Name: creating highly specific chemically induced protein dimerization systems by stepwise phage selection of a combinatorial single-domain antibody library
Uploaded: 2020-01-14T16:00:00
Description: Watch this Scientific Journal Video about Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library at JoVE

这项工作得到了华盛顿大学创新奖（L.G.）的资助，美国国家卫生研究院（1R35GM128918到L.G.）的资助，以及华盛顿大学（L.G.）的启动基金。H.J.得到了华盛顿研究基金会本科生奖学金的支持。K.W.得到了华盛顿大学蛋白质设计研究所的本科奖学金的支持。

1. 图书馆建设
<ol><li>使用合成组合单域抗体库，其多样性为±1.23~7.14 x 109，如前所述19。虽然此协议不包括库构造，但可以应用于其他组合绑定器库。</li>
</ol>2. 配体靶或配体的生物异化
<ol><li>生物素化选定的配体，例如，CBD和四氢大麻酚（THC）19，通过各种化学合成策略，取决于目标的适当生物素化位点。</li>
</ol>3....

<ol>
	<li>Stanton, B. Z., Chory, E. J., Crabtree, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+induced+proximity+in+biology+and+medicine.">Chemically induced proximity in biology and medicine.</a> Science. 359 (6380), (2018).</li><li>Wu, C. Y., Roybal, K. T., Puchner, E. M., Onuffer, J., Lim, 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=Remote+control+of+therapeutic+T+cells+through+a+small+molecule-gated+chimeric+receptor.">Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.</a> Science. 350 (6258), (2015).</li><li>Straathof, K. C., 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=An+inducible+caspase+9+safety+switch+for+T-cell+therapy.">An inducible caspase 9 safety switch for T-cell therapy.</a> Blood. 105 (11), 4247-4254 (2005).</li><li>Di Stasi, 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=Inducible+apoptosis+as+a+safety+switch+for+adoptive+cell+therapy.">Inducible apoptosis as a safety switch for adoptive cell therapy.</a> The New England Journal of Medicine. 365 (18), 1673-1683 (2011).</li><li>Mank, 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+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophysical Journal. 90 (5), 1790-1796 (2006).</li><li>Nagai, T., Sawano, A., Park, E. S., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circularly+permuted+green+fluorescent+proteins+engineered+to+sense+Ca2+.">Circularly permuted green fluorescent proteins engineered to sense Ca2+.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (6), 3197-3202 (2001).</li><li>Hunter, M. M., Margolies, M. N., Ju, A., Haber, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+monoclonal+antibodies+to+the+cardiac+glycoside,+digoxin.">High-affinity monoclonal antibodies to the cardiac glycoside, digoxin.</a> Journal of Immunology. 129 (3), 1165-1172 (1982).</li><li>Bradbury, A. R. M., Sidhu, S., Dubel, S., McCafferty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+natural+antibodies:+the+power+of+in+vitro+display+technologies.">Beyond natural antibodies: the power of in vitro display technologies.</a> Nature Biotechnology. 29 (3), 245-254 (2011).</li><li>Chen, 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=Isolation+of+high-affinity+ligand-binding+proteins+by+periplasmic+expression+with+cytometric+screening+(PECS).">Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS).</a> Nature. Biotechnology. 19 (6), 537-542 (2001).</li><li>Tinberg, C. E., 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=Computational+design+of+ligand-binding+proteins+with+high+affinity+and+selectivity.">Computational design of ligand-binding proteins with high affinity and selectivity.</a> Nature. 501 (7466), 212-216 (2013).</li><li>Spencer, D. M., Wandless, T. J., Schreiber, S. L., Crabtree, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+signal+transduction+with+synthetic+ligands.">Controlling signal transduction with synthetic ligands.</a> Science. 262 (5136), 1019-1024 (1993).</li><li>Ho, S. N., Biggar, S. R., Spencer, D. M., Schreiber, S. L., Crabtree, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimeric+ligands+define+a+role+for+transcriptional+activation+domains+in+reinitiation.">Dimeric ligands define a role for transcriptional activation domains in reinitiation.</a> Nature. 382 (6594), 822-826 (1996).</li><li>Belshaw, P. J., Ho, S. N., Crabtree, G. R., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+protein+association+and+subcellular+localization+with+a+synthetic+ligand+that+induces+heterodimerization+of+proteins.">Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (10), 4604-4607 (1996).</li><li>Farrar, M. A., AlberolaIla, J., Perlmutter, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Raf-1+kinase+cascade+by+coumermycin-induced+dimerization.">Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization.</a> Nature. 383 (6596), 178-181 (1996).</li><li>Erhart, D., 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=Chemical+Development+of+Intracellular+Protein+Heterodimerizers.">Chemical Development of Intracellular Protein Heterodimerizers.</a> Chemistry &amp; Biology. 20 (4), 549-557 (2013).</li><li>Ballister, E. R., Aonbangkhen, C., Mayo, A. M., Lampson, M. A., Chenoweth, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+light-induced+protein+dimerization+in+living+cells+using+a+photocaged+dimerizer.">Localized light-induced protein dimerization in living cells using a photocaged dimerizer.</a> Nature Communications. 17 (5), 5475 (2014).</li><li>Hill, Z. B., Martinko, A. J., Nguyen, D. P., 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=Human+antibody-based+chemically+induced+dimerizers+for+cell+therapeutic+applications.">Human antibody-based chemically induced dimerizers for cell therapeutic applications.</a> Nature Chemical Biology. 14 (2), 112-117 (2018).</li><li>Foight, G. 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=Multi-input+chemical+control+of+protein+dimerization+for+programming+graded+cellular+responses.">Multi-input chemical control of protein dimerization for programming graded cellular responses.</a> Nature Biotechnology. 37 (10), 1209-1216 (2019).</li><li>Kang, 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=COMBINES-CID:+An+efficient+method+for+de+novo+engineering+of+highly+specific+chemically+induced+protein+dimerization+systems.">COMBINES-CID: An efficient method for de novo engineering of highly specific chemically induced protein dimerization systems.</a> Journal of the American Chemical Society. 141 (28), 10948-10952 (2019).</li><li>Muyldermans, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobodies:+natural+single-domain+antibodies.">Nanobodies: natural single-domain antibodies.</a> Annual Review of Biochemistry. 82, 775-797 (2013).</li><li>Fanning, S. W., Horn, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anti-hapten+camelid+antibody+reveals+a+cryptic+binding+site+with+significant+energetic+contributions+from+a+nonhypervariable+loop.">An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop.</a> Protein Science. 20 (7), 1196-1207 (2011).</li><li>Zavrtanik, U., Luken, J., Loris, R., Lah, J., Hadzi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+epitope+recognition+by+heavy-chain+camelid+antibodies.">Structural basis of epitope recognition by heavy-chain camelid antibodies.</a> Journal of Molecular Biology. 430 (21), 4369-4386 (2018).</li><li>Denhardt, D. T., Dressler, D., Ray, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Single-Stranded DNA Phages. , 605-625 (1978).</li><li>Virnekas, B., 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=Trinucleotide+phosphoramidites:+ideal+reagents+for+the+synthesis+of+mixed+oligonucleotides+for+random+mutagenesis.">Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.</a> Nucleic Acids Research. 22 (25), 5600-5607 (1994).</li><li>Gu, L., 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=Multiplex+single-molecule+interaction+profiling+of+DNA-barcoded+proteins.">Multiplex single-molecule interaction profiling of DNA-barcoded proteins.</a> Nature. 515 (7528), 554-557 (2014).</li></ol>

为不同轮的生物泛性选择正确浓度的输入噬菌体库至关重要。我们通常从 #1012#1013噬菌体颗粒的输入库开始，其多样性 >109，允许在下拉测定中呈现每个噬菌体克隆的 100~1，000 份拷贝。如果结合测定中的噬菌体浓度过高或过低，则非特异性结合或阳性克隆损失的可能性将增加。锚点或二聚体粘结剂选择通常包括三到六轮生物盘化，输出噬菌体计数通常从 +104开始，?...

CID系统，其中两种蛋白质仅在小分子配体存在的情况下进行分化（图1），为解剖和操纵代谢、信号和其他生物途径1提供了多功能的工具。他们已经证明了在生物驱动方面的潜力，如药物控制的T细胞活化2和凋亡3，4，以提高采用T细胞治疗的安全性和有效性。此外，它们为体内或体外小分子靶点检测提供了新方法。例如，CID 蛋白可与荧光报告系统（例如荧光共振能量转移 （FRET）5和循环渗透荧光蛋白6进行基因融合，用于实时体内测量，或用作三明治酶链接免疫吸附剂测定 （ELISA） 样的亲和力试剂。
尽管其应用广泛，但创建新的 CID 系统（可通过给定的小分子配体控制）面临重大挑战。已建立的蛋白质粘合剂工程方法包括动物免疫7、体外选择8、9和计算蛋白设计10，可以产生通过二元蛋白-配体相互作用起作用的配体结合蛋白。然而，这些方法很难创建配体引起的三元CID复合物。有些方法通过化学连接两个独立结合相同或不同蛋白质11、12、13、14、15、16的配体而创建CID，或者依靠选择粘合剂蛋白，如针对预先存在的小分子蛋白复合物17、18的抗体，因此配体选择有限。
我们最近开发了一种用于CID系统19的"新工程"的CID（联合体-CID）方法。 该方法可以获得配体诱导的二分化的高特异性（例如，锚点-二聚体解散常数，KD（不含配体）/KD（配体）>1，000）。二聚化特异性是使用具有柔性结合位点的锚点，可在配体结合时引入构象变化，为仅识别配体结合锚活页夹的构象选择性活页夹的选择提供基础。通过创建纳米体大麻二醇（CBD）诱导异体体，从camelid中形成12-15 kDa功能抗体片段，包括一个通用支架和三个灵活的CDR回路（图2）20，从而形成一个可适应小分子表位21、22的结合口袋，从而证明了一种原理证明。值得注意的是，组合蛋白库的体外选择对于CID工程来说应该是经济高效和可通用的，因为相同的高质量库可以应用于不同的配体。
在本协议和视频中，我们重点描述了锚（图3A）和二聚体粘合剂（图3B）的两步体外选择和验证，通过筛选多样性大于109的组合nanobody库，以CBD为目标，但该协议应适用于其他蛋白质库或小分子靶点。CID活页夹的筛选通常需要6~10周（图4）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Ultra TMB ELISA substrate solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>34029</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1423-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter unit (3 kDa cutoff)</td>
			<td>Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Kit II</td>
			<td>Bio-Rad</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>BirA biotin-protein ligase standard reaction kit</td>
			<td>Avidity</td>
			<td>BirA500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CM13 Helper phage</td>
			<td>Antibody Design Labs</td>
			<td>PH020L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose monohydrate</td>
			<td>Alfa Aesar</td>
			<td>A11090</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>11205D</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP120-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N3238S</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest BglI</td>
			<td>Thermo Fisher Scientific</td>
			<td>FD0074</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 200 pg</td>
			<td>GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep 26/10 Desalting Column</td>
			<td>GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr>
			<td>HisTrap-FF-1ml</td>
			<td>GE Healthcare</td>
			<td>11000458</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Alfa Aesar</td>
			<td>161-0718</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher Scientific</td>
			<td>34060</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>M13 Major Coat Protein Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53004</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 96-Well Polypropylene DeepWell Storage Plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>260251</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp</td>
			<td>Thermo Fisher Scientific</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Octet RED96</td>
			<td>ForteBio</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pADL-23c Phagemid Vector</td>
			<td>Antibody Design Labs</td>
			<td>PD0111</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-6000</td>
			<td>Sigma-Aldrich</td>
			<td>81260-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum SuperFi DNA Polymerase</td>
			<td>Invitrogen</td>
			<td>12351010</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink PCR Purification Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K310001</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin M13 Kit</td>
			<td>Qiagen</td>
			<td>27704</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Medium</td>
			<td>Lucigen</td>
			<td>80026-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax Plus 384</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Streptavidin (SSA) Biosensors</td>
			<td>ForteBio</td>
			<td>18-5057</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 75 increase 10/300 GL Column</td>
			<td>GE Healthcare</td>
			<td>28-9909-44</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Fisher Scientific</td>
			<td>15224-025</td>
			<td></td>
		</tr>
		<tr>
			<td>TG1 Electrocompetent Cells</td>
			<td>Lucigen</td>
			<td>60502-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>471283-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP9726-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Column</td>
			<td>Thermo Fisher Scientific</td>
			<td>89882</td>
			<td></td>
		</tr>
	</tbody>
</table>

creating highly specific chemically induced protein dimerization systems by stepwise phage selection of a combinatorial single-domain antibody library

仅在小分子配体存在的情况下发生的蛋白质二聚化事件使小分子生物传感器得以发展，用于解剖和操作生物途径。目前，只有有限数量的化学诱导的二聚体（CID）系统存在，并设计具有特定小分子配体的所需灵敏度和选择性的新系统仍然是蛋白质工程领域的一个挑战。我们在这里描述了一种高通量筛选方法，即一种适用于各种配体的CID系统的"新设计"，即CID（联合体）的选配型。此方法使用噬菌体显示的组合 nanobody 库的两步选择来获得 1） "锚活页夹"，首先绑定到感兴趣的配体，然后 2） 仅绑定到锚结物-配体复合物的"二聚体粘结剂"。为了选择锚固粘合剂，使用生物素化配体筛选出超过109个互补性决定区域（CDR）随机纳米体的组合库，并通过生物层干涉法（BLI）对未标记的配体进行验证。为了获得二分化活页夹，nanobody 库筛选了锚活页夹-配体复合物，作为正筛选的目标，未绑定的锚活页夹用于负筛选。ORS-CID广泛适用于选择具有其他免疫球蛋白、非免疫球蛋白或计算设计的支架的CID粘合剂，以创建用于体外和体内检测药物、代谢物、信号分子等的生物传感器。

我们以CBD为目标筛选多样性大于109的组合nanobody库，描述了锚点和二聚体活化活页夹的两步体选择和验证。在锚点和二聚体粘结剂的连续几轮选择中评估噬菌体生物盘的富集性非常重要。如图5所示，经过 4-6 轮选择后的典型扩充结果很好地表明子库中的潜在命中比率很高，因此可能不需要进一步选择。
单克隆ELISA适用于分析锚点和二聚化活页夹?...

Watch this Scientific Journal Video about Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library at JoVE

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

对于任何给定的小分子配体，创建具有所需亲和力和特异性的化学诱导蛋白二聚系统将具有许多生物传感和驱动应用。在这里，我们描述了一种有效的、可通用的方法，通过逐步选择噬菌体显示的组合单域抗体库来对化学诱导的二聚系统进行新工程。

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 ...

该协议意义重大，因为它为各种小分子的生物传感器提供了一种工程蛋白二化系统的通用方法。此外，它使用一个高度多样化的蛋白质库，以高效且经济高效的方式为不同配体选择生物传感器，而无需专用设备。工程生物传感器可用于化学控制细胞行为和监测细胞代谢物的实时变化。通过这种视觉演示，研究人员可以观察技术的详细指导，对实验输出有一个明确的期望。在每轮选择开始，在6毫升2YT介质中生长一个TG1电穿孔能力细胞群，温度为37摄氏度，每分钟250转，达到600纳米的吸收度，约为0.5。达到最佳光学密度后，将细胞放在冰上。要准备负选择珠子，用一毫升 05%PBS 加 Tween 缓冲液洗三次 300 微升的链球素涂层磁珠，在磁分离机架上每次洗涤一毫升 PBS 两次。在PBS中用1%的1%卡丁的1毫升重新暂停珠子，用生物素使珠子饱和，其含量是珠子所报告结合容量的五倍。在室温下旋转一小时后，用 PBS 加 Tween 清洗珠子五次，用 PBS 洗涤三次，用 PBS 清洗珠子三次。上次洗涤后，将 PBS 中的 13 个噬菌激素和 PBS 中的 1% 牛血清白蛋白加入约 10 倍，在室温下旋转孵育一小时。在孵育结束时，将上经液转移到1.5毫升的管中。对于使用生物基化配体结合链子的正选，在生物素结合配体选择中用于负选择的珠子体积的一半饱和，其含量是手册计算的全部结合容量的五倍。在室温下旋转一小时后，用PBS加Tween洗涤珠子五次，用PBS单独清洗三次，用1%的卡辛和1%牛血清白蛋白用1毫升的旋转阻断珠子一小时。在孵化结束时，在PBS加Tween中清洗三次，在PBS中清洗一次，在PBS中清洗一次，并使用未绑定的噬菌体重新悬浮过链球菌涂层的磁珠。在不干扰珠子的情况下提取不含绑定的噬菌玉，将样品放在一边用作输入。然后用PBS加Tween洗10次，用PBS洗5次，每洗三次后将珠子转移到新管子上，避免将结合的噬菌体与管壁进行非特异性结合。为了竞争地对结合的噬菌体进行旋转，在正选择珠子中加入450微升非生物基基配体的微摩尔浓度，并将珠子旋转30分钟。在孵育结束时，将上经剂转移到一个新的管中，用作输出。对于输入滴定，使用输入噬菌名在 PBS 中准备 10 个串行稀释，最多 10 倍至 9 倍。将10微升输入噬菌体从1倍10转移到7到10到9稀释到70微升的输入噬菌体。在37摄氏度下45分钟后，将受感染的TG1细胞盘上单个90毫米2YT的果子盘上，含有每毫升安他林100微克和2%葡萄糖，在37摄氏度下过夜孵育。第二天早上，使用公式计算噬菌未成年人输入。对于输出感染和滴定，将洗净的噬菌体添加到三毫升的 TG1 细胞中，在 37 摄氏度的水浴中孵育 45 分钟。然后，在新鲜2YT介质中对受感染的细胞进行10次连续稀释，浓度高达1倍10至3次，并在90毫米2YT的阿加盘上进行每盘稀释，在37摄氏度的温度下进行隔夜孵育。第二天早上，根据公式计算噬菌的输出。为了将单个克隆从富集的地下库中分离，将单菌落从隔夜培养的、受噬菌体感染的 TG1 细胞板转移到 250 微升的 2YT 介质中，每孔辅以安西林，在 37 摄氏度的无菌深井板中进行过夜培养。当细胞达到约0.5的600纳米密度时，每毫升CM13帮助器噬菌体的9个斑块形成单位中加入5倍10，并在37摄氏度下以每分钟250次旋转孵育细胞45分钟。在孵育结束时，将500微升2YT介质加用安西林和每毫升卡那霉素50微克添加到每井，并将板在25摄氏度和250次旋转，过夜。第二天早上，通过离心沉淀细胞，以便收集噬菌体颗粒。为了验证 ELISA 的锚点结合，在四摄氏度的涂层缓冲液中，隔夜在涂层缓冲液中涂上 100 微升，每毫升 5 微克。第二天早上，在300微升PBS加Tween中清洗板三次，然后向目标井中加入100微升的一微摩尔生物基化靶点，将100微升的一微摩尔生物素或目标同源物添加到适当的控制井中。在室温下一小时后，用PBS加Tween每次洗涤洗涤5次，并在室温下用300微升1%的卡丁进行阻断任何非特异性结合，在室温下洗涤一小时。在孵育结束时，用新鲜 PBS 和每次洗净 Tween 洗三次盘子，然后加入纯化的噬菌菌。在室温下一小时后，用新鲜的 PBS 加每次洗净的 Tween 清洗盘子 10 次。在每个井中加入100微升的马萝卜过氧化物酶M13主要涂层蛋白抗体，在室温下孵育一小时。然后如所证明的三次清洗板，并在每孔中加入100微升TMB基材，在室温下孵育10分钟，或直到观察到明显的颜色变化。然后停止每井100微升一摩尔盐酸的反应，并在分光光度计上读取450纳米的板。锚定粘结剂验证后，还应使用针对锚点粘结剂配体复合物的相同蛋白质库进行二丁化粘结剂筛选。四到六轮选择后的典型扩充结果很好地表明，在子库中潜在的命中率很高，在这种情况下，可能不需要进行进一轮选择。单克隆ELISA适用于分析锚点和二化粘结剂的相对结合亲和力和选择性，便于比较克隆之间的结合选择性。同样，二丁化活页夹的选择允许识别仅使用固定锚点粘结剂与配体形成异质剂的克隆。这些配体浓度依赖结合数据表明，与控制样品所证明的不良结合相比，经测试的纳米体适合构建化学诱导的二丁体系统。此外，分析大小排除色谱可用于确认锚和二丁体在配体存在的情况下的异质形成。例如，在此实验中，当锚点和二丁二醇与大麻二醇混合时，观察到了二丁二化峰值。相比之下，在没有大麻二醇或将每个活页夹单独加载到柱子上时，未检测到二丁二的二丁极。蛋白质二化系统应在酵母或哺乳动物细胞中进行基因编码，以便进一步验证选定的生物传感器，以开发体内应用。我们期望这种方法将给其他研究人员提供有效和必要的工具，以检测重要的代谢物，信号分子，或药物体内的高时空分辨率。

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

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Measuring Protein Binding to F-actin by Co-sedimentation

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

用 [NiFe] 氢对 H2氧化的研究表明, 蛋白质膜红外电化学

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

蛋白质抗原对羊的免疫和抗原特异性单域抗体的制备

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...