Name: using phage display to develop ubiquitin variant modulators for e3 ligases
Uploaded: 2021-08-27T14:45:00
Description: Watch this Scientific Journal Video about Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases at JoVE.com

泛素变体技术是在Sachdev Sidhu博士（多伦多大学）的实验室中设计的。WZ目前是人类与微生物组计划的CIFAR Azrieli全球学者。这项研究由授予WZ的NSERC发现补助金（RGPIN-2019-05721）资助。

1. 试剂制备
<ol><li>PBS（磷酸盐缓冲盐水）：将50 mL 10x PBS溶液与450 mL超纯H2 O混合。通过过滤灭菌并储存在4°C或室温（〜20-25°C）。</li>
	<li>10%BSA（牛血清白蛋白）：缓慢加入1克BSA至7mL超纯H2 O中，混合直至完全溶解（无团块）。加满超纯 H2O，直至最终体积为 10 mL。过滤灭菌并储存在4°C。</li>
	<li>PB缓冲液（补充有1%BSA的PBS）：缓慢加入5gBSA至400 mL超纯H2O和50 mL PBS中?...

<ol>
	<li>Karlsson, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+PDZbody,+a+bivalent+binder+of+the+E6+protein+from+human+papillomavirus.">Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.</a> Scientific Reports. 5 (1), 9382 (2015).</li><li>Veggiani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+SH2+domains+with+tailored+specificities+and+enhanced+affinities+for+phosphoproteome+analysis.">Engineered SH2 domains with tailored specificities and enhanced affinities for phosphoproteome analysis.</a> Protein Science. 28 (2), 403-413 (2019).</li><li>Kaneko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superbinder+SH2+domains+act+as+antagonists+of+cell+signaling.">Superbinder SH2 domains act as antagonists of cell signaling.</a> Science Signaling. 5 (243), (2012).</li><li>Wiechmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+inhibition+of+the+small+ubiquitin-like+modifier+(SUMO)-conjugating+enzyme+Ubc9+selectively+impairs+SUMO+chain+formation.">Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation.</a> Journal of Biological Chemistry. 292 (37), 15340-15351 (2017).</li><li>Ernst, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strategy+for+modulation+of+enzymes+in+the+ubiquitin+system.">A strategy for modulation of enzymes in the ubiquitin system.</a> Science. 339 (6119), 590-595 (2013).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+selective+inhibition+of+pathogenic+viruses+by+engineered+ubiquitin+variants.">Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants.</a> PLOS Pathogens. 13 (5), 1006372 (2017).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-wide+modulation+of+HECT+E3+ligases+with+selective+ubiquitin+variant+probes.">System-wide modulation of HECT E3 ligases with selective ubiquitin variant probes.</a> Molecular Cell. 62 (1), 121-136 (2016).</li><li>Gabrielsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+strategy+for+discovery+of+inhibitors+and+activators+of+RING+and+U-box+E3+ligases+with+ubiquitin+variants.">A general strategy for discovery of inhibitors and activators of RING and U-box E3 ligases with ubiquitin variants.</a> Molecular Cell. 68 (2), 456-470 (2017).</li><li>Brown, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RING+E3+architectures+regulate+multiubiquitination+and+ubiquitin+chain+elongation+by+APC/C.">Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C.</a> Cell. 165 (6), 1440-1453 (2016).</li><li>Gorelik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SCF+ubiquitin+ligases+by+engineered+ubiquitin+variants+that+target+the+Cul1+binding+site+on+the+Skp1-F-box+interface.">Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.</a> Proceedings of the National Academy of Sciences. 113 (13), 3527-3532 (2016).</li><li>Goldring, J. P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quantification+methods+to+determine+protein+concentration+prior+to+electrophoresis.">Protein quantification methods to determine protein concentration prior to electrophoresis.</a> Protein Electrophoresis. 869, 29-35 (2012).</li><li>Zhang, W., Sidhu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+intracellular+modulators+of+E3+ligases+and+deubiquitinases+from+phage-displayed+ubiquitin+variant+libraries.">Generating intracellular modulators of E3 ligases and deubiquitinases from phage-displayed ubiquitin variant libraries.</a> The Ubiquitin Proteasome System: Methods and Protocols. 1844, 101-119 (2018).</li><li>Sheehan, J., Marasco, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+and+yeast+display.">Phage and yeast display.</a> Microbiology Spectrum. 3 (1), 1-17 (2015).</li><li>Lowman, H. B., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monovalent+phage+display:+A+method+for+selecting+variant+proteins+from+random+libraries.">Monovalent phage display: A method for selecting variant proteins from random libraries.</a> Methods. 3 (3), 205-216 (1991).</li><li>Lowman, H. B., Bass, S. H., Simpson, N., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+high-affinity+binding+proteins+by+monovalent+phage+display.">Selecting high-affinity binding proteins by monovalent phage display.</a> Biochemistry. 30 (45), 1-7 (1991).</li><li>Beaber, J. W., Tam, E. M., Lao, L. S., Rondon, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+helper+phage+for+improved+monovalent+display+of+Fab+molecules.">A new helper phage for improved monovalent display of Fab molecules.</a> Journal of Immunological Methods. 376 (1-2), 46-54 (2012).</li><li>Gal&#225;n, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library-based+display+technologies:+where+do+we+stand.">Library-based display technologies: where do we stand.</a> Molecular BioSystems. 12 (8), 2342-2358 (2016).</li><li>Huang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+display+and+selection+of+single-chain+variable+fragments+effectively+inhibit+growth+and+progression+of+microspheres+in+vitro+and+in+vivo.">Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo.</a> Cancer Science. 109 (5), 1503-1512 (2018).</li><li>Yamaguchi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cDNA+display:+a+novel+screening+method+for+functional+disulfide-rich+peptides+by+solid-phase+synthesis+and+stabilization+of+mRNA-protein+fusions.">cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.</a> Nucleic Acids Research. 37 (16), 1-13 (2009).</li><li>Mochizuki, Y., Kumachi, S., Nishigaki, K., Nemoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+library+size+in+cDNA+display+by+optimizing+purification+procedures.">Increasing the library size in cDNA display by optimizing purification procedures.</a> Biological Procedures Online. 15 (1), 1-5 (2013).</li><li>Odegrip, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIS+display:+In+vitro+selection+of+peptides+from+libraries+of+protein-DNA+complexes.">CIS display: In vitro selection of peptides from libraries of protein-DNA complexes.</a> Proceedings of the National Academy of Sciences. 101 (9), 2806-2810 (2004).</li><li>Reiersen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+antibody+display--an+in+vitro+antibody-DNA+library+selection+system.">Covalent antibody display--an in vitro antibody-DNA library selection system.</a> Nucleic Acids Research. 33 (1), 1-9 (2005).</li><li>Houlihan, G., Gatti-Lafranconi, P., Lowe, D., Hollfelder, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+anti-HER2+DARPins+by+SNAP+display+reveals+stability/function+trade-offs+in+the+selection+process.">Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.</a> Protein Engineering Design and Selection. 28 (9), 269-279 (2015).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+validation+of+intracellular+ubiquitin+variant+inhibitors+for+USP7+and+USP10.">Generation and validation of intracellular ubiquitin variant inhibitors for USP7 and USP10.</a> Journal of Molecular Biology. 429 (22), 3546-3560 (2017).</li><li>Teyra, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+ubiquitin+variant+inhibitors+of+USP15.">Structural and functional characterization of ubiquitin variant inhibitors of USP15.</a> Structure. 27 (4), 590-605 (2019).</li></ol>

如步骤2.1（蛋白质制备）中所述，可以使用多种方法来评估蛋白质浓度，并且每种方法都将基于用于噬菌体显示的特定靶蛋白具有独特的优缺点。以前已经提供了流行方法的详细描述和协议的来源11。
使用上一轮噬菌体展示保留的噬菌体作为后续一轮的输入，通过逐渐去除结合微弱、瞬时或偶然结合的粘结剂来丰富良好的粘结剂。到第四轮和第五轮，理想情...

了解蛋白质 - 蛋白质相互作用的分子细节对于描绘生物过程的信号转导机制至关重要，特别是那些导致临床重要疾病的机制。近年来，噬菌体展示已被用作分离蛋白质/肽的实用且易于使用的方法，其与所需目标蛋白的结合得到了很大的改善1，2，3，4，而靶蛋白相互作用又可用作蛋白 - 蛋白相互作用的细胞内探针。
泛素化是酶促活性（E1激活酶→E2偶联酶→E3连接酶）的级联反应，其共价偶联泛素（Ub）到蛋白质底物以靶向它们进行降解或介导细胞信号传导变化。此外，去泛素酶催化从蛋白质中去除泛素。因此，在细胞中，有成千上万的Ub依赖性蛋白质 - 蛋白质相互作用，其中绝大多数识别具有低亲和力但高特异性的共同表面，以允许通过大而多样化的表面进行弱相互作用。
Ernst等人将突变引入Ub的已知结合区域，以查看它们是否可以增强对目标蛋白质的结合亲和力，同时仍保持高选择性5。开发了一个超过100亿（7.5 x 1010）Ub变体（UbVs）的组合库，这些变体在Ub表面的位置发生了突变，介导了已知的Ub蛋白相互作用。该文库由表达融合到多样化UbV的M13噬菌体pIII涂层蛋白的噬菌体组成。因此，单个UbV可以在表达时通过包衣蛋白显示在噬菌体表面上。在选择过程中，显示与目标蛋白具有显着结合相互作用的UbV的噬菌体将在随后的噬菌体显示轮中被保留和富集，而显示与靶蛋白结合不良的UbV的噬菌体将被冲走并从噬菌体池中去除。保留的噬菌体颗粒含有与其显示的UbV相对应的噬菌体粒，允许它们在分离后进行测序和进一步表征。
利用这种蛋白质工程策略，开发了用于人类去泛素酶5和病毒蛋白酶的UbV抑制剂6。重要的是，我们通过劫持E2结合位点并激活占据HECT结构域上Ub结合外源的UbVs，为人类HECT家族E3连接酶生成了抑制性UbV7。我们还可以通过靶向E2结合位点来抑制单体环家族E3s，并诱导UbV二聚化以激活同源二聚体RING E3s8。对于多亚基RING E3s，UbVs可以通过靶向RING亚基（例如，对于APC / C复合物9）或破坏复合物形成（例如，对于SCF E3s10）来实现抑制。总的来说，可以利用UbV来系统地询问Ub蛋白酶体系统（UPS）中的蛋白质 - 蛋白质相互作用，以便我们可以更好地破译UPS酶的生化机制，并识别和验证治疗干预的功能位点。
以下方案描述了如何利用先前生成的Phage显示的UbV文库来靶向感兴趣的蛋白质，以及如何通过连续几轮噬菌体显示来富集与目标蛋白质相互作用的UbV结合剂。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axygen Mini Tube System (0.65 mL, sterile, 96/Rack, 10 Racks/pack)</td>
			<td>Fisher Scientific</td>
			<td>14-222-198</td>
			<td>Culturing phage outputs after phage display.</td>
		</tr>
		<tr>
			<td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)</td>
			<td>Fisher Scientific</td>
			<td>DF0446-17-3</td>
			<td>Preparing plates for titering.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Fraction V</td>
			<td>BioShop Canada</td>
			<td>ALB001</td>
			<td>Buffer component.</td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt 89.0-100.5% anhydrous</td>
			<td>Millipore-Sigma</td>
			<td>C1389-5G</td>
			<td>Culturing phagemid-infected cells.</td>
		</tr>
		<tr>
			<td>Compact Digital Microplate Shaker</td>
			<td>Fisher Scientific</td>
			<td>11-676-337</td>
			<td>Shaking plates during incubation with the phage library.</td>
		</tr>
		<tr>
			<td>Corning Microplate Aluminum Sealing Tape</td>
			<td>Fisher Scientific</td>
			<td>07-200-684</td>
			<td>Sealing phage glycerol stocks.</td>
		</tr>
		<tr>
			<td>Dehydrated Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140-01-0</td>
			<td>Preparing plates for titering.</td>
		</tr>
		<tr>
			<td>DS-11 Spectrophotometer/Fluorometer</td>
			<td>DeNovix</td>
			<td>DS-11 FX+</td>
			<td>Protein concentration measurement.</td>
		</tr>
		<tr>
			<td>Greiner Bio-One CellStar 96-Well, Non-Treated, U-Shaped-Bottom Microplate</td>
			<td>Fisher Scientific</td>
			<td>7000133</td>
			<td>Storing phage glycerol stocks.</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Phage elution.</td>
		</tr>
		<tr>
			<td>Invitrogen One Shot OmniMAX 2 T1R Chemically Competent E. coli</td>
			<td>Fisher Scientific</td>
			<td>C854003</td>
			<td>Bacterial strain for phage infection.</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>AAJ1792406</td>
			<td>Culturing M13K07 helper phage-infected cells.</td>
		</tr>
		<tr>
			<td>M13KO7 Helper Phage</td>
			<td>New England Biolabs</td>
			<td>&#160;N0315S</td>
			<td>Permit phagemid packing and secretion.</td>
		</tr>
		<tr>
			<td>MaxQ 4000 Benchtop Orbital Shaker</td>
			<td>Fisher Scientific</td>
			<td>11-676-076</td>
			<td>Bacterial cell culture.</td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp 96 well microplate, flat bottom</td>
			<td>Life Technologies</td>
			<td>44-2404-21</td>
			<td>Immobilizing proteins.</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 10X Solution</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>Buffer component/phage resuspension medium.</td>
		</tr>
		<tr>
			<td>Polyester Films for ELISA and Incubation</td>
			<td>VWR</td>
			<td>60941-120</td>
			<td>Covering the microplates during incubation.</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 8000 (PEG)</td>
			<td>Fisher Scientific</td>
			<td>BP233-1</td>
			<td>Phage precipitation.</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore-Sigma</td>
			<td>S3014</td>
			<td>Phage precipitation.</td>
		</tr>
		<tr>
			<td>Sterile Plastic Culture Tubes: Translucent Polypropylene</td>
			<td>Fisher Scientific</td>
			<td>14-956-1D</td>
			<td>Culturing phage inputs.</td>
		</tr>
		<tr>
			<td>Tetracycline Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP912-100</td>
			<td>Culturing E. coli OmniMax cells.</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP1525</td>
			<td>Neutralizing eluted phage solution.</td>
		</tr>
		<tr>
			<td>Tryptone Powder</td>
			<td>Fisher Scientific</td>
			<td>BP1421-2</td>
			<td>Cell growth media component.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337500</td>
			<td>Buffer component.</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td>Cell growth media component.</td>
		</tr>
	</tbody>
</table>

using phage display to develop ubiquitin variant modulators for e3 ligases

泛素是一种8.6 kDa的小蛋白，是泛素蛋白酶体系统的核心成分。因此，它可以与具有高特异性但低亲和力的各种蛋白质结合。通过噬菌体展示，可以设计泛素变体（UbV），使它们表现出比野生型泛素更好的亲和力，并保持与靶蛋白的结合特异性。噬菌体展示利用噬菌体文库，其中丝状M13噬菌体的pIII包衣蛋白（选择它是因为它显示在噬菌体表面上）与UbV融合。人类野生型泛素的特定残基是软的和随机的（即，偏向于天然野生型序列）以产生UbVs，从而避免蛋白质构象的有害变化，同时引入促进与靶蛋白的新相互作用所需的多样性。在噬菌体展示过程中，这些UbV在噬菌体涂层蛋白上表达和显示，并针对感兴趣的蛋白质进行平移。与靶蛋白表现出有利结合相互作用的UbV被保留，而较差的结合剂被冲走并从文库池中除去。保留的UbV附着在含有UbV相应噬菌体颗粒上的UbV被洗脱，扩增和浓缩，以便它们可以在另一轮噬菌体展示中与相同的靶蛋白平移。通常，最多进行五轮噬菌体展示，在此期间，对弱结合和/或混杂结合的UbV施加强大的选择压力，以便集中和富集具有较高亲和力的UbV。最终，分离出比野生型对应物对靶蛋白具有更高特异性和/或亲和力的UbV，并且可以通过进一步的实验进行表征。

噬菌体展示产生的粘合剂可以通过多种方式进行验证和分析。建议首先使用引物对噬菌体进行测序，引物位于噬菌体文库中多样化插入片段的两侧。理想的噬菌体展示实验将显示对多个序列的明显偏倚（图1）。其他序列也将存在，但计数较低，更多地显示为背景噪声。在提供的示例中，在泛素变体（UbV）和野生型UBE4B之间进行噬菌体显示时，存在对序列#1-4的特别偏倚。提供...

Watch this Scientific Journal Video about Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases at JoVE.com

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

泛素化是翻译后修饰的关键蛋白质，其失调与许多人类疾病有关。该协议详细介绍了如何利用噬菌体显示来分离新的泛素变体，这些变体可以结合和调节控制泛素特异性，效率和模式的E3连接酶的活性。

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 ...

噬菌体展示不仅有助于我们研究临床上重要酶的结合特性，而且还开发出这些酶和相应疾病的调节剂。噬菌体显示可用于开发各种靶蛋白的新型粘合剂，并且比其他常规显示方法更容易执行。这个过程涉及很多重复的动作，所以关键是不要自动驾驶，并始终保持专注。首先接种30毫升2YT四环素肉汤和200微升种子培养物。在37摄氏度下孵育约三个小时，200 RPM的轨道振荡，直到细菌处于对数中期。将涂层溶液从板中丢弃到水槽中。用纸巾轻轻擦干。然后向每个包被孔中加入200微升PB缓冲液。从盘子中丢弃PB缓冲液，并在纸巾上干燥。向板的每个包被孔中再加入200微升PB缓冲液。在室温下孵育一小时，300 RPM的轨道振荡。在冰上解冻噬菌体库，然后将其稀释至PBS中文库多样性的100倍。以稀释的文库体积的五分之一加入聚乙二醇氯化钠溶液，并将溶液在冰上孵育30分钟。接下来，将溶液以11， 000倍G在4摄氏度下离心30分钟。弃去上清液并再离心两分钟以拉下剩余的上清液并浓缩噬菌体沉淀。将噬菌体沉淀轻轻重悬于一毫升PBT缓冲液中，每个要分析的目标蛋白质，通常总共四个。向对照板中的每个包衣孔中加入100微升噬菌体文库。在室温下孵育一小时，300 RPM的轨道振荡。将目标板中的PB缓冲液丢弃到水槽中，并在纸巾上干燥板。将对照板中噬菌体文库的所有100微升转移到目标板的每个包衣孔中。在室温下孵育一小时，300 RPM的轨道振荡。接下来，从板中取出噬菌体文库，并用PT缓冲液洗涤包被的孔四次。倒置盘子并点击纸巾以除去最后一滴缓冲液。向每个包被孔中加入100微升0.1摩尔盐酸以洗脱噬菌体。将板在室温下孵育五分钟，300 RPM轨道振荡。之后，通过向每个包被的孔中加入12.5微升pH 11摩尔三盐酸盐来中和pH值。将所有八个孔中洗脱的噬菌体转移到单个1.5毫升微量离心管中。在转移过程中上下移液，使溶液均匀，并从孔中吸出所有液体。向洗脱的噬菌体中加入10%BSA，使最终浓度为1%，将微量离心管储存在4摄氏度。这是第一轮输出。准备种子培养物，通过用从琼脂平板中分离的大肠杆菌菌落接种5毫升2YT四环素种子培养物，用于培养下一轮选择的噬菌体投入物。在37摄氏度下孵育过夜，200 RPM轨道振荡。用适量的PBS稀释第二轮和第三管中的目标蛋白。取第二轮和第三轮试管内容物的一半，并在96孔结合板中为每个目标蛋白质涂覆四孔，以备下一轮使用。接下来，使用第一轮输出的一半接种三毫升的中间对数相细胞。在37摄氏度下孵育30分钟，200 RPM轨道振荡。将M13KO7辅助噬菌体加入每毫升100亿PFU的终浓度。在37摄氏度下再次孵育一小时，200 RPM轨道振荡。一小时后，将整个三毫升培养物转移到30毫升2YT羧苄青霉素卡那霉素溶液中。在37摄氏度下生长过夜，200 RPM轨道振荡。这里显示了针对UBE4B的泛素变体选择的代表性结果。UBV按从最高到最低频率排序。序列代表UBV中具有所有随机残基的多样化泛素区域。结合剂与野生型靶蛋白，突变靶蛋白以及脱靶蛋白的相对结合亲和力通过酶联免疫吸附测定或ELISA测量。所有ELISA吸光度均根据96个平均BSA ELISA评分和96个平均GST ELISA评分进行标准化。深绿色表示较强的相对结合。GST作为非特异性结合的对照包括在内，因为目标蛋白被GST标记。ELISA结果以图形方式显示在此处。x 轴表示 UBV，y 轴表示相应的归一化吸光度。在尝试此过程时，重要的是在添加盐酸后不要将溶液丢弃在板中。噬菌体现在悬浮在溶液中，您希望保留这些噬菌体，以便您可以执行后续的富集最终分析或两者兼而有之。按照此过程，应进行测序以确定UBV频率。ELISA和IC50测定可以分别确定结合功效和抑制功效，也有助于确定进一步表征哪些UBV。

Research

JoVE Journal

Biochemistry

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

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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

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. ...

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

用酮抑制剂 PD0325901 和维生素 C 维持小鼠胚胎干细胞基因组低甲基化的一种替代培养方法

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

RING-E3型无二体气体在Vitro和Planta中的功能特性

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...