重组β-catenin的降解<em>爪蟾</em>卵提取物
1Department of Cell and Developmental Biology and Program in Developmental Biology,Vanderbilt University Medical Center, 2Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology,Cincinnati Children's Hospital Medical Center, 3Vanderbilt Ingram Cancer Center,Vanderbilt University School of Medicine
Summary
的方法被描述为使用放射性标记和荧光素酶融合蛋白在非洲蟾蜍卵提取物和其适应高通量筛选用于蛋白质降解的小分子调节剂进行分析的蛋白质降解。
Abstract
非洲爪蟾卵提取物是一种良好的特点，强大的系统为研究不同细胞过程的生物化学。 爪蟾卵提取物已被用于研究蛋白质周转的许多细胞环境，包括细胞周期和信号转导通路1-3。在此，描述了一种方法用于隔离爪蟾卵提取物进行了优化，以促进关键Wnt信号通路组分，β-连环蛋白的降解。两种不同的方法进行了说明，以评估在非洲爪蟾卵提取物β-连环蛋白降解。一种方法是在视觉信息（[35 S] -放射性标记的蛋白质），而另一种是更容易按比例用于高通量测定法（萤火虫荧光素标记的融合蛋白）。所描述的技术可用于，但不限于，评估β-连环蛋白周转和识别分子组分贡献的营业额。此外，快速的学习性净化大量的同质爪蟾卵提取物结合荧光素标记的蛋白质的定量和轻便读出允许这个系统可以很容易地适用于高通量筛选β-catenin降解的调节剂。
Introduction
非洲爪蟾卵提取物已被广泛用于研究许多细胞生物学过程，包括细胞骨架动力学，核组装和进口，细胞凋亡，泛素代谢，细胞周期进程，信号转导和蛋白质周转1-17。在爪蟾卵提取系统是适合于细胞过程的一个军团的生化分析，因为卵提取物本质上代表未稀释的细胞质包含所有必要执行这些过程，使调查必要的细胞质成分。大量的卵提取物中可同时适用于需要大量的材料（ 例如 ，蛋白纯化或高通量筛选）18-20生化操作做好准备。另一个优点是，在非洲爪蟾卵提取物的特定蛋白质的浓度可以通过加入重组蛋白和/或endog的免疫耗竭的精确调整源性蛋白质相反的质粒DNA，其中感兴趣的蛋白质的表达是难以控制的转染。此外，由于缺乏可用的重组蛋白可通过加入转录编码目的蛋白质的被克服，取新鲜制备的非洲爪蟾卵提取物的高容量翻译外源添加的mRNA的优势。
蛋白质降解的调控是许多细胞途径的关键控制和处理21。 爪蟾卵提取物已被广泛用于研究蛋白质降解为系统允许多个方法来监测蛋白质周转不转录和翻译的混杂影响。 Wnt信号通路是一个高度保守的信号传导途径中起着发育和疾病的关键作用。 β-连环蛋白，Wnt信号通路的主要效应的营业额，受到严格的监管，并增加稳态升β-连环蛋白的伊维尔基尼是对Wnt靶基因的活化是至关重要的。 β-连环蛋白降解的重要性通过以下事实，在Wnt途径的突变抑制β-连环蛋白降解发现在〜90％的结直肠癌22都散发病例的高亮显示。通过Wnt信号通路的组成部分β-catenin降解，可以忠实地概括在非洲爪蟾卵提取物，研究其营业额的机制，以及确定其降解2，19，20，23-29的新型小分子调节剂。
方法，用于制备非洲爪蟾卵提取物用于研究细胞周期的前一朱庇特的出版物30-32进行了描述。当前协议描述了这些方法的变型，并且对降解的优化[35 S] -放射标记的β-连环蛋白和荧光素酶标记的β-连环蛋白在爪蟾卵提取物。放射性标记的降解测定允许蛋白水平的放射自显影通过直接观察。 [35 S]甲硫氨酸掺入到感兴趣的地方使用，然后可以直接添加到降解反应在体外翻译反应的蛋白质。此外，放射性标记的蛋白质周转测定法不需要对感兴趣的蛋白质或表位标签，这可以影响蛋白质稳定性的抗体。因为在蛋白水平的微小变化，这反映在变化放射性标记的蛋白条带的强度，很容易放射自显影时，[35 S]-放射性标记的降解测定代表蛋白质周转2的可视化一个非常有用的方法。
β-连环蛋白，以萤火虫荧光素酶的融合（以下简称为“荧光素酶”）允许对蛋白水平的精确和轻便的定量测量，以来确定β-连环蛋白周转19,20的动力学性质。萤光素酶测定法的一个主要优点是它提供了一个强有力的定量系统，很容易按比例放大。下面的协议提供了简单的方法，用于测定β-连环蛋白降解和健壮，高效且有效的方法的新的β-连环蛋白调节剂的高通量筛选。
Protocol
非洲爪蟾卵提取物1。准备注：每个青蛙产生大约1毫升可用卵提取物。从10青蛙提取物通常被制备在同一时间，并如下所述缓冲器的容量是用于执行10蛙爪蟾卵提取物制备。缓冲体积可以相应于更大或更小卵提取物的制剂来调节。以这种方式产生的Preps始终得到蛋白质浓度≥50毫克/毫升。当由两个人进行收集鸡蛋，将它们加工成提取物的过程中是最有效的。 （对于?…
Representative Results
在非洲爪蟾卵提取物β-连环蛋白降解的示意图示于图2A中 。35 S-标记的β-连环蛋白孵育在非洲爪蟾卵提取物，等分试样（1毫升提取当量）除去在适当的时间，并且样品进行SDS-PAGE后的放射自显影。通过Wnt通路的组分β-连环蛋白降解是通过遍在蛋白-蛋白酶体系统2介导的和[35 S] -放射标记的β-连环蛋白在非洲爪提取物的降解通过加入蛋白酶体?…
Discussion
爪蟾卵提取物是一种强大的生化系统的研究β-catenin的营业额。 β-连环蛋白在非洲蟾蜍卵提取物的浓度为〜25nM的2。在最佳条件下，卵提取物是能够在50-100纳米/小时的速度降低β-连环蛋白，并且是半最大，在200 nM的24。有β-catenin的降解利用非洲爪蟾卵提取物成功重建的几个关键步骤。这些包括：1）生成高品质的爪蟾卵提取物和由卵提取物的制备方式，2?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢李劳里的手稿的批判性阅读。三元催化器是由美国心脏协会博士前奖学金（12PRE6590007）的支持。 MRB是由美国国家癌症研究所的训练津贴（T32 CA119925）的支持。 SSH是由美国国立卫生研究院（R01DK078640）的支持。 EL是由美国国立卫生研究院（R01GM081635和R01GM103926）的支持。
Materials
| Name of Reagent/ Material | Company | Catalog Number | Comments/Description |
| Pregnant Mare Serum Gonadotropin (PMSG) | ProSpec | hor-272-A | Reconstituted with distilled water before use. |
| Human Chorionic Gonadotropin | Sigma | CG10-10VL | |
| Potassium Chloride | Fisher | BP366-1 | |
| Sodium Chloride | Research Products International | S23020-5000.0 | |
| Magnesium Chloride | Fisher | BP214-500 | |
| Calcium Chloride | Acros Organics | AC42352-5000 | |
| HEPES | Fisher | BP310-1 | |
| Cysteine | Acros Organics | AC17360-1000 | |
| Leupeptin | Sigma | L2884-10MG | |
| Aprotinin | Sigma | A1153-10MG | |
| Pepstatin | Sigma | P4265-5MG | |
| Cytochalasin B | Sigma | C8273-10MG | |
| 3 mL syringe: Luer Lock tip | Becton Dickinson | 309657 | |
| 27G1 needle | Becton Dickinson | 305109 | |
| 96 well solid white polystyrene microplate, round bottomed | Corning | 3605 | |
| Steady-Glo Luciferase assay system | Promega | E2520 | Store long-term at -80, can store for up to 1 month at -20 |
| TNT Sp6 coupled reticulocyte lysate system | Promega | L4600 | |
| TNT Sp6 High-yield Wheat Germ protein expression system | Promega | L3260 | Generally higher yield than reticulocyte lysate |
| EasyTag Express Protein Labeling Mix [S35] | Perkin Elmer | NEG772007MC | |
| Creatine phosphate | Sigma | 27920-5G | |
| ATP | Sigma | A2383-5G | |
| Creatine phosphokinase | Sigma | C3755-35KU | |
| Dimethyl Sulfoxide (DMSO) | Sigma | D8418-50ML | |
| Dual-Glo luciferase assay system | Promega | E2920 | Same storage conditions as Steady-Glo |
| 50 mL Centrifuge tubes | Fisher Scientific | 0556214D | |
| Sorvall SS-34 fixed angle rotor | Thermo Scientific | 28020 | |
| 115 V 50/60 Hz Minicentrifuge | Fisher Scientific | 05-090-128 | |
| mMessage mMachine Sp6 kit | Ambion | AM1340 | |
| Anti-Firefly Luciferase antibody | Abcam | ab16466 | |
| Anti-GSK3 antibody | BD Transduction Laboratories | 610201 | |
| Name of Equipment | Company | ||
| FLUOStar Optima | BMG Labtech | ||
| Sorvall RC-6 Plus Centrifuge | Thermo Scientific | ||
| 16°C Incubator | Percival Scientific |
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Citazione di questo articolo
Chen, T. W., Broadus, M. R., Huppert, S. S., Lee, E. Reconstitution Of β-catenin Degradation In Xenopus Egg Extract. J. Vis. Exp. (88), e51425, doi:10.3791/51425 (2014).