Name: quartz-crystal microbalance biosensor-based biopanning to study protein-drug interactions
Uploaded: 2023-06-29T19:11:40.000+00:00
Duration: 1 min 48 s
Description: Source: Takakusagi, Y. Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein ...

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NOTE: The following are the steps for screening drug-recognizing T7 phages using a QCM biosensor and recovering the screened phages via E. coli (BLT5615) infection. The protocols for the synthesis of a derivative of a small molecule that forms a self-assembled monolayer (SAM) and for the construction of the T7 phage-displayed 15-mer random peptide library can be found elsewhere.
1. Preparation of the QCM sensor chip
<ol>
	<li>Attach a ceramic sensor chip on the oscillator of a 27-MHz QCM apparatus, and record the intrinsic frequency (Hz) in the air phase before small molecule immobilization.</li>
	<li>Detach the chip and drop 20 &#956;L of a solution (1 mM in 70% ethanol) of a small molecule derivative that forms SAM onto the gold electrode of the sensor chip using a pipette. 
	CAUTION: The sensor chip crystal where the gold electrode (Au, 0.1 mm thick, 2.5 mm i.d., 4.9 mm2) is located is extremely thin and may crack easily (SiO2, 0.06 mm thick, 9 mm i.d.). Hence, pipette carefully.</li>
	<li>Leave for 1 h at room temperature (around 20 &#176;C) in a Petri dish with moistened tissue and shielded from room lights.</li>
	<li>Wash the electrode surface gently with ultrapure water; then, remove the water drops by blowing air with a syringe or air duster.</li>
	<li>Set up the sensor chip for the QCM apparatus and record the reduction in frequency in the air phase to measure the amount of the small molecule that has been immobilized. 
	NOTE: At least, 100 Hz of intrinsic frequency is necessary for successful small molecule immobilization (1 Hz immobilizes 30 pg).</li>
</ol>
2. Biopanning of the T7 phage library using a QCM biosensor (Figure 1)
<ol>
	<li>Set a cuvette with a dedicated magnetic stirrer on the QCM biosensor and pour 8 mL of the reaction buffer (10 mM Tris-HCl, 200 mM NaCl, pH 7-8) into the cuvette.</li>
	<li>Attach the QCM sensor chip to the oscillator and pull down the arm of the oscillator to immerse the chip into the buffer being stirred at 1000 rpm.</li>
	<li>Start monitoring the QCM frequency on the personal computer (PC) and wait until the sensorgram equilibrates to around 3 Hz/min of the frequency drift.</li>
	<li>Inject 8 &#956;L of a T7 phage library (1&#8212;2 &#215; 1010 pfu/mL) into the cuvette (final concentration: 1&#8212;2 &#215; 107 pfu/mL) and mark the injection point on the sensor.</li>
	<li>Monitor the frequency reduction caused by the binding of T7 phages to the small molecule immobilized on the gold electrode surface for 10 min.</li>
	<li>Stop the QCM frequency monitor, dislodge the sensor chip from the oscillator, and remove the buffer by blowing air and/or wicking away with wipes.</li>
	<li>Put the sensor chip into a humid Petri dish and drop the 20 &#956;L of the suspension of E. coli (BLT5615) (OD600 = 0.5&#8212;1.0 after shaking at 37 &#176;C by adding IPTG to 1 mM) host cells in the log phase onto the gold electrode.</li>
	<li>Close the lid of the dish and cover it with aluminum foil to block out light.</li>
	<li>Incubate the dish at 37 &#176;C for 30 min on a 96-well microplate mixer (1000-1500 rpm) for enhancing the recovery of the bound T7 phages.</li>
	<li>Recover the 20 &#956;L of the solution and suspend it into 200 &#956;L of LB medium. 
	NOTE: The samples obtained in this step can be preserved at 4 &#176;C for one week.</li>
	<li>Prepare a dilution series of the phage solution for plaque isolation and DNA sequencing according to the general procedure described in the manufacturer&#39;s instructions.</li>
</ol>

<table><tbody><tr><td>AFFINIXQN</td><td>ULVAC, Inc. (Tokyo, Japan)</td><td>QCM2008-STKIT</td><td>Contains Glass cuvette, stir magnet, operation and analysis software with a Windows PC</td></tr><tr><td>Ceramic Sensor Chip</td><td>ULVAC, Inc. (Tokyo, Japan)</td><td>QCMST27C</td><td>4 sensor chips/package</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich&lt;br/&gt; (St. Louis, MO, USA)</td><td>D8418</td><td></td></tr><tr><td>Ethanol</td><td>Merck (Kenilworth, NJ, USA)</td><td>09-0850</td><td></td></tr><tr><td>Immobilization kit for AFFINIX</td><td>ULVAC, Inc. (Tokyo, Japan)</td><td>QCMIMKT</td><td>SAM reagent and amine-coupling reagent</td></tr><tr><td>Liquid LB medium</td><td>Sigma-Aldrich&lt;br/&gt; (St. Louis, MO, USA)</td><td>L3522</td><td>Autoclave for 20 min</td></tr><tr><td>NaCl</td><td>Merck (Kenilworth, NJ, USA)</td><td>S3014</td><td></td></tr><tr><td>Tris</td><td>Merck (Kenilworth, NJ, USA)</td><td>252859</td><td></td></tr></tbody></table>

quartz-crystal microbalance biosensor-based biopanning to study protein-drug interactions

Source: Takakusagi, Y. <a href="https://app.jove.com/v/61873">Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions.</a> J. Vis. Exp. (2020)
This video demonstrates a method to study protein-drug interactions using QCM biosensor-based biopanning of the T7 phage-displayed peptide library. The biopanning of phages displaying a specific peptide on the QCM biosensor allows specific isolation of high-affinity peptides, followed by amino acid sequencing, which helps to identify specific interaction sites on the target protein.

<img alt="Figure 1" src="/files/ftp_upload/21447/21447_Figure1.jpg" /> 
Figure 1: Schematic representation of the QCM biosensor-based biopanning of the T7 phage-displayed peptide library. A T7 phage library that displays random peptides is injected into the cuvette containing the buffer (under stirring) where the QCM biosensor chip is immersed and the frequency is stabilized. After monitoring the frequency reduction due to the binding of T7 phages to...

Quartz-Crystal Microbalance Biosensor-Based Biopanning to Study Protein-Drug Interactions

Source: Takakusagi, Y. Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein ...

To study protein-drug interactions, begin with a quartz-crystal microbalance, QCM, sensor chip comprising piezoelectric ceramic material coated with a thin gold layer acting as an electrode.
Overlay the electrode with test drug solution, forming a uniform layer.
Insert the sensor chip into the QCM apparatus. Immerse it into a buffer-containing cuvette. Apply an electric field, causing ceramic crystals to oscillate at a fixed frequency based on the drug molecules&#39; mass over the sensor chip.
Add a T7 phage library containing a mixture of genetically engineered phages displaying peptide variants fused with the phages&#39; capsid protein. The phages displaying a specific peptide bind to drug molecules with high affinity and get immobilized on the sensor chip. This panning of T7 phages increases the mass on the sensor chip, resulting in crystal oscillation decrease over time.
Post-equilibration, transfer the phage-bound sensor chip into a humid petri dish. Overlay the chip with a bacterial solution and incubate. Phages infect the bacteria and replicate, producing specific peptide-expressing phages that get released in the solution.
Recover these phages and analyze the drug-affinity-selected peptides&#39; amino acid sequence. Compare this peptide&#39;s amino acid sequence with the target protein.
The region with high similarity represents the potential drug interaction site crucial for the drug-protein interaction.

quartz-crystal-microbalance-biosensor-based-biopanning-to-study

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

130 次观看. 来源:Takakusagi， Y. 基于生物传感器的高通量生物淘选和生物信息学分析策略，用于药物-蛋白质相互作用的全球验证。J. Vis. Exp. （2020 年） 该视频演示了一种使用基于 QCM 生物传感器的 T7 噬菌体展示肽库生物淘选来研究蛋白质-药物相互作用的方法。对在 QCM 生物传感器上显示特定肽的噬菌体进行生物淘选，可以特异性分离高亲和力肽，然后进行氨基酸测序，这有助于识别靶蛋?...

视频: 基于石英晶体微天平生物传感器的生物淘选研究蛋白质-药物相互作用 - 概念

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

科研

JoVE Journal

生物化学

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.

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.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Quartz-Crystal Microbalance Biosensor-Based Biopanning to Study Protein-Drug Interactions

成績單

Quartz-Crystal Microbalance Biosensor-Based Biopanning to Study Protein-Drug Interactions