This work was supported by the University of Washington Innovation Award (to L.G.), a grant from the U.S. National Institutes of Health (1R35GM128918 to L.G.), and a startup fund of the University of Washington (to L.G.). H.J. was supported by a Washington Research Foundation undergraduate fellowship. K.W. was supported by an undergraduate fellowship from the University of Washington Institute for Protein Design.

1. Library construction
<ol>
	<li>Use a synthetic combinatorial single-domain antibody library with a diversity of ~1.23&#8211;7.14 x 109, as previously described19. While this protocol does not include library construction, it can be applied to other combinatorial binder libraries.</li>
</ol>
2. Biotinylation of ligand target or ligand
<ol>
	<li>Biotinylate the selected ligand, for example, CBD and tetrahydrocannabinol (THC)19, via va...

<ol>
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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. 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It is critical to choose the correct concentrations of input phage libraries for different rounds of biopanning. We typically started from an input library of ~1012&#8211;1013 phage particles with a diversity &#62;109, allowing ~100&#8211;1,000 copies of each phage clone to be presented in the pull-down assay. If the phage concentration in a binding assay is too high or low, the likelihood of nonspecific binding or loss of positive clones will increase. The anchor or dimerization binder s...

CID systems, in which two proteins dimerize only in the presence of a small-molecule ligand (Figure 1), offer versatile tools for dissecting and manipulating metabolic, signaling, and other biological pathways1. They have demonstrated the potential in biological actuation, such as drug-controlled T cell activation2 and apoptosis3,4, for improving the safety and efficacy of adoptive T cell therapy. Additionally, they provide a new methodology for in vivo or in vitro detection of small-molecule targets. For example, CID proteins can be genetically fused with fluorescence reporter systems (e.g., fluorescence resonance energy transfer (FRET)5 and circularly permuted fluorescent proteins)6 for real-time in vivo measurements, or serve as affinity reagents for sandwich enzyme-linked immunosorbent assay (ELISA)-like assays.
Despite their wide applications, creating new CID systems that can be controlled by a given small-molecule ligand has major challenges. Established protein binder engineering methods including animal immunization7, in vitro selection8,9, and computational protein design10 can generate ligand binding proteins that function via binary protein-ligand interactions. However, these methods have difficulties creating a ligand-induced ternary CID complex. Some methods create CID by chemically linking two ligands that independently bind to the same or different proteins11,12,13,14,15,16 or rely on selecting binder proteins such as antibodies targeting preexisting small molecule-protein complexes17,18, and thus have a limited choice of ligands.
We recently developed a combinatorial binders-enabled selection of CID (COMBINES-CID) method for de novo engineering of CID systems19. This method can obtain the high specificity of ligand-induced dimerization (e.g., an anchor-dimerization binder dissociation constant, KD (without ligand)/KD (with ligand) &#62; 1,000). The dimerization specificity is achieved using anchor binders with flexible binding sites that can introduce conformational changes upon ligand binding, providing a basis for the selection of conformationally selective binders only recognizing ligand-bound anchor binders. We demonstrated a proof-of-principle by creating cannabidiol (CBD)-induced heterodimers of nanobodies, a 12&#8211;15 kDa functional antibody fragment from camelid comprising a universal scaffold and three flexible CDR loops (Figure 2)20, which can form a binding pocket with adaptable sizes for small-molecule epitopes21,22. Notably, the in vitro selection of a combinatorial protein library should be cost-effective and generalizable for CID engineering because the same high-quality library can be applied to different ligands.
In this protocol and video, we focus on describing the two-step in vitro selection and validation of anchor (Figure 3A) and dimerization binders (Figure 3B) by screening the combinatorial nanobody library with a diversity higher than 109 using CBD as a target, but the protocol should be applicable to other protein libraries or small-molecule targets. The screening of CID binders usually takes 6&#8211;10 weeks (Figure 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

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.

We describe the two-step in vitro selection and validation of anchor and dimerization binders by screening the combinatorial nanobody library with a diversity higher than 109 using CBD as a target. Assessing the enrichment of the phage biopanning during the successive rounds of selection for both anchor and dimerization binders is important. Typical enrichment results after 4&#8211;6 rounds of selection as shown in Figure 5 are a good indication that there is a high ratio of poten...

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

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.

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

This protocol is significant because it provides a general approach to engineering protein dimerization systems as biosensors for a variety of small molecules. In addition, it uses one highly diverse protein library to select biosensors for different ligands in an efficient and cost-effective manner without the need for specialized equipment. The engineered biosensor can be used to chemically control cell behavior and to monitor real-time changes in cell metabolites.Through this visual demonstration, researchers can observe a detailed instruction of the techniques with a clear expectation of the experimental output. Begin every round of selection by growing a single TG1 electroporation-competent cell colony in six milliliters of 2YT medium at 37 degrees Celsius and 250 revolutions per minute to a 600-nanometer absorbance of approximately 0.5. When the optimal optical density has been reached, place the cells on ice.To prepare the negative selection beads, wash 300 microliters of streptavidin-coated magnetic beads three times with one milliliter of 05%PBS plus Tween buffer and two times with one milliliter of PBS per wash on a magnetic separation rack. Resuspend the beads with one milliliter of 1%casein in PBS, and saturate the beads with biotin at five times the reported binding capacity of the beads. After one hour at room temperature with rotation, wash the beads five times with PBS plus Tween and three times with PBS as demonstrated.After the last wash, add approximately one times 10 to the 13 phage particles in 1%casein and 1%bovine serum albumin in PBS to the beads for a one-hour incubation at room temperature with rotation. At the end of the incubation, transfer the supernatant into a 1.5-milliliter tube. For positive selection with the biotinylated ligand-bound streptavidin beads, saturate half of the volume of beads used for the negative selection in the biotinylated ligand of choice at five times the full binding capacity as calculated according to the manual.After a one-hour incubation at room temperature with rotation, wash the beads five times with PBS plus Tween and three times with PBS alone as demonstrated, and block the beads with one milliliter of 1%casein and 1%bovine serum albumin for one hour with rotation. At the end of the incubation, wash the streptavidin-coated magnetic beads three times in PBS plus Tween and one time in PBS alone as demonstrated, and use unbound phage to resuspend the streptavidin-coated magnetic beads. Extract the unbound phage-containing supernatant without disturbing the beads, and set the sample aside to be used as input.Then wash the beads 10 times with PBS plus Tween and five times with PBS as demonstrated, transferring the beads to a new tube after every three washes to avoid nonspecific binding of the bound phages to the tube walls. To competitively elute the bound phages, add 450 microliters of a micromolar concentration of non-biotinylated ligand to the positive selection beads, and place the beads on rotation for 30 minutes. At the end of the incubation, transfer the supernatant into a new tube to be used as output.For input titration, prepare 10 serial dilutions in PBS up to one times 10 to the ninefold with the input phage. Transfer 10 microliters of input phage from the one times 10 to the seven to 10 to the nine dilutions to 70-microliter aliquots of the prepared TG1 cells. After 45 minutes at 37 degrees Celsius, plate the infected TG1 cells onto individual 90-millimeter 2YT agar dishes containing 100 micrograms per milliliter of ampicillin and 2%glucose for an overnight incubation at 37 degrees Celsius.The next morning, use the formula to calculate the phage input. For output infection and titration, add the eluted phages to three milliliters of TG1 cells for a 45-minute incubation in a 37-degree Celsius water bath. Then prepare 10 serial dilutions of the infected cells in fresh 2YT medium up to a one times 10 to the third concentration, and plate each dilution on 90-millimeter 2YT agar dishes for their overnight incubation at 37 degrees Celsius.The next morning, calculate the phage output according to the formula. To isolate individual clones from an enriched sublibrary, transfer single colonies from the overnight-cultured, phage-infected TG1 cell plates into 250 microliters of 2YT medium supplemented with ampicillin per well in sterile deep-well plates for their overnight culture at 37 degrees Celsius. When the cells reach a 600-nanometer density of approximately 0.5, add five times 10 to the nine plaque-forming units per milliliter of CM13 helper phage to each well, and incubate the cells for 45 minutes at 37 degrees Celsius at 250 rotations per minute.At the end of the incubation, add 500 microliters of 2YT medium supplemented with ampicillin and 50 micrograms per milliliter of kanamycin to each well, and place the plate at 25 degrees Celsius and 250 rotations per minute overnight. The next morning, sediment the cells by centrifugation to allow collection of the phage particles. To validate the anchor binding by ELISA, coat 96-well ELISA plates with 100 microliters of five micrograms per milliliter streptavidin in coating buffer at four degrees Celsius overnight.The next morning, wash the plates three times in 300 microliters of PBS plus Tween before adding 100 microliters of one-micromolar biotinylated target to the target wells and 100 microliters of one-micromolar biotin or target homolog to the appropriate control wells. After one hour at room temperature, wash the plates five times with PBS plus Tween per wash, and block any nonspecific binding with 300 microliters of 1%casein per well for one hour at room temperature. At the end of the incubation, wash the plates three times in fresh PBS plus Tween per wash, and add the purified phage supernatant.After one hour at room temperature, wash the plates 10 times with fresh PBS plus Tween per wash. Add 100 microliters of horseradish peroxidase M13 major coat protein antibody to each well for a one-hour incubation at room temperature. Then wash the plates three times as demonstrated, and add 100 microliters of TMB substrate to each well for a 10-minute incubation at room temperature or until a visible color change is observed.Then stop the reaction with 100 microliters of one-molar hydrochloric acid per well, and read the plate at 450 nanometers on a spectrophotometer. After anchor binder validation, dimerization binder screening should also be performed using the same protein library targeting the anchor binder-ligand complex. Typical enrichment results after four to six rounds of selection are a good indication that there is a high ratio of potential hits in the sublibraries, in which case further rounds of selection may not be necessary.Single-clone ELISA is suitable for analyzing the relative binding affinity and selectivity of anchor and dimerization binders and facilitates the comparison of binding selectivity between clones. Likewise, dimerization binder selection allows the identification of clones that form a heterodimer with the immobilized anchor binder only with or without the ligand. These ligand concentration-dependent binding data suggest that the tested nanobodies are suitable for constructing a chemically induced dimerization system compared to the poor binding demonstrated by the control samples.Further, analytical size-exclusion chromatography can be used to confirm the heterodimer formation between anchor and dimerization binders in the presence of the ligand. For example, in this experiment, a dimerization peak was observed when the anchor and dimerization binders and cannabidiol were mixed. In contrast, no dimerization peak was detected in the absence of cannabidiol or when each binder was loaded to the column alone.The protein dimerization system should be genetically encoded in yeast or mammalian cells to allow further verification of the selected biosensors for developing in vivo applications. We expect that this method will give other researchers the effective and necessary tools for detecting important metabolites, signaling molecules, or drugs in vivo with a high spatiotemporal resolution.

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JoVE Journal

Biochemistry

7.6K visualizações.  University of Washington. Este protocolo é significativo porque fornece uma abordagem geral para sistemas de dimerização de proteínas de engenharia como biosensores para uma variedade de moléculas pequenas. Além disso, utiliza uma biblioteca de proteínas altamente diversificada para selecionar biosensores para diferentes ligantes de forma eficiente e econômica sem a necessidade de equipamentos especializados. O biosensor projetado pode ser usado para controlar quimicamente o comportamento celular e monitorar m...

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