The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Podsumowanie

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid β-lactamase technology. This method relies on release of protons upon hydrolysis of β-lactams.

Streszczenie

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used β-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP β-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the β-lactamase activity. This suggests that the nanobody and β-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid β-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

Wprowadzenie

Biosensors are analytical devices that combine a bio-molecular interaction with physical or chemical signaling devices referred to as transducers¹. The recorded signals can then be interpreted and converted to monitor the interactions between the immobilized and free partners. Most of the biosensors involve the use of an antibody to detect analytes such as hormones or different pathogen markers². Different sensor formats can be used and include mass-based, magnetic, optical or electrochemical biosensors. The latter are among the most commonly used sensors, and function by converting a binding event into an electrical signal. The performances and sensitivities of all antibody-based biosensors are strongly dependent on essentially two parameters: i) the quality of the antibody and ii) the properties of the system used to generate the signal².

Antibodies are high-molecular mass dimeric proteins (150–160 kDa) that are composed of two heavy chains and two light chains. The interaction between the light and heavy chains is mostly stabilized by hydrophobic interactions as well as a conserved disulfide bond. Each chain includes a variable domain that interacts with the antigen essentially via three hypervariable regions named Complementary Determining Regions (CDR1-2-3). Despite numerous advances in the field, the large-scale expression of full-length antibodies with low-cost expression systems (e.g., E. coli) often leads to the production of unstable and aggregated proteins. This is why various antibody fragments have been engineered such as single-chain variable fragments³ (ScFvs ≈ 25 kDa). They consist of the variable domains of respectively one heavy and one light chains that are covalently linked by a synthetic amino acid sequence. However, these fragments often display a poor stability and have the tendency to aggregate, since they expose a large portion of their hydrophobic regions to the solvent⁴. In this context, single chain camelid antibody fragments, referred to as nanobodies or VHHs, seem to be excellent alternatives to ScFvs. These domains correspond to the variable domains of camelid single-chain antibodies. In contrast to conventional antibodies, camelid antibodies are devoid of light chains and only contain two heavy chains⁵. Therefore, nanobodies are the smallest monomeric antibody fragments (12 kDa) able to bind to an antigen with an affinity similar to that of conventional antibodies⁶. In addition, they present improved stability and solubility compared to other full-length antibodies or antibody fragments. Finally, their small sizes and their extended CDR3 loops allow them to recognize cryptic epitopes and bind to enzyme active sites^7,8. Nowadays, these domains are receiving considerable attention and have been combined to the biosensor technology. For example, Huang et al. have developed a nanobody-based biosensor for the detection and quantification of human prostate-specific antigen (PSA)⁹.

As mentioned-above, an important parameter in biosensor assays is the efficiency of the system used to generate the electric signal. For this reason, enzyme-based electrochemical biosensors have attracted ever-increasing attention and have been used widely for various applications such as health care, food safety, and environmental monitoring. These biosensors rely on the catalytic hydrolysis of a substrate by an enzyme to generate the electrical signal. In this context, β-lactamases were shown to be more specific, more sensitive and easier to implement experimentally than many other enzymes such as alkaline phosphatase or horseradish peroxidase¹⁰. β-lactamases are enzymes that are responsible for bacterial resistance to β-lactam antibiotics by hydrolyzing them. They are monomeric, very stable, efficient, and of small size. Moreover, domain/peptide insertions into β-lactamases generate bi-functional chimeric proteins that were shown to be efficient tools to study protein-ligand interactions. Indeed, recent studies have shown that insertion of antibody variable fragments into the TEM1 β-lactamase results in a chimeric protein that remains able to bind with high affinity to its target antigen. Interestingly, the antigen binding was shown to induce allosteric regulation of TEM1 catalytic activity^11,12. Furthermore, we showed in several studies that protein domain insertion into a permissive loop of the Bacillus licheniformis BlaP β-lactamase generates functional chimeric proteins that are well suited to monitor protein-ligand interactions^13,14. We recently inserted a nanobody, named cAb-Lys3, into this permissive insertion site of BlaP¹⁵. This nanobody was shown to bind to hen-egg-white lysozyme (HEWL) and to inhibit its enzymatic activity¹⁶. We showed that the generated hybrid protein, named BlaP-cAb-Lys3, retained a high specificity / affinity against HEWL while the β-lactamase activity remained unchanged. Then we successfully combined the hybrid β-lactamase technology to an electrochemical biosensor and showed that the amount of generated electric signal was dependent of the interaction between BlaP-cAb-Lys3 and HEWL immobilized on an electrode. Indeed, hydrolysis of β-lactam antibiotics by BlaP induces a proton release that can be converted into a quantitative electric signal. This combination of the hybrid β-lactamase technology with an electrochemical biosensor is fast, sensitive, quantitative, and allows real-time measurement of the generated signal. This methodology is described herein.

Protokół

1. Protein Sample Preparation

1. Produce and purify the hybrid protein BlaP-cAb-Lys3 as reported in our previous study¹⁵. Store the protein in 50 mM phosphate buffer pH 7.4 with the following composition: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄  and 0.24 g of KH₂PO₄ dissolved in 800 mL of distilled water Fix the pH of the solution to 7.4 before adjusting the final volume of the solution to 1 L. Filter sterilize the protein solution.
2. Prepare a hen egg white lysozyme (HEWL) stock solution. Dissolve 100 mg (40,000 units/mg) of commercially purchased HEWL in 10 mL of phosphate buffer saline (PBS see step 2.1.1). Sterilize the protein solution by filtration using filters with a 0.22 μm cutoff.

2. Biosensor Assays

1. Solution and buffer preparation
   1. Prepare 50 mM PBS by dissolving 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, and 0.24 g of KH₂PO₄ in 800 mL of distilled water. Adjust to pH 7.4 with 1 N HCl or 1 N NaOH before adjusting the volume of the solution to 1 L. Filter sterilize and store at 4 °C.
   2. Prepare a saturation/blocking solution by dissolving 3 g of casein hydrolysate in 100 mL of PBS prepared as described above (see step 2.1.1). Filter sterilize and store at 4 °C.
   3. Prepare a binding solution by dissolving 1 g of casein hydrolysate in 100 mL of PBS prepared as described above (see step 2.1.1). Filter sterilize and store at 4 °C.
   4. Prepare a washing solution (0.1% Tween - PBS) by adding 100 μL of Tween 20 (100%) in 100 mL of PBS prepared as described above (see step 2.1.1). Store at 4 °C.
   5. Prepare an electrode preparation solution (1% Triton X-100 - PBS) by adding 1 mL of Triton X-100 (100%) in 100 mL of PBS prepared as described above (see step 2.1.1). Store at 4 °C.
   6. Prepare an electrode regeneration solution (3.5 M KCl) by dissolving 26 g of KCl in distilled water to a final volume 100 mL. Filter sterilized and store at 4 °C.
   7. Prepare a 5 mM NaCl solution by dissolving 0.29 g of NaCl in distilled water to a final volume of 1 L. Filter sterilize and store at 4 °C. Then, prepare a detection solution (4 mM benzylpenicillin) by dissolving 26.7 mg of benzylpenicillin in 20 mL of 5 mM NaCl solution. Filter sterilize and store at -20 °C.
2. Sensor preparation and regeneration
   Note: The polyaniline coated sensor chips were developed and kindly provided by Dr. P. Bogaerts, Dr. S. Yunus and Prof. Y. Glupczynski’s (Catholic University of Louvain-la-Neuve - CHU Mont-Godinne). The description of the sensor as well as the polyaniline electro-polymerization protocols used to synthesize these sensors are detailed in their previous work¹⁷. Briefly, this system uses re-usable sensors of eight individual chips that were manufactured by classical printed circuit board (PCB) techniques. Individual chips are composed of three electrode round spots. The top one is the working electrode on which polyaniline was electro-synthesized. The middle one is the reference electrode and the bottom electrode constitutes the counter electrode. Both, the reference and the counter electrodes are functionalized using solid Ag/AgCl amalgam on top of the carbon layer.
   1. Perform 3 washes of the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of electrode preparation solution (1% Triton X-100 - PBS, see step 2.1.5.). Perform each wash for 2 min with gentle mixing at room temperature.
   2. Rinse the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of distilled water for 2 min with gentle mixing at room temperature.
   3. Regenerate the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of regeneration solution (3.5 M KCl, see step 2.1.6) overnight at 4 °C or 1 h at room temperature.
   4. Perform 3 washes of the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of phosphate buffer saline (see step 2.1.1.). Perform each wash for 2 minutes with gentle mixing at room temperature.
3. Binding assay performed on the sensor
   1. Coat HEWL onto the PANI (polyaniline) surface of the electrode by depositing a 15 µL drop of 40 µg/mL HEWL prepared in PBS onto the electrode surface. Incubate overnight at 4 °C or 1 hour at room temperature.
   2. Perform three washes of the electrodes with phosphate buffer saline (see step 2.1.1) by dipping the electrode parts of the sensor chips into wells of a 96-well plate containing 300 µL/well of phosphate buffer saline. Perform each wash for 2 min with gentle mixing at room temperature.
   3. Saturate the electrodes by adding a 50 µL drop of the blocking solution (see step 2.1.2) onto the electrode surface. Incubate for 1 h at room temperature. Then wash three times as described in the previous step (see step 2.3.2).
   4. Dilute the BlaP-cAb-Lys3 solution to 20 µg/mL in binding solution (see step 2.1.3) and apply a 15 µL drop of this diluted solution onto the electrodes. Incubate for 10 min at room temperature. After antigen-nanobody reaction, wash three times as described in the previous step using the wash solution (see step 2.1.4). Then rinse the electrode once with PBS (see step 2.1.1).
   5. For detection, plug the sensor chip via the copper-circuitry part to a digital multimeter. Then, initiate the sensor response by applying a 50 µL drop of detection solution (see step 2.1.7) onto the positive electrodes and applying a 50 µL drop of NaCl 5 mM solution onto the negative electrodes (see step 2.1.7). Incubate for 30 min at room temperature. Monitor the conductance with a digital multimeter.
      Note: The multimeter was provided by Dr. P. Bogaerts, Dr. S. Yunus and Prof. Y. Glupczynski’s (Catholic University of Louvain-la-Neuve - CHU Mont-Godinne. This potentiostat is computer-controlled via a USB port and analyzes the eight different chips of the sensor simultaneously. The software created by Yunus and colleagues¹⁷ creates a real-time plot that represents the measurements of the conductance difference between the reference and sample electrodes against time.

Wyniki

Design and engineering of the chimeric protein BlaP-cAb-Lys3

Figure 1 represents the insertion of cAb-Lys3 into a permissive loop of the BalP class A β-lactamase from Bacillus licheniformis. The insertion was performed between residues Asp198 and Lys199. A thrombin cleavage site was introduced on each side of cAb-Lys3. Cells transformed with a constitutive expression plasmid encoding the BlaP-cAb-Lys3 chimeric protein...

Dyskusje

In this work we present a method to functionalize a nanobody using the BlaP β-lactamase as a carrier protein and we show that we can successfully implement the resulting hybrid protein in a potentiometric sensor assay. The main innovation aspect of our work compared to other biosensor assays is the covalent coupling of the antibody part to the enzymatic activity that generates the electrical signal. This so-called protein insertion technology presents advantages and limitations that will be the main focus of this se...

Materiały

Name	Company	Catalog Number	Comments
Reagents
KH2PO4	Sigma-Aldricht	V000225
K2HPO4	Sigma-Aldricht	1551128
NaCl	Sigma-Aldricht	S7653
Tris–HCl	Roche	10812846001
EDTA	Sigma-Aldricht	E9884
KCl	Sigma-Aldricht	P9541
Na2HPO4	Sigma-Aldricht	NIST2186II
2-mercaptoethanol	Sigma-Aldricht	M6250
alanine	Sigma-Aldricht	A7627
HClO4	Fluka	34288	1M HClO4 solution, distributor : Sigma-Aldricht
casein hydrolysate	Sigma-Aldricht	22090
benzylpenicillin sodium	Sigma-Aldricht	B0900000
hen egg white lysozyme	Roche	10837059001
heptane	Sigma-Aldricht	246654
methanol	Sigma-Aldricht	322415
ammonium hydroxide solution	Sigma-Aldricht	380539	28% NH3 in H2O, purified by double-distillation (concentrated?)
Laboratory consumables
6-well plate	Greiner Bio-One	657165	CELLSTAR 6-Well Plate
Equipment
pH meter	WTW	1AA110	Lab pH meter inoLab pH 7110
vacuum and filtration system	Nalgene	NALG300-4100	Filter holders with receiver, distributor : VWR
potentiometric sensor chips			manufactured by Yunus and colleagues (ref 16)
PGSTAT30 Autolab	Metrohm Autolab		discontinued, succesor Autolab PGSTAT302N
digital multimeter, METRAHit 22M	Gossen Metrawatt		discontinued, successor Metrahit Base