Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs

Abstract

The fundamental importance of membrane protein (MP) targets in central biological and cellular events has driven a marked increase in the use of membrane mimetics for exploring these proteins as therapeutic targets. The main challenge associated with biophysical analysis of membrane protein is the need for detergent extraction from the bilayer environment, which in many cases causes the proteins to become insoluble, unstable or display altered structure or activity. Recent technological advances have tried to limit the exposure of purified membrane protein to detergents. One such method involves the amphipathic co-polymer of styrene and maleic acid (SMA), which can release lipids and integral membrane proteins into water soluble native particles (or vesicles) termed SMALPs (Styrene Maleic Acid Lipid Particles). In this study, assay conditions that leverage SMA for membrane protein stabilization were developed to perform kinetic analysis of antibody binding to integral membrane protein and complexes in SMALPs in both purified and complex mixture settings using multiple biosensor platforms. To develop a robust and flexible platform using SMALPs technology, we optimized various SPR assay formats to analyze SMALPs produced with cell membrane pellets as well as whole cell lysates from the cell lines overexpressing membrane protein of interest. Here we emphasize the extraction of model membrane proteins of diverse architecture and function from native environments to encapsulate with SMALPs. Given the importance of selected membrane targets in central biological events and therapeutic relevance, MP-specific or tag-specific antibodies were used as a proof-of-principal to validate the SMALPs platform for ligand binding studies to support drug discovery or tool generation processes. MP-SMALPs that retain specific binding capability in multiple assay formats and biosensors, such as waveguide interferometry and surface plasmon resonance, would be a versatile platform for a wide range of downstream applications.

Keywords: Antibodies and SMALPs (Styrene Maleic Acid Lipid Particles); Binding Kinetics; Detergent Free; Grating-coupled Interferometry (GCI); Label Free; Membrane Proteins (MP); Surface Plasmon Resonance (SPR).

Graphical abstract

Fig. 1

Purification and Characterization of SMALPs produced from whole cell pellets and isolated membranes. SMALPs produced from isolated membranes or whole-cell pellets were analyzed by both denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE). (A) MPR1/MPA1 SMALPs purified from whole cells using either anti-FLAG or Ni-NTA resin. The SMALPs were analyzed by SDS-PAGE and SMA-PAGE. (B) MPR2 SMALPs purified from isolated membranes using either anti-FLAG or Ni-NTA resin. The SMALPs were analyzed by SDS-PAGE and SMA-PAGE. Blue stars identify SMALPs containing membrane proteins of various quaternary structures or protein complex stoichiometries.

Fig. 2

Purified SMALPs bind specifically to surface captured α-Target antibodies. Increasing concentrations of purified MPR1/MPA1 SMALPs in solution were interacted with surface bound α-target antibodies on a Creoptix Waveguide GCI system. (A) SDS-PAGE analysis of MPR1/MPA1 SMALPs and the assay configuration for binding analysis shown in panel B. (B) Referenced dose-dependent binding response sensorgrams of purified MPR1/MPA1 SMALPs and α-MPR1 antibody captured to surface coupled α-Hu Fc antibody (C) No binding detection from sensorgram responses of increasing concentrations of purified MPR1/MPA1 SMALPs and a directly surface coupled α-MPR2 antibody. (D) Sensorgram binding responses of increasing concentrations of purified MPR1/MPA1 SMALPs and a directly surface coupled α-MPR1 antibody.

Fig. 3

Comparison of assay setups for binding analysis of antibodies and membrane proteins stabilized in SMALPs. Assay configurations for SMALPs vs antibody binding studies using amine coupling or complementary DNA strands are visualized. (A) Assay design for amine coupling of captured antibodies are shown using PCH Creoptix chips. (B) Assay design for streptavidin immobilization of biotinylated capture antibodies are shown using the Biacore CAP chips.

Fig. 4

Antibody binding responses after SMALPs capture from total cellular SMALPs. Total cellular SMALPs produced with cell membrane pellets from cell lines expressing MPR2-His-and MPR2-FLAG were used to capture MPR2 SMALPs with surface captured α-TAG antibodies. Subsequent captured SMALPs were then used for antibody binding studies. (A) Assay configuration for SMALPs capture and target-specific antibody binding. (B) Capture levels for all flow cells using total SMALPs from overexpressing cell lines and parental cell lines as a background control. (C) Dose-dependent binding response levels of α-MPR2 antibody with surface captured α-His-or α -FLAG MPR2 SMALPs.

Fig. 5

Comparison of assay formats for antibody binding to FLAG captured MPR2 SMALP. Total cellular SMALPs produced with cell membrane pellets from a cell line expressing MPR2 -FLAG was used to capture MPR2 SMALPs with surface captured α-FLAG antibody in two separate formats. Subsequent captured SMALPs were then used for antibody binding studies. (A) Assay configuration for SMALPs capture and target- specific antibody binding using a direct surface capture of a biotinylated α-FLAG antibody. (B) Assay configuration for SMALPs capture and target-specific antibody binding using a stacked surface capture of biotinylated α-Mu Fc antibody with α-FLAG antibody. (C) Capture levels for all flow cells using total SMALPs from an overexpressing MPR2-FLAG cell line and the parental cell lines as a background control. (D) Dose-dependent binding response levels of α-MPR2 antibody with surface captured α-FLAG MPR2 SMALPs using the two different capture formats.

Fig. 6

Demonstration of antibody binding specificity with MPR2 and MPR1/MPA1 SMALPs using target-specific antibodies and SMALPs α-FLAG capture. Total cellular SMALPs produced with cell membrane pellets from a cell line expressing MPR2-FLAG and total cell pellets from a cell line overexpressing MPR1/MPA1 were used to demonstrate the specificity of target-specific antibodies and the various SMALPs species. SMALPs were captured on the chip surface using a cell surface captured biotinylated α-FLAG antibody and the Biacore CAP chip. (A) Assay configuration for SMALPs capture and target-specific antibody binding using a direct surface capture of a biotinylated α-FLAG antibody using the Biacore CAP chip. (B) Capture levels for all flow cells using total SMALPs from an overexpressing MPR2 -FLAG cell line and the parental cell lines as a background control. (C) Dose-dependent binding response levels of α-MPR2 antibody to α-FLAG captured MPR2 and MPR1/MPA1 SMALPs. (D) Dose-dependent binding response levels of α-MPR1 antibody to α-FLAG captured MPR2 and MPR1/MPA1 SMALPs.