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 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 ...

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

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JoVE Journal

Biochemistry

7.6K Views.  University of Washington. 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.

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 ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

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

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

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

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

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

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

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

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

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

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

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

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

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

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

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

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

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

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.