Lipoparticles comprising proteins, methods of making, and using the same
Inventors
Doranz, Benjamin J. • Willis, Sharon • Ross, Eric • Greene, Tiffani Anne
Assignees
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Publication Number
US-8574590-B2
Publication Date
2013-11-05
Expiration Date
Abstract
The present invention relates to lipoparticles. The invention also relates to producing lipoparticles. The invention further relates to lipoparticles comprising a viral structural protein. The invention further relates to a lipoparticle comprising a membrane protein, and the lipoparticle can be attached to a sensor surface. The invention further relates to methods of producing and using the lipoparticle to, inter alia, assess protein binding interactions.
Core Innovation
The invention relates to lipoparticles that comprise an external lipid bilayer, an enveloped retroviral structural protein, and a multiple membrane spanning protein. The enveloped retroviral structural protein is an uncleaved gag protein, and the lipoparticle is defined so that only viral proteins present are structural proteins. In some embodiments, the gag protein is unmodified and does not comprise a heterologous tag, and in one embodiment the multiple membrane spanning protein does not bind to the gag protein.
The disclosure further describes incorporation of membrane proteins in a way that supports imaging, quantification, preservation of native membrane protein structure, and receptor integrity. Fluorescent-reporter fusion constructs associated with Gag and membrane proteins include Gag-GFP, CXCR4-GFP, and CCR5-GFP, and the assay context includes correlation across fluorescent imaging, DLS, and spectroscopy. It also describes receptor quantification using V5-tagged GPCRs with Western blot and other detection formats, radioligand binding, and antibody-based detection including conformation-specific antibodies in ELISA formats such as VELISA and AVELISA.
Additional embodiments include biosensor or sensor surface detection, including binding and affinity/on-off behavior analysis on sensor surfaces in array format. The disclosure also mentions multiplexed antigen detection, structural integrity assays using conformation-dependent monoclonal antibodies, reporter systems such as fluorescence, luminescence, radioactivity, and LipoProbe architecture for real-time signaling and antigen detection, as well as immunofluorescence, flow cytometry, and other sensor surface detection contexts.
Claims Coverage
The provided independent claim set contains 3 independent claims. They focus on lipoparticles with an external lipid bilayer, an enveloped retroviral structural protein, and a multiple membrane spanning protein, with three principal inventive-feature themes: uncleaved gag with no binding to the multiple membrane spanning protein, unmodified uncleaved gag, and uncleaved gag without a heterologous tag.
Separated gag protein and non-binding multiple membrane spanning protein
A lipoparticle comprising an external lipid bilayer, an enveloped retroviral structural protein that is an uncleaved gag protein, and a multiple membrane spanning protein, wherein the multiple membrane spanning protein incorporated into the lipoparticle does not bind to the gag protein, provided that the only viral proteins in the lipoparticle are structural proteins.
Unmodified, uncleaved gag protein limitation
A lipoparticle comprising an external lipid bilayer, an enveloped retroviral structural protein that is an unmodified, uncleaved gag protein, provided that the only viral proteins in the lipoparticle are structural proteins.
Uncleaved gag protein without heterologous tag
A lipoparticle comprising an external lipid bilayer, an enveloped retroviral structural protein that is an uncleaved gag protein and wherein the gag protein does not comprise a heterologous tag, provided that the only viral proteins in the lipoparticle are structural proteins.
Across the independent claims, the lipoparticles are defined by an external lipid bilayer, an enveloped uncleaved gag protein together with a multiple membrane spanning protein, under the limitation that only viral structural proteins are present. One independent claim further requires that the multiple membrane spanning protein does not bind to the gag protein, while the other independent claims additionally specify the gag protein as unmodified and/or tag-free.
Stated Advantages
Maintains receptor integrity while enabling measurement of weak binding interactions.
Supports binding and affinity/on-off behavior analysis on sensor surfaces in array format.
Enables real-time signaling and/or antigen detection with reporter systems, including LipoProbe architecture.
Supports multiplexed antigen detection and structural integrity assays using conformation-dependent monoclonal antibodies.
Enables efficient imaging and quantification of lipoparticles via fluorescent reporter fusion constructs.
Provides quantitative assay principles to determine lipoparticle protein concentration and to correlate readouts across fluorescent imaging, DLS, and spectroscopy.
Allows receptor quantification per lipoparticle and supports assessment of receptor binding and structural integrity using conformation-specific antibodies and receptor-detection formats.
Preserves native membrane protein structure.
Enables biosensor-based binding and kinetics measurements using sensor surfaces.
Enables ligand and modulator screening for receptors such as GPCRs.
Enables immune responses, including monoclonal antibody generation against native membrane proteins and conformation-sensitive antibodies.
Enables detection of infectious pathogens using arrays of lipoparticles on sensor surfaces.
Documented Applications
Biosensor-based measurement of weak binding interactions by presenting membrane proteins as arrays on a biosensor surface, including platforms such as BIACORE and other sensor surfaces.
Infectious disease receptor biosensor use cases.
Water quality receptor biosensor use cases.
Antigen detection and multiplexed detection.
Structural integrity assays using conformation-dependent monoclonal antibodies.
Detection and quantification using fluorophore-labeled particles and reporter architectures, including real-time signaling and antigen detection with LipoProbe architecture.
Ion channel/transporter assays using fluorescent voltage- and ion-sensitive dyes.
Microfluidics and nano-scale sensing contexts.
Antigen presentation and antibody generation approaches, including immunogens/vaccines and tethered ligand/receptor examples.
Eliciting an immune response by introducing the lipoparticle into an animal.
Imaging and quantification of lipoparticles using GFP- and fluorescent-reporter fusion constructs.
Quantitative characterization of lipoparticles including protein concentration, size and purity, and receptor quantification per lipoparticle.
Assessment of structural integrity of incorporated membrane proteins using VELISA and AVELISA and antibody-based detection with conformation-specific antibodies.
Receptor binding assessment using radioligand binding for CXCR4 ligand binding and receptor concentration.
Detection via biosensor or sensor surface detection.
Biosensor-based binding and kinetics measurements on sensor surfaces.
Ligand and modulator screening for receptors such as GPCRs.
Immune response generation against multiple membrane spanning proteins, including monoclonal antibody generation and conformation-sensitive antibodies against native membrane proteins.
Detection of infectious pathogens using arrays of lipoparticles on sensor surfaces.
Eliciting an immune response to a multiple membrane spanning protein by introducing the lipoparticle into an animal.
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