Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs
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 n...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-01-01
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| Series: | BBA Advances |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667160321000211 |
| _version_ | 1819097976952324096 |
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| author | Pooja Sharma Matthew Plant Sheung Kwan Lam Qing Chen |
| author_facet | Pooja Sharma Matthew Plant Sheung Kwan Lam Qing Chen |
| author_sort | Pooja Sharma |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T00:23:39Z |
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| id | doaj.art-ec1e472128ac483db74b592db91ec414 |
| institution | Directory Open Access Journal |
| issn | 2667-1603 |
| language | English |
| last_indexed | 2024-12-22T00:23:39Z |
| publishDate | 2021-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | BBA Advances |
| spelling | doaj.art-ec1e472128ac483db74b592db91ec4142022-12-21T18:45:06ZengElsevierBBA Advances2667-16032021-01-011100022Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPsPooja Sharma0Matthew Plant1Sheung Kwan Lam2Qing Chen3Discovery Attribute Sciences, Amgen Research, Amgen Inc., Thousand Oaks, CA, 91320; Corresponding author.Discovery Attribute Sciences, Amgen Research, Amgen Inc., Cambridge, MA, 02141Biologics, Amgen Research, Amgen Inc., South San Francisco, CA 94080Discovery Attribute Sciences, Amgen Research, Amgen Inc., Thousand Oaks, CA, 91320The 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.http://www.sciencedirect.com/science/article/pii/S2667160321000211Grating-coupled Interferometry (GCI)Surface Plasmon Resonance (SPR)Antibodies and SMALPs (Styrene Maleic Acid Lipid Particles)Membrane Proteins (MP)Binding KineticsLabel Free |
| spellingShingle | Pooja Sharma Matthew Plant Sheung Kwan Lam Qing Chen Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs BBA Advances Grating-coupled Interferometry (GCI) Surface Plasmon Resonance (SPR) Antibodies and SMALPs (Styrene Maleic Acid Lipid Particles) Membrane Proteins (MP) Binding Kinetics Label Free |
| title | Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs |
| title_full | Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs |
| title_fullStr | Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs |
| title_full_unstemmed | Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs |
| title_short | Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs |
| title_sort | kinetic analysis of antibody binding to integral membrane proteins stabilized in smalps |
| topic | Grating-coupled Interferometry (GCI) Surface Plasmon Resonance (SPR) Antibodies and SMALPs (Styrene Maleic Acid Lipid Particles) Membrane Proteins (MP) Binding Kinetics Label Free |
| url | http://www.sciencedirect.com/science/article/pii/S2667160321000211 |
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