Name: a protocol for phage display and affinity selection using recombinant protein baits
Uploaded: 2014-02-16T14:00:00
Description: Watch this Scientific Journal Video about A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits at JoVE.com

哈奇，麦金太尔 - 斯坦尼斯（AD421 CRIS），美国农业部种子格兰特（2011-04375）和弗雷德里克·麦克马斯特研究奖学金爵士ABD;该项目部分由美国国家科学基金会的IOS（0849230）资助。

下面（ 图1）中描述的方法的图形描述突出使用噬菌体展示文库的两个主要组成部分为亲和选择：A）一种噬菌体展示文库很可能与蛋白质的诱饵亲和力编码和，B）的纯化的重组诱饵蛋白质。生产诱饵（重组蛋白）已被广泛研究和文献概述了从大肠杆菌可溶性安全，积极重组蛋白的最佳实践12-13 大肠杆菌 ，真核酵母14，昆虫15-16，17-18植物或哺乳动...

<ol>
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通过运行该实验在3复制的井，同一蛋白结合到所述诱饵的独立获得的噬菌体可以区分，即使它们是相同的克隆（ 即在已经获得的编码区的碱基序列没有差异，但这些已从独立的水井中检索）。否则，噬菌体编码相同的蛋白质结合到所述诱饵之间区分的唯一途径是如果它们是独立地扭转这种不同的核苷酸序列中的某些部分转录区域。 使用1费恩巴赫烧瓶中，在其中在扩?...

为什么使用，而不是用于发现和研究蛋白质相互作用与其他分子的无数其他技术噬菌体展示和亲和选择？噬菌体展示可以声称对检测蛋白质-配体相互作用1-3包括下列其他方法的一些独特的优势： 诱饵分子非常广泛的曲目最重要的原因是能够充当诱饵的亲和选择4分子的多样性。噬菌体展示是一种非常强大的分离蛋白片段与其它蛋白，核苷酸，碳水化合物， 等等。5交互本质上说，如果聚合物/分子可以连接到一个可恢复的支持，它可以进行筛选，与噬菌体展示的蛋白质的亲和手段。此外，诱饵分子可被访问以确定它是否保留生物活性时固定6，如果使用对r的条件唤起/消除其活性是有效的6或，引入之前亲和选择的翻译后修饰的诱饵。 外在因素噬菌体抗性第二个原因为使用噬菌体展示的是，它可能是一些诱饵可能需要环境应力（热，高浓度渗压剂，具体的辅因子， 等等 ）来捕捉其相互作用的蛋白质，或在噬菌体展示的蛋白质，可能需要以某种方式改变之前，亲和选择。正在研究中的主要因素是饲料和蛋白质，而不是允许的相互作用发生，或者如果它是致死的试验有机体的条件之间的相互作用。在T7噬菌体是特别适合这种研究，因为它可以承受恶劣的实验条件下既完整且可行的（ 例如已公布的最高温度为T7生存能力〜60℃7）。为改变噬菌体的例子显示亲teins，研究拟南芥种子蛋白质组的修复酶的蛋白质底物时，蛋白质异门冬氨酰甲基Transferease（PIMT），在这些研究中所使用的病毒已经“老化”一个星期之前，每个亲和选择轮，鼓励引进isoaspartate的（isoAsp）残基的蛋白质易感6，这是不可能的生物，可以识别并修复/这种代谢异常。 代谢惰性此外，噬菌体通常是抗代谢毒物和干扰的小分子，将，至少，导致在代谢活性试验有机体多效性效应。继严格清洗，毒药被大量细菌被引入感染，因此毒稀释到一系列无害的细菌或噬菌体随后复制之前删除。在调查的PIMT修复蛋白靶酶，S-腺苷甲硫氨酸（转移酶的）被用来激活微滴定板孔的固定化酶以允许靶蛋白捕获同时依靠S-腺苷高半胱氨酸（AdoHcy）使酶失活，并提供了有用的阴性对照固定在该病毒不会受到任何一方转移酶的或AdoHcy 6影响的知识。此外，某些胚胎发育晚期丰富（LEA）的蛋白家族，研究了该实验室的成员，被称为改变其形状的添加剂如蔗糖8，可以在该点达到浓度高达200毫米的大豆种子的存在生理成熟9。该病毒是没有预料到可通过加入200mM的蔗糖在每个亲和选择圆形，可能需要一定的LEA-客户蛋白相互作用，这不是可行的自主测试生物体10的情况下的影响。 该实验室一直专注于discoverin克蛋白质-蛋白质相互作用中所依据存储的蛋白质保护的脱水11或维修所存储的蛋白质易受isoAsp形成，一旦种子已吸胀6的部件的过程的机理成熟，脱水或发芽的种子。因此，需要作为诱饵中的活动状态的重组蛋白的生产和纯化，并确保它们保持这样，它们被固定化之前和之后，虽然常常难，是一个基础，以我们的工作。然而，由于各重组蛋白生产的情况是不同的，为生产重组蛋白的条件优化这里将不讨论。用户务必尝试尽可能以确定固定的蛋白质仍是功能性（ 例如 ，如果它是一种酶，尽在微孔板酶测定法）。这将提供一些信心，诱饵是生物活性，因此，任何发现的相互作用有些更可能有生物相关性。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>Becton Dickinson Co.</td>
 <td>211705</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>Becton Dickinson</td>
 <td>212750</td>
 <td></td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Becton Dickinson</td>
 <td>214010</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>BP358-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Research Products International</td>
 <td>A20090</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocking Reagent</td>
 <td>EMD Chemicals Inc.</td>
 <td>69064</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Fisher Scientific</td>
 <td>BP337-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Hydroxide</td>
 <td>Fisher Scientific</td>
 <td>BP359-212</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Dodecyl Sulfate</td>
 <td>Fisher Scientific</td>
 <td>BP166-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Fisher Scientific</td>
 <td>BP152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Fisher Scientific</td>
 <td>BP1145-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Escherichia coli BLT5403</td>
 <td>EMD Chemicals Inc.</td>
 <td>BLT5403</td>
 <td>Genotype: F- ompT hsdSB (rB - mB -) gal dcm pAR5403 (AmpR)</td>
 </tr>
 <tr>
 <td>ampicillin, Sodium Salt</td>
 <td>Research Products International</td>
 <td>A40040</td>
 <td></td>
 </tr>
 <tr>
 <td>ethidium bromide</td>
 <td>Fisher Scientific</td>
 <td>BP102-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin (BSA)</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>A-9647</td>
 <td></td>
 </tr>
 <tr>
 <td>penta-HIS primary antibody</td>
 <td>Qiagen Inc.</td>
 <td>34660</td>
 <td></td>
 </tr>
 <tr>
 <td>Goat anti mouse alkaline phosphatise conjugate</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>A-5153</td>
 <td></td>
 </tr>
 <tr>
 <td>para-nitrophenylphosphate</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>N-7653</td>
 <td></td>
 </tr>
 <tr>
 <td>T7-UP primer</td>
 <td>EMD Chemicals Inc.</td>
 <td></td>
 <td>5'-GGAGCTGTCGTATTCCAGTC-3'</td>
 </tr>
 <tr>
 <td>T7-DOWN primer</td>
 <td>EMD Chemicals Inc.</td>
 <td></td>
 <td>5'-AACCCCTCAAGACCCGTTTA-3'</td>
 </tr>
 <tr>
 <td>Qiaquick PCR Purification kit</td>
 <td>Qiagen Inc.</td>
 <td>28104</td>
 <td></td>
 </tr>
 <tr>
 <td>Qiaquick Gel Extraction kit </td>
 <td>Qiagen Inc.</td>
 <td>28706</td>
 <td></td>
 </tr>
 <tr>
 <td>Big Dye Terminator v3.1 Cycle Sequencing Kit</td>
 <td>Invitrogen Life Technologies</td>
 <td>4336917</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Block</td>
 <td>Pierce Chemical Co. (Thermo Fisher Scientific Co.)</td>
 <td>18780</td>
 <td></td>
 </tr>
 <tr>
 <td>96 Well Cell Culture Plates</td>
 <td>Corning</td>
 <td>Costar 3590</td>
 <td></td>
 </tr>
 <tr>
 <td>Isotemp Incubator Oven</td>
 <td>Fisher Scientific</td>
 <td>516D</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur Pipets, 5 &frac34;"</td>
 <td>Fisher Scientific</td>
 <td>13-678-20A</td>
 <td></td>
 </tr>
 <tr>
 <td>ChromaView Transilluminator</td>
 <td>UVP Inc.</td>
 <td>TS-15</td>
 <td></td>
 </tr>
 <tr>
 <td>UV Shield</td>
 <td>Oberon</td>
 <td>071AF</td>
 <td></td>
 </tr>
 <tr>
 <td>Gloves, Nitrile</td>
 <td>Fisher Scientific</td>
 <td>19-170-010C</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile 1.5 ml tubes</td>
 <td>USA Scientific Inc</td>
 <td>1615-5500</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml borosilicate Pipets</td>
 <td>Fisher Scientific</td>
 <td>13-678-25E</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate culture tubes</td>
 <td>Fisher Scientific</td>
 <td>14-961-27</td>
 <td></td>
 </tr>
 <tr>
 <td>Uniskan I, ELISA plate reader</td>
 <td>Labsystem and Flow Laboratories, Helsinki, Finland</td>
 <td>Type 362</td>
 <td></td>
 </tr>
 </tbody>
</table>

a protocol for phage display and affinity selection using recombinant protein baits

利用重组噬菌体作为支架提出了定向克隆cDNA文库中编码为固定诱饵分子的各种蛋白部分是一种有效的手段来发现相互作用。该技术已经在很大程度上被用于发现蛋白质 - 蛋白质相互作用，但诱饵分子受到挑战不必限于蛋白质。这里提出的协议进行了优化，允许重复进行筛选的诱饵数量适中，最大限度地独立克隆呈现相同的蛋白质的鉴定。这允许更大的信心，确定相互作用的蛋白质都是诱饵分子的合法干扰作用。监测噬菌体效价每亲和选择后轮提供了有关如何在亲和选择进展以及对阴性对照组的疗效信息。滴定噬菌体，以及如何的一种手段，什么要提前准备，使这一进程取得进展的效率，因为可能的话，呈现。检索以下的独立噬斑分离扩增子的属性被特别指出，可以用于确定如何很好的亲和选择进展。故障排除技术，以减少误报或绕过持续恢复的噬菌体进行了解释。减少病毒污染动火的方式进行了讨论。

能够削弱诱饵与噬菌体展示蛋白质相互作用（代谢中毒诱饵）的能力提供了一个有力的阴性对照这一技术。这也是可取的，以确定该诱饵，当结合到微量滴定板孔，保持其功能。这两个检查将增加信心，由nonpoisoned诱饵回收相互作用，噬菌体展示的蛋白质是合法的。 从各孔中取样3式三份大大增加参与了亲和选择的时间和工作，但提供的滴度内每孔一个更准确的估计比采样一?...

Watch this Scientific Journal Video about A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits at JoVE.com

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

噬菌体展示是一种强大的技术，以捕获与感兴趣的固定化分子相互作用的蛋白质或蛋白质的部分。一旦噬菌体cDNA文库的类型来创建和屏幕的决定已经作出，这里所描述的协议允许高效亲和选择，导致识别干扰作用的。

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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A Method For Production of Recombinant mCD1d Protein in Insect Cells.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Primer-Free Aptamer Selection Using A Random DNA Library

引物的适体的选择，使用一个随机DNA文库

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

从噬菌体抗体库的肽调节间质细胞的基因表达和使可能在Unicortical骨缺损的成骨

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

A Protocol for Computer-Based Protein Structure and Function Prediction

基于计算机的蛋白质结构与功能预测议定书

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...