β-Lactamase-Based Conductimetric Biosensor Assay for Protein-Protein Interactions

Abstract

Source:  Vandevenne, M., et al. The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions. J. Vis. Exp. (2018)

This video demonstrates a conductimetric biosensor assay to study the interaction of an antibody with a target antigen immobilized on a transducer chip. The biosensor is engineered to have a bifunctional chimeric protein — a β-lactamase enzyme with a functional catalytic site fused to a nanobody for binding to the immobilized target antigen. Upon binding, the catalytic hydrolysis of an antibiotic substrate by the β-lactamase enzyme generates an electrical signal — confirming the antigen-antibody interaction.

Protocol

1. Protein Sample Preparation Produce and purify the hybrid protein BlaP-cAb-Lys3 as reported in our previous study. 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. Prepare a hen egg …

Materials

Reagents
KH2PO4	Sigma-Aldrich	V000225
K2HPO4	Sigma-Aldrich	1551128
NaCl	Sigma-Aldrich	S7653
Tris–HCl	Roche	10812846001
EDTA	Sigma-Aldrich	E9884
KCl	Sigma-Aldrich	P9541
Na2HPO4	Sigma-Aldrich	NIST2186II
2-mercaptoethanol	Sigma-Aldrich	M6250
Alanine	Sigma-Aldrich	A7627
HClO4	Fluka	34288	1M HClO4 solution, distributor : Sigma-Aldrich
Casein hydrolysate	Sigma-Aldrich	22090
Benzylpenicillin sodium	Sigma-Aldrich	B0900000
Hen egg white lysozyme	Roche	10837059001
Heptane	Sigma-Aldrich	246654
Methanol	Sigma-Aldrich	322415
Ammonium hydroxide solution	Sigma-Aldrich	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, successor Autolab PGSTAT302N
Digital multimeter, METRAHit 22M	Gossen Metrawatt		discontinued, successor Metrahit Base