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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.
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.
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....
1. Membrane protein reconstitution
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.......
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.......
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.).
....Name | Company | Catalog Number | Comments |
1-octadecanethiol solution | Sigma Aldrich | O1858-25ML | |
1,2-diphytanoyl-sn-glycero-3-phosphocholine | Avanti Polar Lipids | 850356C-25mg | |
Bio-Beads SM-2 Adsorbent (Polystyrene adsorbent beads) | BioRad | #152-3920 | |
PD 10 Desalting Columns | GE Healthcare | GE17-0851-01 | |
Filter 200 nm membrane | Whatman Nucleopore | WHA800282 | |
2-Propanol | Merck | 33539-1L-R | |
n-Decane | Sigma Aldrich | 8034051000 | |
n-dodecyl-ß-D-maltoside (DDM) | Avanti Polar Lipids | 850520P-25g | |
Sodium Chloride | AppliChem | 131659.1211 | |
(SSM setup) SURFE2R N1 | Nanion | ----- | |
SURFE2R N1 Single Sensor Chips | Nanion | # 161001 | |
Trizma Base | Sigma Aldrich | T1503 | |
E. coli Polar Lipid Extract | Avanti Polar Lipids | 100600C | |
Egg PC L-α-phosphatidylcholine | Avanti Polar Lipids | 840051C |
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