Name: creating highly specific chemically induced protein dimerization systems by stepwise phage selection of a combinatorial single-domain antibody library
Uploaded: 2020-01-14T16:00:00
Description: 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

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

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

<ol>
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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 ...

Research

JoVE Journal

Biochemistry

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 ...

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 ...

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, ...

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

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 ...

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 ...

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 ...

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

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 ...

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 ...

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 ...