Name: bacterial inner-membrane display for screening a library of antibody fragments
Uploaded: 2016-10-15T15:00:00.000+00:00
Duration: 12 min 28 s
Description: Watch this Scientific Journal Video about Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments at JoVE.com

We thank Tomer Zohar for work on scFv screening and characterization assays. A portion of this work was supported by award number F32CA150622 from the National Cancer Institute (to AJK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

<ol>
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The authors declare that they have no competing financial interests.

工程抗体的细胞质活动是一个艰巨的任务，由于细胞质，阻碍稳定的二硫键6,7-形成的还原环境。这会导致最抗体，除非它们被设计为在细胞质中的稳定性和溶解度，除了被设计为结合亲和力是细胞质无效。噬菌体展示，细菌表面展示，酵母表面展示方法的现有方法都使用分泌途径14-16工程抗体的显示，但这些方法都没有办法工程师胞内折叠。因为Tat途径的折叠质量控制阻止了不良折叠并在细胞质中不稳定的抗体的转用内膜显示工程化抗体具有改善的细胞质的稳定性和溶解性。此方法简化了工程细胞内抗体的迭代过程对亲和性的第二溶解度，因为这两个性质在一个步骤工程改造。虽然这种方法被设计用于工程抗体与降低细胞内环境的溶解度，它也可以适用于工程抗体在非还原条件下发挥作用，因为蛋白质使用这种方法维持其在周质的氧化环境的折叠设计。 尽管该技术简化了工程以高亲和力和高细胞质溶解度抗体的过程中，一些限制是重要的使用该协议时要考虑的。在分析了二次筛选的ELISA信号，以确定有前途的单链抗体变体，潜在的有趣的变体和那些可能不会出现足够的抗原结合之间挑剔的门槛并不容易，直到经过多次克隆得到了进一步的特点是显而易见的。重要的是要寻找改良过的亲本抗体结合是很重要的;然而，异常高的信号可以是指示亲和力29或聚集效应30，其不是唯一的内膜显示筛选方法的挑战。要记住一个关键限制使用此协议时，是无法平移后的恢复原生质球，因为他们都是非可行的（未公布数据）。这就需要DNA扩增和改造步骤来恢复编码抗体的质粒。 该协议的几个关键步骤，使折叠和抗体结合的同步工程。用于筛选是成功的，所述scFv文库被筛选必须表达为融合到ssTorA信号肽。没有该序列中，抗体将不被引导到Tat途径，因而将不被转运到周质19。另外，至关重要的是，C-末端表位标签融合至抗体以允许在bin显示抗体的检测鼎检测。显然， 大肠杆菌大肠杆菌菌株用于表达的scFv还必须有必要的Tat途径机械，但是这是在通常使用的大肠杆菌的真大肠杆菌菌株。 修改这个协议是可能的，以改善其电势以分离具有所需特性的抗体。减色层叠步骤可以淘洗针对靶抗原耗尽非所需成分的抗体库之前完成。库球状体，可以单独用或涂覆涂覆有BCCP用非所需的蛋白的磁珠孵育，并结合于那些珠的球状体可筛选其余未结合的球状体结合于期望的目标之前被丢弃。如在代表结果所提到的，一个方法来改善的分离的scFv的亲和力是包括在平移反应的可溶性竞争者与对球状体显示的scFv竞争。由于可溶性补偿etitor是纯化的蛋白质，没有DNA从它放大，所以只对球状体显示的scFv的序列将在PCR反应中回收。此外，这种方法可以扩展到工程其他类型的抗体或非抗体结合蛋白。 大肠杆菌内的膜显示为工程抗体以高亲和力和高含量的胞内溶解度的有力的平台。这种方法特别适用于设计成在细胞内的环境中发挥作用的抗体的有效的工程。这些细胞内的抗体已经被探索作为在许多领域的潜在治疗剂，包括神经变性疾病，癌症和病毒感染31。这种技术可以使更广泛地使用胞内抗体作为在这些领域的研究和医学并且其中正在学原位蛋白靶标是所期望的任何其他字段的工具。

能够折叠，并在细胞内环境中起作用的抗体可用于研究和治疗应用中有希望的工具。它们具有通过在细胞内结合到靶蛋白，以防止蛋白质-蛋白质相互作用，破坏蛋白质-核酸相互作用，或阻止对酶1-5基底入口调节蛋白活性的能力。 尽管抗体具有细胞内的应用程序多的潜力，工程他们在细胞内环境正确折叠和溶解度，同时维持对结合靶抗原的能力是具有挑战性的。的还原性的细胞质环境防止通常所需的全长抗体和抗体片段，包括单链可变片段（scFv）抗体6,7的稳定折叠的二硫键的形成。许多定向进化方法已被用来工程用H抗体对于目标IGH亲和力抗原8-10。这些方法通常使用噬菌体展示，酵母表面展示或细菌表面展示筛选抗体11-13的大型图书馆。这些方法是有力及有效用于鉴定结合靶的抗体，但它们依赖于分泌途径运输将要显示14-16蛋白质。分泌途径易位从还原细胞质折叠蛋白成在酵母或进入细菌的周质中的内质网腔。然后将蛋白质在氧化条件下折叠并显示在细胞表面上或包装入噬菌体颗粒来筛选结合亲和力17,18。其结果是，使用这些技术中分离的抗体不一定会在细胞质折好，和细胞内的溶解度通常必须单独设计，如果抗体将在细胞内的应用中使用。 为了改善那些在细胞质以及折叠工程抗体的效率，我们先前报道的MAD-TRAP（对于基于康达识别相关联蛋白的膜锚定显示器）的成功，用于筛选使用大肠杆菌党内的scFv抗体文库的方法膜显示19。细菌内膜显示依赖于双精氨酸易位（TAT）途径用于传输显示抗体，而相比之下，使用分泌途径其他常见的显示方法。 Tat途径包含质量控制机制，只允许可溶的，正确折叠的蛋白从大肠杆菌运大肠杆菌胞质中，穿过内膜并进入周质20,21。 （定位到具有N末端融合到Tat信号肽ssTorA Tat途径即 ，蛋白质），其在细胞质以及折叠过表达的Tat基板形成长寿命易位与N-末端中间体In中的细胞质和周质19中的C末端。这允许正确折叠的Tat基材，包括抗体片段的显示，在大肠杆菌的周质面大肠杆菌内膜。通过酶消化除去外膜以产生原生质球后，抗体被暴露于细胞外空间（ 图1）。这允许对内部隔膜显示达衬底被筛选用于结合到一个特定的目标。重要的是，利用对细胞表面显示Tat途径确保只有在该库被在细胞质以及折叠抗体将被询问的结合，允许结合亲和力和细胞内折叠同步工程。在这个协议中，我们描述了如何显示在E.一个单链抗体库大肠杆菌内膜，平移针对靶抗原的文库，并进行二次筛选，以确定该文库的最有希望的成分。虽然我们关注<html>关于scFv的协议，该方法可以应用于工程的应用要求绑定和细胞内折叠任何蛋白质。 <img alt="图1" src="/files/ftp_upload/54583/54583fig1.jpg" /> 图1.达内膜显示，在E.大肠杆菌，被表达为融合到ssTorA信号序列和正确地在细胞质折叠的scFv抗体穿过内膜运输。 A易位中间形式，其中所述的scFv在与在细胞质N端和在周质的C-末端的内膜锚。 大肠大肠杆菌外膜被酶消化，以形成球状体，从而暴露锚定的抗体与细胞外的空间，使它们可用于检测通过使用结合于所显示的抗体的C-末端融合表位标签的抗体。负载/ 54583 / 54583fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

<table><tbody><tr><td>scFv library</td><td>Varies</td><td></td><td>A suitable scFv library should be obtained from a commercial or academic source.</td></tr><tr><td>MC4100 &lt;em&gt;E. coli&lt;/em&gt; cells</td><td>Coli Genetic Stock Center</td><td>6152</td><td>Cells need to be chemically competent or electrocompetent, depending on the selected transformation method.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Difco dehydrated culture media LB Broth, Miller (Luria-Bertani)</td><td>BD</td><td>244610</td><td></td></tr><tr><td>Chloramphenicol (Cm)</td><td>Fisher Scientific</td><td>BP904-100</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Sodium phosphate, dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Potassium phosphate, monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP9706-100</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>BP220-1</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP1521</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA), 0.5 M</td><td>Fisher Scientific</td><td>BP2482-500</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma Aldrich</td><td>L3790-10X1ML</td><td></td></tr><tr><td>Vortex mixer</td><td>VWR</td><td>97043-564</td><td></td></tr><tr><td>Bicine</td><td>Fisher Scientific</td><td>BP2646100</td><td></td></tr><tr><td>D-Biotin</td><td>Fisher Scientific</td><td>BP232-1</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside</td><td>Fisher Scientific</td><td>BP1755-1</td><td></td></tr><tr><td>BugBuster Master Mix (cell lysis detergent)</td><td>EMD Millipore</td><td>71456</td><td></td></tr><tr><td>Vivaspin 2 MWCO, 3,000&amp;nbsp;daltons</td><td>GE Healthcare Sciences</td><td>28932240</td><td></td></tr><tr><td>Target antigen</td><td>Varies</td><td>N/A</td><td>Purified target antigen may be purchased or produced/purified.</td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td>Invitrogen</td><td>65601</td><td></td></tr><tr><td>Dynamag-2 magnet</td><td>Invitrogen</td><td>12321D</td><td></td></tr><tr><td>Tube rotator</td><td>VWR</td><td>13916-822</td><td></td></tr><tr><td>PCR primers</td><td>IDT</td><td>N/A</td><td>Primer sequences are as described in the protocol.</td></tr><tr><td>10x T4 DNA ligase reaction buffer</td><td>New England BioLabs</td><td>B0202S</td><td></td></tr><tr><td>T4 Polynucelotide kinase (PNK)</td><td>New England BioLabs</td><td>M0201S</td><td>Make sure the T4 ligase buffer used in the primer phosphorylation reaction contains 1 mM ATP.&amp;nbsp;</td></tr><tr><td>5x Phusion HF buffer pack</td><td>New England BioLabs</td><td>B0518S</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) solution mix, 10 mM each dNTP</td><td>New England BioLabs</td><td>N0447L</td><td></td></tr><tr><td>Phusion DNA polymerase</td><td>New England BioLabs</td><td>M0530S</td><td>Other high-fidelity polymerases may be used as an alternative, but the annealing temperature in &lt;strong&gt;Table 3&lt;/strong&gt; must be adjusted.</td></tr><tr><td>C1000 Touch thermal cycler with dual 48/48 fast reaction module</td><td>Bio-Rad</td><td>185-1148</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3121</td><td></td></tr><tr><td>SYBR Safe DNA gel stain</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Wizard SV gel and PCR clean-up system</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>T4 DNA ligase</td><td>New England BioLabs</td><td>M0202S</td><td></td></tr><tr><td>Microdialysis membrane filter</td><td>EMD Millipore</td><td>VSWP04700</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214030</td><td></td></tr><tr><td>96-well polystyrene round-bottom cell culture plates</td><td>VWR</td><td>10062-902</td><td></td></tr><tr><td>Costar general polystyrene assay plate lids</td><td>Corning</td><td>3931</td><td></td></tr><tr><td>Microtitre plate shaker</td><td>VWR</td><td>12620-926</td><td></td></tr><tr><td>Costar 96 well EIA/RIA Easy Wash clear flat bottom polystyrene high bind microplate</td><td>Corning</td><td>3369</td><td></td></tr><tr><td>Bel-blotter polycarbonate 96-well replicating tool</td><td>Bel-Art Products</td><td>378760002</td><td></td></tr><tr><td>Instant nonfat dry milk</td><td>Quality Biological</td><td>A614-1000</td><td></td></tr><tr><td>Tween 20 (polysorbate 20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>PopCulture reagent (concentrated cell lysis detergent)</td><td>EMD Millipore</td><td>71092-3</td><td></td></tr><tr><td>Monoclonal ANTI-FLAG M2-Peroxidase(HRP) antibody produced in mouse</td><td>Sigma Aldrich</td><td>A8592</td><td></td></tr><tr><td>SigmaFast OPD</td><td>Sigma Aldrich</td><td>P9187-50SET</td><td></td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;), 10&amp;nbsp;N solution</td><td>Fisher Scientific</td><td>SA200-1</td><td></td></tr><tr><td>Reynolds Wrap aluminum foil</td><td>VWR</td><td>89079-075</td><td></td></tr><tr><td>BioTek Epoch microplate spectrophotometer</td><td>Fisher Scientific</td><td>11120570</td><td></td></tr></tbody></table>

bacterial inner-membrane display for screening a library of antibody fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

在大肠杆菌 Tat途径的细胞内蛋白质折叠质量控制机制大肠杆菌限制穿过内细胞膜转运到在还原细胞质环境良好折叠的蛋白质。通过超表达的scFv的ssTorA信号序列（从TORA蛋白，这是自然的Tat途径20传送的信号序列）的融合，易位被停止，从而导致在内侧膜19 scFv的显示。外膜的酶破坏之后，所显示的抗体用于筛选抗原结合活性可用。取Tat途径为单链抗体显示的优点的能力是由Karlsson 等所示的19（ 图6）。所述scFv抗体scFv13和scFv13.R4被融合到缺少由公认的精氨酸 - 精氨酸残基对天然ssTorA序列或修饰的ssTorATat途径。 scFv13.R4通过马丁纽等人通过四轮定向进化工程。从scFv13并且已知在细胞质9折好。这种单链抗体被显示在内膜，但只有当作为融合到天然ssTorA信号序列表达（ 图6）。反之，scFv13没有很好地折叠细胞质9，所以它不是在内部膜显示良好，无论信号序列其所稠合。此外，如果的scFv被在于缺乏TATC蛋白，康达机械20,28的一个重要组成部分，显示没有观察到，显示内膜显示和Tat途径之间的重要链接细胞中表达。这些结果表明，仅包含Tat信号肽和蛋白质正确折叠在细胞质中被显示在内膜，允许传输通过Tat途径充当细胞内FOL一个屏幕鼎。 <img alt="图6" src="/files/ftp_upload/54583/54583fig6.jpg" /> 图6.检测上内膜显示scFv的流式细胞仪分析，以检测在其内的膜折叠很差scFv13和良好的折叠scFv13.R4的显示。的scFv进行融合到天然ssTorA或ssTorA（KK），其中精氨酸 - 精氨酸对在ssTorA序列修饰成Lys赖氨酸。用异硫氰酸荧光素（FITC）检测上的scFv的C-末端FLAG表位标签缀合抗FLAG抗体。没有TATC蛋白（ΔtatC）和ssTorA-scFv13没有FLAG标记细胞进行了测试作为对照。 M表示中间荧光值。从权限参考19转载。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54583/54583fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 19。为了证明这一点，根据scFv13，其具有对β半乳糖苷酶结合亲和力的低电平的易错PCR文库，是针对靶抗原β半乳糖苷酶使用显示器和平移在协议中所述方法平移。 scFv的1-4被一轮诱变和淘选后分离，并表现出较高的结合亲和力β-半乳糖苷比scFv13（ 图7A）和细胞质溶解度更高水平（ 图7B）。 一个新的图书馆，以单链抗体1-4，使用易错PCR制成，以及对第二代文库淘β半乳糖苷酶使用所描述的协议的变形完成。第二轮演进抗β半乳糖苷酶的声像是在纯化的，可溶性scFv 14的存在下完成的作为竞争对手，以改善与比的scFv 1-4更高的亲和性分离的克隆的可能性。第二轮诱变和平移后，单链抗体2-1和2-3单链抗体使用基于ELISA的二次筛选分离。这些scFv的不仅表现出对β半乳糖苷酶比scFv13更高的结合亲和力，但也表现出比第一轮克隆的scFv 1-4更好的结合。的scFv 2-1表现出β半乳糖苷酶结合比得上scFv13.R4（ 图7A）的。的scFv 2-3还示出了在细胞质溶解度进一步增加相比的scFv 14，突出的溶解性和抗原结合的同步工程。由于scFv的亲和力和可溶性表达筛选，用于同时，可能的是一个选定的scFv已国防部中心提供全方位溶解性，但高结合，反之亦然。例如，单链抗体2-1具有比单链抗体2-3可溶表达低，但它具有较高的结合亲和力β-半乳糖苷。 <img alt="图7" src="/files/ftp_upload/54583/54583fig7.jpg" /> 图7.目标结合和scFv细胞质表达变异体内部采用膜分离出的显示（A）的scFv在大肠杆菌的细胞质中表达大肠杆菌细胞（ 例如 ，没有ssTorA信号序列）与六组氨酸（6×-His）标签，并使用镍-氮川三乙酸自旋柱纯化。对β-半乳糖苷酶的结合的纯化的scFv的用的ELISA测定。纯化的scFv被装载到β半乳糖苷酶包被ELISA板，并用抗6×-His抗体检测结合的scFv的。的数据是六个重复的平均，误差棒表示平均值的标准误差。（B）中的细胞裂解物的从表达细胞质通过用抗6×-His抗体探测Western印迹分析的scFv细胞的可溶和不溶组分。总蛋白质浓度用于标准化样品的加载。转载（A）和经许可的参考19改编（B）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54583/54583fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments at JoVE.com

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

bacterial-inner-membrane-display-for-screening-library-antibody

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.4K 次观看.  University of Maryland. We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

视频: Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

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

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 ...

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

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

通过抗体介导的实时成像增强蛋白质可视化

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

发育生物学

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

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 ...

细菌内膜显示筛查抗体片段的图书馆

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此视频中的章节

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

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

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

可扩展的高通量选择从噬菌体展示合成抗体库

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

细菌肽显示用于选择新型生物仿生酶