journal

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A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

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生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

酶的结晶和结构确定：多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE 

高通量筛选以获得用于蛋白质晶体学的晶体命中

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots (Video) | JoVE

一种分析脑血凝块细胞成分的综合方法

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...