Abstract
Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.
Highlights
Multi-pass membrane proteins are important targets of biologic medicines
Upon successful purification and incorporation of voltage-sensing domain 4 (VSD4)-Nav from Arcobacter butzleri (NavAb) into nanodiscs, we demonstrate their use in Fluorescence-activated cell sorting (FACS) of antigen-specific hybridoma
In the bacterial sequence of Nav from Arcobacter butzleri (NavAb), the outward facing half of the voltage sensing domain was replaced with human sequences from domain IV of human Nav1.7 channel yielding the VSD4-NavAb construct (Fig. 1)
Summary
Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins. Removal of the detergent results in the target membrane protein assembling into a phospholipid bilayer held together by two molecules of MSP encircling it as a helical protein belt In this form, the intracellular and extracellular domains of the membrane protein are exposed to solvent with the hydrophobic transmembrane domains interacting with the lipid acyl chains which in turn interact with the amphipathic helices of the MSP14. We sought to determine whether nanodisc technology offers a means to FACS hybridoma cells specific to target multi-pass integral membrane proteins. The use of target-loaded nanodiscs for FACS allows us to circumvent the need for polyclonal plating of hybridoma and vastly improves the efficiency of identifying antigen-specific hybridoma
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