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 various chemical synthesis strategies, depending on the suitable biotinylation sites of a target.</li>
</ol>
3. Anchor binder screening
<ol>
	<li>Beginning of selection
	<ol>
		<li>Begin every round of selection by inoculating a single TG1-cell colony, freshly grown in 6 mL of 2YT at 37 &#730;C and 250 revolutions per minute (rpm) to a 600 nm (OD600) absorbance of ~0.5. Incubate the cells on ice for the use in step 3.5.1.</li>
	</ol>
	</li>
	<li>Negative selection with biotin-bound streptavidin beads
	<ol>
		<li>Prepare the &#34;negative selection beads&#34; by washing 300 &#181;L of streptavidin-coated magnetic beads using a magnetic separation rack, 3x with 0.05% phosphate-buffered saline with Tween buffer (PBST, 1 x PBS with 0.05% vol/vol Tween 20%) and 2x with 1 x PBS.</li>
		<li>Resuspend the beads with 1 mL of 1% casein in 1 x PBS (pH = 7.4), and saturate the beads by adding 5x the reported binding capacity using biotin. Incubate at room temperature (RT) on a rotator for 1 h.</li>
		<li>Wash the beads 5x using 0.05% PBST and 3x using 1 x PBS, for a total of eight washes.</li>
		<li>Add ~1013 phage particles in 1% casein/1% BSA in 1 x PBS (pH = 7.4) and incubate at RT on a rotator for 1 h.</li>
		<li>After incubation, collect the supernatant to be used in step 3.3.6.</li>
	</ol>
	</li>
	<li>Positive selection with biotinylated ligand-bound streptavidin beads
	<ol>
		<li>Prepare the &#34;positive selection beads&#34; using 1/2 the volume of the beads used for the &#34;negative selection beads&#34; following steps 3.2.1.</li>
		<li>Re-suspend the beads with 1 mL 1% casein in 1 &#215; PBS, pH 7.4 and saturate the beads by adding 5x the full binding capacity calculated based on the manual using the biotinylated ligand of choice. Incubate at RT on a rotator for 1 h.</li>
		<li>Wash the beads 5x using 0.05% PBST and 3x using 1 x PBS, for a total of eight washes.</li>
		<li>Block the beads with 1 mL of 1% casein/1% BSA in 1 x PBS (pH = 7.4) and incubate at RT on a rotator for 1 h to prevent nonspecific binding between the phages and the streptavidin-coated magnetic beads.</li>
		<li>Wash the streptavidin-coated magnetic beads 3x using 0.05% PBST and one time using 1 x PBS, for a total of four washes.</li>
		<li>Resuspend the streptavidin-coated magnetic beads using the unbound phages taken from step 3.2.5 and incubate at RT on a rotator for 1 h.</li>
		<li>Extract the supernatant without disturbing the magnetic beads. Save the unbound phages as input, to be used in step 3.5.1.</li>
		<li>Wash the beads 10x using 0.05% PBST and 5x using 1 x PBS. In between every three washes transfer them to a new tube to avoid phages nonspecifically bound to the tube walls.</li>
	</ol>
	</li>
	<li>Elution of phage-displayed nanobodies
	<ol>
		<li>Competitively elute bound phages by adding 450 &#181;L of the non-biotinylated ligand, using a concentration in the micromolar range (e.g., 10&#8211;50 &#181;M) and incubating at RT on a rotator for 30 min. The selected ligand concentration for the competitive elution of bound phages is dependent on desired KD of the &#34;anchor binder&#34;. Ligand concentrations can be relatively high in initial selection rounds and then decreased in later rounds.</li>
		<li>Collect supernatant and save the eluted phages as output, to be used in step 3.5.2.</li>
	</ol>
	</li>
	<li>Input/output titrations and infection
	<ol>
		<li>For input titration, prepare 10x serial dilutions in 1 x PBS up to 109-fold with the input phage from step 3.3.7. Use the 107&#8211;109 serial dilutions to do infections by transferring 10 &#181;L input phage from each dilution to 70 &#181;L TG1 cells (OD600 of ~0.5). Incubate at 37 &#730;C for 45 min, plate the infected TG1 cells on three 90 mm 2YT-agar dishes containing 100 &#956;g/mL ampicillin and 2% (wt/vol) glucose, and incubate overnight at 37 &#730;C. From the overnight plates, phage input can be calculated as follows: 
		<img alt="Equation 1" src="/files/ftp_upload/60738/60738eq1.jpg" /></li>
		<li>For output infection and titration, transfer the eluted phages from step 3.4.2 to 3 mL of TG1 cells (OD600 of ~0.5). Incubate in a water bath at 37 &#730;C for 45 min. Then prepare 10x serial dilutions in 2YT up to 103-fold, plate each dilution on 90 mm 2YT-agar dishes, and incubate overnight at 37 &#730;C. From the overnight plates, phage output can be calculated as follows: 
		<img alt="Equation 1" src="/files/ftp_upload/60738/60738eq1.jpg" /></li>
		<li>Divide the remaining infected TG1 cells on three 150 mm 2YT-agar plates containing 100 &#956;g/mL ampicillin and 2% (wt/vol) glucose. Incubate plates overnight at 37 &#730;C.</li>
	</ol>
	</li>
	<li>Library amplification and recovery for further rounds of selection
	<ol>
		<li>Add 3 mL of 2YT per plate, scrape with a sterile cell scraper and collect all cells in a 50 mL conical tube. Mix the collected cells with sterile glycerol (20% wt/vol final concentration). Measure the OD600 of the mixture and make 3&#8211;5 stock aliquots. Store at -80 &#730;C for long-term storage.</li>
		<li>For phage rescue, dilute the phagemid-containing TG1 bacterial mixture using 25 mL of 2YT media supplemented with 2% glucose and 100 &#956;g/mL ampicillin to an OD600 of ~0.1. Culture cells at 37 &#730;C and 250 rpm to an OD600 of ~0.5.</li>
		<li>Superinfect the cells by adding CM13 helper phage at 5 x 109 pfu/mL and incubate at 37 &#730;C and 250 rpm for 45 min. The CM13 helper phage provides required phage coat proteins for the assembly of complete phage particles.</li>
		<li>Centrifuge the culture at 8,000 x g for 10 min to remove the glucose. Resuspend the cells using 50 mL of 2YT media supplemented with 100 &#956;g/mL ampicillin and 50 &#956;g/mL kanamycin and incubate at 25 &#730;C and 250 rpm overnight.</li>
		<li>Centrifuge the cells from the overnight culture at 9,000 x g, 4 &#730;C for 30 min. Transfer supernatant to a new tube and precipitate phages in the supernatant using 1/5 volume PEG/NaCl solution (20% wt/vol polyethylene glycol-6,000 and 2.5 M NaCl). Mix gently and place on ice for 1 h.</li>
		<li>Collect phage particles by centrifugation using 12,000 x g at 4 &#730;C for 30 min. Resuspend the pellets using 1 mL of 1 x PBS, and transfer the suspension to a microcentrifuge tube. Centrifuge the tube at 20,000 x g and 4 &#730;C for 10 min to remove residual bacteria.</li>
		<li>Transfer the supernatant to a new microcentrifuge tube without disturbing the bacterial pellet. Use a 1:100 dilution to measure the absorption at 269 nm and 320 nm. The total number of phages can be calculated using the following formula23: 
		<img alt="Equation 3" src="/files/ftp_upload/60738/60738eq3.jpg" /></li>
		<li>Store phage library at 4 &#730;C for short-term use or with 25% glycerol at -80 &#730;C for long-term storage.</li>
		<li>Repeat rounds of selection (steps 3.1&#8211;3.6) for 3&#8211;6 rounds or until desired enrichment is observed (refer to Results section). Plate and pick single clones (section 4) in order to characterize their affinity and specificity to the ligand (sections 5&#8211;7).</li>
	</ol>
	</li>
</ol>
4. Single clone isolation
<ol>
	<li>To isolate individual clones from an enriched sublibrary, prepare 10x serial dilutions of the phage-infected TG1 cells (step 3.5.2). Plate serial dilutions on 90 mm 2YT-agar dishes containing 100 &#956;g/mL ampicillin and 2% (wt/vol) glucose and incubate at 37 &#730;C overnight.</li>
	<li>From the overnight plates, pick single colonies into 250 &#956;L of 2YT media supplemented with 100 &#956;g/mL ampicillin per well in sterile deep-well plates and grow at 37 &#730;C overnight.</li>
	<li>From the overnight cultures, inoculate 10 &#181;L into 500 &#181;L of fresh 2YT media supplemented with 100 &#956;g/mL ampicillin.</li>
	<li>Grow cells to an OD600 of ~0.5, add CM13 helper phage at 5 x 109 pfu/mL and incubate at 37 &#730;C and 250 rpm for 45 min.</li>
	<li>Add 500 &#181;L of 2YT media supplemented with 100 &#956;g/mL ampicillin and 50 &#956;g/mL kanamycin. Incubate at 25 &#730;C and 250 rpm overnight.</li>
	<li>Centrifuge the deep-well plates from the overnight cultures at 3,000 x g for 10 min. Collect the supernatant containing the phage particles without disturbing the cell pellet.</li>
	<li>Phage particles can be used for ELISA to determine the specificity of the selected clones to the ligand. Biotin or a structural homolog of the target can be used as a negative control.</li>
</ol>
5. Anchor binder validation by ELISA
<ol>
	<li>Coat 96 well ELISA plates using 100 &#956;L of 5 &#956;g/mL streptavidin in coating buffer (100 mM carbonate buffer, pH = 8.6) at 4 &#730;C overnight.</li>
	<li>Wash the ELISA plates 3x using 0.05% PBST and add 100 &#956;L of 1 &#956;M biotinylated target to the target wells. Add 100 &#956;L of 1 &#956;M biotin or target homolog to the control wells. Incubate at RT for 1 h.</li>
	<li>Wash plates 5x using 0.05% PBST and block nonspecific binding by adding 300 &#181;L of 1% casein in 1 x PBS. Incubate at RT for 1 h.</li>
	<li>Wash the ELISA plates 3x using 0.05%-PBST and add the purified phage supernatant. Incubate for 1 h at RT.</li>
	<li>Wash the ELISA plates 10x using 0.05% PBST and add 100 &#956;L horseradish peroxidase (HRP)-M13 major coat protein antibody (1:10,000 dilution with 1 x PBS with 1% casein). Incubate at RT for 1 h.</li>
	<li>Wash the ELISA plates 3x using 0.05% PBST and add 100 &#956;L tetramethylbenzidine (TMB) substrate. Incubate for 10 min or until a visible color change is observed. Stop the reaction by adding 100 &#956;L of 1 M HCl. Read the plate at 450 nm on a spectrophotometer.</li>
	<li>For protein expression and purification, choose the clones showing high affinity and specificity for the target (see Discussion).</li>
</ol>
6. Protein expression, purification, and biotinylation
<ol>
	<li>As previously reported19, subclone selected clones from section 5 and express as C-terminal Avi-tagged and His-tagged nanobodies.</li>
	<li>Express selected nanobodies in the periplasm of E. coli WK6 cells (typically in 1 L culture), release by osmotic shock, and purify using a nickel-NTA column (see Table of Materials).</li>
	<li>Exchange buffer with a desalting column (1 x PBS with 5% glycerol; see Table of Materials).</li>
	<li>Biotinylate nanobodies using a commercial kit (see Table of Materials) for further use.</li>
</ol>
7. Anchor binder characterization by BLI
<ol>
	<li>Analyze the binding affinity and kinetics of selected anchor binders by immobilizing 200 nM biotinylated anchor binders on streptavidin biosensors (see Table of Materials) with binding assay buffer (1 x PBS (pH = 7.4), 0.05% Tween 20, 0.2% BSA, 3% methanol).</li>
	<li>Calculate dissociation constants (KD) of anchor binder-ligand interactions by steady-state analysis using data analysis software (see Table of Materials). Obtained KD values typically range from single- to double-digit micromolar.</li>
</ol>
8. Dimerization binder screening
NOTE: The biopanning screening of &#34;dimerization binders&#34; is similar to that of anchor binders, except for two critical steps: 1) Dimerization binders are selected using a selected biotinylated anchor binder and the anchor binder-ligand complex for the negative and positive selections, respectively. 2) During the elution step, 100 mM triethylamine is used to elute positively selected phages that were only bound to the anchor binder--ligand target complex. The 100 mM trimethylamine solution (pH = 11.5) is used to elute positive clones by disrupting the protein interactions.
<ol>
	<li>Beginning of selection
	<ol>
		<li>Begin every round of selection by inoculating a single TG1 cell colony, freshly grown on a minimal media, in 6 mL 2YT at 37 &#730;C and 250 rpm to an OD600 of ~0.5. Incubate cells on ice.</li>
	</ol>
	</li>
	<li>Removal of negatively selected nanobodies
	<ol>
		<li>Prepare the &#34;subtraction tube&#34; by using 400 &#181;L of streptavidin-coated magnetic beads and follow step 3.2. However, instead of saturating with biotin, add 5x the calculated full binding capacity using the selected biotinylated anchor binder and save the unbound phages to be used in step 8.3.3.</li>
	</ol>
	</li>
	<li>Selection of positively selected nanobodies
	<ol>
		<li>Prepare the &#34;capturing tube&#34; by using 1/2 the volume of streptavidin-coated magnetic beads used for the &#34;subtraction tube&#34; and following steps 3.3.2 to 3.3.3. However, instead of saturating with the biotinylated ligand, add five times the calculated full binding capacity using the selected biotinylated anchor binder.</li>
		<li>To form the anchor binder-ligand complex for the positive dimerization binder selection, add a high enough concentration of non-biotinylated ligand. This will allow most streptavidin-bound anchor binder to form the ligand-bound complex.</li>
		<li>Follow steps 3.3.3 to 3.3.8, using the unbound phages taken from the &#34;subtraction tube&#34;.</li>
	</ol>
	</li>
	<li>Elution of positively selected nanobodies
	<ol>
		<li>Elute the phages bound to the anchor binder-ligand complex by adding 450 &#181;L of 100 mM triethylamine, and incubating at RT on a rotator for 10 min.</li>
		<li>Collect the competitively eluted phages and follow steps 3.4.1 to 3.4.2.</li>
	</ol>
	</li>
	<li>Further rounds of dimerization binder selection
	<ol>
		<li>Follow steps 3.5 and 3.6 to amplify and recover the library in order to perform further rounds of selection. Repeat rounds of selection for 3&#8211;6 rounds or until desired enrichment is observed. Plate and pick single clones (refer to section 4) in order to characterize their affinity and specificity to the target.</li>
	</ol>
	</li>
</ol>
9. Dimerization binder characterization by ELISA
<ol>
	<li>Follow the steps in section 4 to isolate individual clones for characterization via ELISA.</li>
	<li>To test the affinity of dimerization binder candidates to the anchor binder-ligand complex, coat the ELISA target plate using 100 &#956;L of 100 nM biotinylated anchor binder. After incubation for 1 h, add 1 &#956;M of the ligand target to form the anchor binder-ligand complex.</li>
	<li>The control plate should be coated using the biotinylated anchor binder alone to screen out clones that can also bind to the free anchor binder. Add 100 &#956;L of 100 nM biotinylated anchor binder and incubate at RT for 1 h.</li>
	<li>Follow sections 5.3&#8211;5.7.</li>
</ol>
10. Dimerization binder characterization by BLI
<ol>
	<li>The binding affinity and kinetics of dimerization binders for the anchor binder--ligand complex can be analyzed by immobilizing biotinylated dimerization binders on streptavidin (SA) biosensors with the binding assay buffer and then assayed with 1 &#956;M anchor binder pre-equilibrated with serial dilutions of the ligand. The KD, kon, and koff of the interactions can be calculated using our reported method19.</li>
</ol>

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A provisional patent related to this work has been filed by the University of Washington.

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><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 ...

creating-highly-specific-chemically-induced-protein-dimerization

Research

JoVE Journal

Biochemistry

7.7K Views.  University of Washington. Dit protocol is belangrijk omdat het een algemene benadering biedt voor engineering eiwit dimerization systemen als biosensoren voor een verscheidenheid van kleine moleculen. Bovendien gebruikt het één zeer diverse eiwitbibliotheek om biosensoren voor verschillende liganden op een efficiënte en kosteneffectieve manier zonder de behoefte aan gespecialiseerde apparatuur te selecteren. De ontworpen biosensor kan worden gebruikt om het celgedrag chemisch te controleren en om real-time verander...

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

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.

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

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

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

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

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of 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.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Immunology and Infection

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.

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 ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Bioengineering

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.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Biology

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.

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.

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 ...

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

Samenvatting

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