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....

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<....

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