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。

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作者什么都没有透露。

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><tbody><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>&lt;em&gt;Escherichia coli&lt;/em&gt; BLT5403</td><td>EMD Chemicals Inc.</td><td>BLT5403</td><td>Genotype: F- &lt;em&gt;ompT hsdS&lt;/em&gt;&lt;strong&gt;&lt;sub&gt;B&lt;/sub&gt;&lt;/strong&gt; (r&lt;strong&gt;&lt;sub&gt;B&lt;/sub&gt; - m&lt;sub&gt;B&lt;/sub&gt; -) &lt;/strong&gt;&lt;em&gt;gal dcm&lt;/em&gt; pAR&lt;strong&gt;&lt;sub&gt;5403&lt;/sub&gt;&lt;/strong&gt; (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>&lt;em&gt;para&lt;/em&gt;-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&#039;-GGAGCTGTCGTATTCCAGTC-3&#039;</td></tr><tr><td>T7-DOWN primer</td><td>EMD Chemicals Inc.</td><td></td><td>5&#039;-AACCCCTCAAGACCCGTTTA-3&#039;</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 Pipettes, 5 &amp;#190; in</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 Pipettes</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 ...

a-protocol-for-phage-display-affinity-selection-using-recombinant

Research

JoVE Journal

Biology

33.8K 次观看.  University of Kentucky. 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。 

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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

科研

生物化学

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

免疫与感染

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

使用噬菌体显示开发用于E3连接酶的泛素变体调节剂

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

具有量身定做多样性的合成噬菌体展示库的构建

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

基于生物传感器的高通量生物跨越和生物信息学分析战略，用于全球药物-蛋白质相互作用验证

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

生物工程

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

生物学

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

细菌肽显示用于选择新型生物仿生酶

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

使用 D11 scFv-碱性磷酸酶融合蛋白和免疫荧光直接检测组织中的异左旋蛋白

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to ...