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

Summary

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.

Abstract

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) "anchor binders" that first bind to a ligand of interest and then 2) "dimerization binders" 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.

Introduction

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 apoptosis^3,4, 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 selection^8,9, 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 proteins^{11,12,13,14,15,16} or rely on selecting binder proteins such as antibodies targeting preexisting small molecule-protein complexes^17,18, 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, K_D (without ligand)/K_D (with ligand) > 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 12–15 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 epitopes^21,22. 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 6–10 weeks (Figure 4).

Protocol

1. Library construction

1. Use a synthetic combinatorial single-domain antibody library with a diversity of ~1.23–7.14 x 10⁹, as previously described¹⁹. While this protocol does not include library construction, it can be applied to other combinatorial binder libraries.

2. Biotinylation of ligand target or ligand

1. 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.

3. Anchor binder screening

1. Beginning of selection
   1. Begin every round of selection by inoculating a single TG1-cell colony, freshly grown in 6 mL of 2YT at 37 ˚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.
2. Negative selection with biotin-bound streptavidin beads
   1. Prepare the "negative selection beads" by washing 300 µL 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.
   2. 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.
   3. Wash the beads 5x using 0.05% PBST and 3x using 1 x PBS, for a total of eight washes.
   4. 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.
   5. After incubation, collect the supernatant to be used in step 3.3.6.
3. Positive selection with biotinylated ligand-bound streptavidin beads
   1. Prepare the "positive selection beads" using 1/2 the volume of the beads used for the "negative selection beads" following steps 3.2.1.
   2. Re-suspend the beads with 1 mL 1% casein in 1 × 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.
   3. Wash the beads 5x using 0.05% PBST and 3x using 1 x PBS, for a total of eight washes.
   4. 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.
   5. Wash the streptavidin-coated magnetic beads 3x using 0.05% PBST and one time using 1 x PBS, for a total of four washes.
   6. 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.
   7. Extract the supernatant without disturbing the magnetic beads. Save the unbound phages as input, to be used in step 3.5.1.
   8. 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.
4. Elution of phage-displayed nanobodies
   1. Competitively elute bound phages by adding 450 µL of the non-biotinylated ligand, using a concentration in the micromolar range (e.g., 10–50 µM) 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 K_D of the "anchor binder". Ligand concentrations can be relatively high in initial selection rounds and then decreased in later rounds.
   2. Collect supernatant and save the eluted phages as output, to be used in step 3.5.2.
5. Input/output titrations and infection
   1. 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 10⁷–10⁹ serial dilutions to do infections by transferring 10 µL input phage from each dilution to 70 µL TG1 cells (OD₆₀₀ of ~0.5). Incubate at 37 ˚C for 45 min, plate the infected TG1 cells on three 90 mm 2YT-agar dishes containing 100 μg/mL ampicillin and 2% (wt/vol) glucose, and incubate overnight at 37 ˚C. From the overnight plates, phage input can be calculated as follows:
      
   2. 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 ˚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 ˚C. From the overnight plates, phage output can be calculated as follows:
      
   3. Divide the remaining infected TG1 cells on three 150 mm 2YT-agar plates containing 100 μg/mL ampicillin and 2% (wt/vol) glucose. Incubate plates overnight at 37 ˚C.
6. Library amplification and recovery for further rounds of selection
   1. 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 3–5 stock aliquots. Store at -80 ˚C for long-term storage.
   2. For phage rescue, dilute the phagemid-containing TG1 bacterial mixture using 25 mL of 2YT media supplemented with 2% glucose and 100 μg/mL ampicillin to an OD₆₀₀ of ~0.1. Culture cells at 37 ˚C and 250 rpm to an OD₆₀₀ of ~0.5.
   3. Superinfect the cells by adding CM13 helper phage at 5 x 10⁹ pfu/mL and incubate at 37 ˚C and 250 rpm for 45 min. The CM13 helper phage provides required phage coat proteins for the assembly of complete phage particles.
   4. 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 μg/mL ampicillin and 50 μg/mL kanamycin and incubate at 25 ˚C and 250 rpm overnight.
   5. Centrifuge the cells from the overnight culture at 9,000 x g, 4 ˚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.
   6. Collect phage particles by centrifugation using 12,000 x g at 4 ˚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 ˚C for 10 min to remove residual bacteria.
   7. 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:
      
   8. Store phage library at 4 ˚C for short-term use or with 25% glycerol at -80 ˚C for long-term storage.
   9. Repeat rounds of selection (steps 3.1–3.6) for 3–6 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 5–7).

4. Single clone isolation

1. 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 μg/mL ampicillin and 2% (wt/vol) glucose and incubate at 37 ˚C overnight.
2. From the overnight plates, pick single colonies into 250 μL of 2YT media supplemented with 100 μg/mL ampicillin per well in sterile deep-well plates and grow at 37 ˚C overnight.
3. From the overnight cultures, inoculate 10 µL into 500 µL of fresh 2YT media supplemented with 100 μg/mL ampicillin.
4. Grow cells to an OD₆₀₀ of ~0.5, add CM13 helper phage at 5 x 10⁹ pfu/mL and incubate at 37 ˚C and 250 rpm for 45 min.
5. Add 500 µL of 2YT media supplemented with 100 μg/mL ampicillin and 50 μg/mL kanamycin. Incubate at 25 ˚C and 250 rpm overnight.
6. 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.
7. 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.

5. Anchor binder validation by ELISA

1. Coat 96 well ELISA plates using 100 μL of 5 μg/mL streptavidin in coating buffer (100 mM carbonate buffer, pH = 8.6) at 4 ˚C overnight.
2. Wash the ELISA plates 3x using 0.05% PBST and add 100 μL of 1 μM biotinylated target to the target wells. Add 100 μL of 1 μM biotin or target homolog to the control wells. Incubate at RT for 1 h.
3. Wash plates 5x using 0.05% PBST and block nonspecific binding by adding 300 µL of 1% casein in 1 x PBS. Incubate at RT for 1 h.
4. Wash the ELISA plates 3x using 0.05%-PBST and add the purified phage supernatant. Incubate for 1 h at RT.
5. Wash the ELISA plates 10x using 0.05% PBST and add 100 μL 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.
6. Wash the ELISA plates 3x using 0.05% PBST and add 100 μL tetramethylbenzidine (TMB) substrate. Incubate for 10 min or until a visible color change is observed. Stop the reaction by adding 100 μL of 1 M HCl. Read the plate at 450 nm on a spectrophotometer.
7. For protein expression and purification, choose the clones showing high affinity and specificity for the target (see Discussion).

6. Protein expression, purification, and biotinylation

1. As previously reported¹⁹, subclone selected clones from section 5 and express as C-terminal Avi-tagged and His-tagged nanobodies.
2. 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).
3. Exchange buffer with a desalting column (1 x PBS with 5% glycerol; see Table of Materials).
4. Biotinylate nanobodies using a commercial kit (see Table of Materials) for further use.

7. Anchor binder characterization by BLI

1. 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).
2. Calculate dissociation constants (K_D) of anchor binder-ligand interactions by steady-state analysis using data analysis software (see Table of Materials). Obtained K_D values typically range from single- to double-digit micromolar.

8. Dimerization binder screening

NOTE: The biopanning screening of "dimerization binders" 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.

1. Beginning of selection
   1. Begin every round of selection by inoculating a single TG1 cell colony, freshly grown on a minimal media, in 6 mL 2YT at 37 ˚C and 250 rpm to an OD₆₀₀ of ~0.5. Incubate cells on ice.
2. Removal of negatively selected nanobodies
   1. Prepare the "subtraction tube" by using 400 µL 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.
3. Selection of positively selected nanobodies
   1. Prepare the "capturing tube" by using 1/2 the volume of streptavidin-coated magnetic beads used for the "subtraction tube" 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.
   2. 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.
   3. Follow steps 3.3.3 to 3.3.8, using the unbound phages taken from the "subtraction tube".
4. Elution of positively selected nanobodies
   1. Elute the phages bound to the anchor binder-ligand complex by adding 450 µL of 100 mM triethylamine, and incubating at RT on a rotator for 10 min.
   2. Collect the competitively eluted phages and follow steps 3.4.1 to 3.4.2.
5. Further rounds of dimerization binder selection
   1. 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 3–6 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.

9. Dimerization binder characterization by ELISA

1. Follow the steps in section 4 to isolate individual clones for characterization via ELISA.
2. To test the affinity of dimerization binder candidates to the anchor binder-ligand complex, coat the ELISA target plate using 100 μL of 100 nM biotinylated anchor binder. After incubation for 1 h, add 1 μM of the ligand target to form the anchor binder-ligand complex.
3. 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 μL of 100 nM biotinylated anchor binder and incubate at RT for 1 h.
4. Follow sections 5.3–5.7.

10. Dimerization binder characterization by BLI

1. 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 μM anchor binder pre-equilibrated with serial dilutions of the ligand. The K_D, k_on, and k_off of the interactions can be calculated using our reported method¹⁹.

Results

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 4–6 rounds of selection as shown in Figure 5 are a good indication that there is a high ratio of potential h...

Discussion

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 ~10¹²–10¹³ phage particles with a diversity >10⁹, allowing ~100–1,000 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...

Materials

List of materials used in this article

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