作者赞赏 Sidhu 实验室的弗雷德里克 Fellouse 博士对安德鲁·库恩克尔方法的综合构建。作者赞赏 Alevtina Pavlenco 夫人和 Sidhu 实验室的其他成员, 为制备高效的电子能力的大肠杆菌细胞和高质量的杜 ssDNA 提供了宝贵的帮助。这项工作得到中国国家自然科学基金 (批准号: 81572698, 31771006) 的支持, 并由 ShanghaiTech 大学 (批准号: F-0301-13-005) 到抗体工程实验室。

<ol>
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作者没有什么可透露的。

构建高多样性、噬菌体展示的工厂图书馆, 需要质量控制检查点来监测施工过程的各个阶段, 包括电控细胞的能力、ssDNA 模板的质量、效率dsDNA 合成、电穿孔后滴度、晶圆折叠、CDRs 氨基酸多样性的序列分析。
ssDNA 的高产和纯度是高诱变率的必要条件。在我们的经验, 噬菌体诱导在25°c 隔夜可以产生更多的杜 ssDNA 比从噬菌体诱导在37摄氏度过夜。这与以前的报表19<...

抗体具有广泛的应用范围, 从基础研究到疾病诊断和治疗。截至 2016年, 美国食品药品监督管理局 (USFDA) 批准了60多抗体, 用于临床治疗自身免疫性疾病、癌症和传染性疾病1,2。
在 1975年, 科勒和米尔斯坦报告了一种技术, 连续产生抗体的单一克隆特异性从细胞源称为 ' 杂交瘤 ', 这一技术后来成为医药和工业的基石 3 ,4。这种方法产生的抗体需要各种步骤, 包括抗原生产、小鼠免疫、b 淋巴细胞的提取、b 细胞与骨髓瘤细胞的融合, 形成不朽的杂交瘤细胞、克隆选择和治疗应用,人性化是需要避免人类抗鼠抗体 (哈马)4,5。然而, 对于这种技术, 抗原, 包括毒素, 病原体和高度保守的蛋白质是相对无效的触发体内免疫应答的单克隆生产5。
在 1978年, 和记黄埔et报告使用寡核苷酸直接诱变的残留在单链噬菌体病毒6。在 1985年, 史密斯报告说, 外国基因片段可以融合在框架内的基因编码噬菌体涂层蛋白 III, 从而可以显示在噬菌体表面, 而不损害其传染性7。这些开创性的工作奠定了基础, 随后构建噬菌体显示抗体库的免疫, 幼稚, 和合成形式的单链可变片段 (抗体) 和抗原结合片段 (工厂) 的格式, 以治疗单克隆开发8,9。从技术角度看, 基于噬菌体的抗体开发提供了一种互补的方法, 以杂交瘤为基础的单克隆细胞的发展, 可以帮助规避的局限性, 一些抗原可以构成和人性化的过程,杂交瘤衍生的抗体通常需要5。截至 2016年, 已在市场上批准了6种噬菌体显示衍生抗体, 其中 Humira 是治疗类风湿性关节炎最成功的抗体之一, 许多噬菌体显示抗体候选者目前处于不同的临床阶段。调查10。
对于免疫和幼稚的噬菌体抗体库, 在光和重链中互补决定区域 (CDRs) 的多样性来源于自然免疫汇 (即, 从 B 细胞)。与此相反, 合成噬菌体抗体库中 CDRs 的多样性是完全人工的。图书馆建设的综合方法对序列多样性的设计提供了精确的控制, 为抗体结构和功能的机械研究提供了机会11,12。此外, 在图书馆建设之前, 可以优化合成库框架, 以方便下游、大型工业发展11、12。
在 1985年, 安德鲁·库恩克尔报告了单链 DNA (ssDNA) 基于模板的突变方法, 将站点定向突变引入到 M13 噬菌体中, 有效地13。这种方法后来被广泛应用于噬菌体展示库的建设。化学合成的 DNA 寡核苷酸设计, 以引入多样性的 CDRs, 被纳入一个 phagemid 与抗体骨干模板。在这个过程中, phagemid 被表达为一个尿的 ssDNA (杜 ssDNA), 寡核苷酸被退火到 CDRs, 并扩展到合成双链 dna (dsDNA) 在存在 T7 dna 聚合酶和 T4 dna 连接酶。最后, 生成的 ds DNA 可以通过电穿孔引入到大肠杆菌中. 
为高多样性, 噬菌体展示图书馆建设, 应仔细准备的两组分混合物的电主管细胞和共价键闭环 dsDNA (CCC-dsDNA) 的高压电穿孔。Sidhu et修改了传统方法制备的电控细胞和 DNA, 大大提高了图书馆的多样性14。
在本协议中, 我们描述了一种构建的合成噬菌体显示的工厂图书馆的多样性 109-1010获得单电穿孔。图 1显示了库结构的概述, 其中包括: 1) 高效电控电池的制备;2) 提取杜 ssDNA;3) 基于寡核苷酸定向诱变的安德鲁·库恩克尔方法;4) 电穿孔及库尺寸计算;5) 蛋白质 A/l-基 ELISA 用于折叠和功能多样性评价;和 6) DNA 序列分析的多样性。所有试剂、菌株和设备都列在材料的表中。表 1显示了试剂设置。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.0 M H&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>AC29570</td><td></td></tr><tr><td>1.0 M Tris, pH 8.0</td><td>Invitrogen</td><td>15568-025</td><td></td></tr><tr><td>10 mM ATP</td><td>Invitrogen</td><td>18330-019</td><td></td></tr><tr><td>100 mM dithiothreitol</td><td>Fisher</td><td>BP172</td><td></td></tr><tr><td>100 mM dNTP mix</td><td>GE Healthcare</td><td>28-4065-60</td><td>solution containing 25 mM each of dATP, dCTP, dGTP and dTTP.</td></tr><tr><td>3,3&amp;rsquo;,5,5&amp;rsquo;-tetramethylbenzidine (TMB)</td><td>Kirkegaard &amp;amp; Perry Laboratories Inc</td><td>50-76-02</td><td></td></tr><tr><td>50X TAE</td><td>Invitrogen</td><td>24710030</td><td></td></tr><tr><td>Agarose</td><td>Fisher</td><td>BP160</td><td></td></tr><tr><td>Carbenicillin, carb</td><td>Sigma</td><td>C1389</td><td>100 mg/mL in water,&amp;nbsp; 0.22 &amp;mu;m filter-sterilize, work concentration: 100 &amp;mu;g/mL.</td></tr><tr><td>Chloramphenicol, cmp</td><td>Sigma</td><td>C0378</td><td>100 mg/mL in ethanol, 0.22 &amp;mu;m filter-sterilize, work concentration: 10 &amp;mu;g/mL.</td></tr><tr><td>EDTA 0.5 M, pH 8.0</td><td>Invitrogen</td><td>AM9620G</td><td></td></tr><tr><td>Granulated agar</td><td>VWR</td><td>J637-500G</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; peroxidase substrate</td><td>Kirkegaard &amp;amp; Perry Laboratories Inc</td><td>50-65-02</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>795488</td><td></td></tr><tr><td>Kanamycin, kan</td><td>Fisher</td><td>AC61129</td><td>50 mg/mL in water,&amp;nbsp; 0.22 &amp;mu;m filter-sterilize, work concentration: 50 &amp;mu;g/mL.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P2222</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>94046</td><td></td></tr><tr><td>NaCl</td><td>Alfa Aesar</td><td>U19C015</td><td></td></tr><tr><td>Nanodrop</td><td>Fisher</td><td>ND2000C</td><td></td></tr><tr><td>NaOH</td><td>Fisher</td><td>SS256</td><td>! CAUTION NaOH causes burns.</td></tr><tr><td>NON-Fat Powdered Milk</td><td>Sangon Biotech</td><td>A600669</td><td></td></tr><tr><td>PEG-8000</td><td>Fisher</td><td>BP233</td><td></td></tr><tr><td>Protein A-HRP conjugate</td><td>Invitrogen</td><td>101123</td><td></td></tr><tr><td>QIAprep Spin M13 Kit</td><td>Qiagen</td><td>22704</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28706</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Recombinant Protein L</td><td>Fisher</td><td>77679</td><td></td></tr><tr><td>T4 DNA polymerase</td><td>New England Biolabs</td><td>M0203S</td><td></td></tr><tr><td>T4 polynucleotide kinase</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>T7 DNA polymerase</td><td>New England Biolabs</td><td>M0274S</td><td></td></tr><tr><td>Tetracycline, tet</td><td>Sigma</td><td>T7660</td><td>50 mg/mL in water, 0. 22 &amp;mu;m filter-sterilize, work concentration: 10 &amp;mu;g/mL.</td></tr><tr><td>Tryptone</td><td>Fisher</td><td>0123-07-5</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>Ultrapure glycerol</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3750</td><td>25 mg/mL in ethanol, work concentration: 0.25 &amp;mu;g/mL.</td></tr><tr><td>Yeast extract</td><td>VWR</td><td>DF0127-08</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company &lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E.coli&lt;/em&gt; CJ236</td><td>New England Biolabs</td><td>E4141</td><td>Genotype: &lt;em&gt;dut&lt;sup&gt;-&lt;/sup&gt;&amp;nbsp; ung&lt;sup&gt;-&lt;/sup&gt;&amp;nbsp; thi-1 relA1 spoT1 mcrA/pCJ105&lt;/em&gt;(&lt;em&gt;F&#039; cam&lt;sup&gt;r&lt;/sup&gt;&lt;/em&gt;). Used for preparation of dU-ssDNA.</td></tr><tr><td>&lt;em&gt;E.coli&lt;/em&gt; SS320</td><td>Lucigen</td><td>60512</td><td>Genotype: [&lt;em&gt;F&#039;proAB+lacIq lacZ&amp;Delta;M15 Tn10 (tet&lt;sup&gt;r&lt;/sup&gt;)&lt;/em&gt;] &lt;em&gt;hsdR mcrB araD139 &amp;Delta;(araABC-leu)7679 &amp;Delta;lacX74 galUgalK rpsL thi&lt;/em&gt;. Optimized for high-efficiency electroporation and filamentous bacteriophage production.</td></tr><tr><td>M13KO7</td><td>New England Biolabs</td><td>N0315S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company &lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2-cm gap electroporation cuvette</td><td>BTX</td><td></td><td></td></tr><tr><td>96-well 2mL Deep-well plates</td><td>Fisher</td><td>278743</td><td></td></tr><tr><td>96-well Maxisorp immunoplates</td><td>Nunc</td><td>151759</td><td></td></tr><tr><td>Baffled flasks</td><td>Corning</td><td></td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>5811000096</td><td></td></tr><tr><td>Centrifuge bottles</td><td>Nalgene</td><td></td><td></td></tr><tr><td>ECM-630 electroporator</td><td>BTX</td><td></td><td></td></tr><tr><td>Magnetic stir bars</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Thermo Fisher centrifuge</td><td>Fisher</td><td></td><td></td></tr><tr><td>High speed shaker</td><td>TAITEK</td><td>MBR-034P</td><td></td></tr><tr><td>Microplate shaker</td><td>QILINBEIER</td><td>QB-9002</td><td></td></tr><tr><td>Liquid handler for 96 and 384 wells</td><td>RAININ</td><td></td><td></td></tr><tr><td>Mutil-channel pipette</td><td>RAININ</td><td>E4XLS</td><td></td></tr><tr><td>Amicon concentrator</td><td>Merck</td><td>UFC803096</td><td></td></tr></tbody></table>

construction of synthetic phage displayed fab library with tailored diversity

基础研究和医学中单克隆抗体 (抗体) 的需求逐年增加。自1975年第一次报告以来, 杂交瘤技术一直是单克隆抗体发展的主要方法。作为一种替代技术, 噬菌体显示方法为单克隆的发展越来越有吸引力, 因为 Humira, 第一种噬菌体抗体和最畅销的抗体之一, 被批准用于临床治疗类风湿关节炎在2002年。噬菌体展示作为一种非动物性的单克隆技术, 它绕过了传统的基于杂交瘤技术的抗体开发所需要的抗原免疫原性、人性化和动物维护。在本协议中, 我们描述了一种构建的合成噬菌体显示的工厂图书馆的多样性 109-1010获得单电穿孔。本协议包括: 1) 高效电控细胞制剂;2) 提取含尿单链 DNA (杜 ssDNA);3) 基于寡核苷酸定向诱变的安德鲁·库恩克尔方法;4) 电穿孔及库尺寸计算;5) 蛋白质 A/l-基酶联免疫吸附试验 (ELISA) 用于折叠和功能多样性评价;和 6) DNA 序列分析的多样性。

按照工厂库结构的流程图 (参见图 1), 我们准备了 M13KO7 帮助噬菌体预感染的大肠杆菌SS320 电子主管细胞。当使用 phagemid 库构造骨干 (图 4) 时, 这些电子主管单元的效率估计为 2 X 109 cfu/µg。
通过对大肠杆菌CJ236 和大肠杆菌SS320 细胞的效价比较, 尿合并效率...

Watch this Scientific Journal Video about Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity at JoVE.com

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

本议定书描述了一个详细的程序, 以构建一个噬菌体显示合成抗体库, 具有量身定做的多样性。合成抗体具有广泛的应用, 从基础研究到疾病诊断和治疗。

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 ...

construction-synthetic-phage-displayed-fab-library-with-tailored

Research

JoVE Journal

Immunology and Infection

13.5K 次观看.  ShanghaiTech University. 本议定书描述了一个详细的程序, 以构建一个噬菌体显示合成抗体库, 具有量身定做的多样性。合成抗体具有广泛的应用, 从基础研究到疾病诊断和治疗。

视频: Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

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.

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 ...

科研

生物化学

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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. ...

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 ...

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

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

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 ...

生物学

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 ...

Using Phage Display to Select Proteins with High Affinity to a Target Protein

Source: Zhang, W., et al., <a href="https://app.jove.com/v/62950">Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the phage display technique to isolate pre-engineered phages expressing proteins with high affinity to the target protein.

使用 Phage Display 选择对靶蛋白具有高亲和力的蛋白质

Source: Zhang, W., et al., Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases. J. Vis. Exp. (2021)
In this video, we demonstrate ...