Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

Abstract

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Introduction

Antibodies are composed of two identical heavy chains and two light chains that are responsible for the antigen binding. Camelids have heavy-chain only antibodies that exhibit similar affinity for their cognate antigen compared to conventional antibodies1,2. The single variable domain (VHH) of heavy-chain only antibodies retain the full antigen-binding potential and has been shown to be very stable1,2. These isolated VHH molecules or "nanobodies" have been implemented in studies related to membrane proteins biochemistry as tools for stabilizing....

Protocol

1. Membrane protein reconstitution

  1. Mix 3 mL of E. coli polar lipids with 1 mL of phosphatidylcholine in a round bottom flask under a ventilated hood.
  2. Dry the lipid mixture for 20 min under vacuum using a rotary evaporator and a water bath at 37 °C to remove chloroform. If needed, dry further under nitrogen or argon gas.
  3. Using TS buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl) containing 2 mM β-mercaptoethanol, resuspend lipids to 25 mg/mL.
  4. Aliquot lipids in 500 .......

Representative Results

SSM-based electrophysiology has been extensively used for the characterization of electrogenic transporters. In the protocol presented here, we show how to use SSM-based electrophysiology to classify nanobodies targeting a secondary transporter (here a bacterial choline symporter) based on their inhibitory and non-inhibitory properties. One of the most useful features of this technique is that it allows for the high-throughput screening of multiple buffer conditions. This particular characteristic is beneficial for the a.......

Discussion

The technique presented here classifies nanobodies with inhibitory and non-inhibitory properties targeting electrogenic transporters. Assessing the substrate transport is possible due to the detection of the movement of charges through the transporter embedded in the membrane of proteoliposomes. Some of the critical steps during the setup of an experiment are reconstitution of active protein in liposomes, preparation of stable monolayers on SSM chips, and recovering of initial conditions after the application of the wash.......

Acknowledgements

We thank Cedric A. J. Hutter and Markus A. Seeger from the Institute of Medical Microbiology at the University of Zurich, and Gonzalo Cebrero from Biozentrum of the University of Basel for collaboration in the generation of synthetic nanobodies (sybodies). We thank Maria Barthmes and Andre Bazzone from NANION Technologies for technical assistance. This work was supported by the Swiss National Science Foundation (SNSF) (PP00P3_170607 and NANION Research Grant Initiative to C.P.).

....

Materials

NameCompanyCatalog NumberComments
1-octadecanethiol solutionSigma AldrichO1858-25ML
1,2-diphytanoyl-sn-glycero-3-phosphocholineAvanti Polar Lipids850356C-25mg
Bio-Beads SM-2 Adsorbent (Polystyrene adsorbent beads)BioRad#152-3920
PD 10 Desalting ColumnsGE HealthcareGE17-0851-01
Filter 200 nm membraneWhatman NucleoporeWHA800282
2-PropanolMerck33539-1L-R
n-DecaneSigma Aldrich8034051000
n-dodecyl-ß-D-maltoside (DDM)Avanti Polar Lipids850520P-25g
Sodium ChlorideAppliChem131659.1211
(SSM setup) SURFE2R N1Nanion-----
SURFE2R N1 Single Sensor ChipsNanion# 161001
Trizma BaseSigma AldrichT1503
E. coli Polar Lipid ExtractAvanti Polar Lipids100600C
Egg PC L-α-phosphatidylcholine Avanti Polar Lipids840051C

References

  1. Braden, B. C., Goldman, E. R., Mariuzza, R. A., Poljak, R. J. Anatomy an antibody molecule: structure, kinetics, thermodynamics, and mutational studies of the antilysozyme antibody D1.3. Immunology Reviews. 163, 45-57 (1998).
  2. Hamers-Casterman, C., et al.

Explore More Articles

Transporter targeted Inhibitory NanobodiesSolid supported membrane SSM based ElectrophysiologyHigh throughput ScreeningElectrogenic TransportersProteoliposomesMembrane VesiclesNanobodiesMedical ApplicationsElectrogenic TransportersSSM BufferActivating SSM BuffersProteoliposome coated ChipConductivityCapacitanceActivating SolutionsNonactivating BufferBAB SequenceProtein free Liposomes

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved