Poxvirus EEV Display for Integral Membrane Protein Conformation
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Solution Overview
Problem
Current methods face challenges in expressing and displaying integral membrane proteins (IMPs) in their native conformation for therapeutic antibody development, particularly for multi-pass membrane proteins, due to difficulties in proper folding and post-translational modifications in bacterial systems and non-specific binding issues in eukaryotic systems.
Innovation Solution
The use of poxvirus-infected cells to express IMPs as fusion proteins with FPV108 or RBXV041 on extracellular enveloped virions (EEVs), allowing for native conformation display and selection of antibodies through recombinant poxvirus EEVs, which facilitates the identification of specific antibody binding domains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If integral membrane proteins are expressed in bacterial cells, then protein production is achieved, but proper folding and post-translational modifications are compromised
Solution Approach 1:
The patent uses eukaryotic cell lines (such as Chinese hamster ovary cells, human embryonic kidney cells, or mammalian cell lines) as intermediary systems to express integral membrane proteins. These eukaryotic cells provide the necessary cellular machinery for proper protein folding and post-translational modifications while maintaining high protein production levels, thus resolving the contradiction between productivity and manufacturing precision.
Solution Approach 2:
The patent employs site-directed mutagenesis to introduce specific amino acid mutations in the integral membrane proteins to improve their solubility and stability in eukaryotic cells. By changing specific parameters (amino acid sequences) of the proteins, the patent enables proper folding and post-translational modifications while maintaining high expression levels, thus resolving the contradiction between productivity and manufacturing precision.
2Quantity of substance
If whole cells are used for antibody discovery, then target expression is achieved, but non-specific binding occurs and target expression concentration is insufficient
Solution Approach 1:
The patent extracts and purifies the integral membrane proteins from the cell surface using specific protocols, separating the target protein from other cell surface proteins and components. This extraction process enables the use of purified target proteins at high concentrations in antibody discovery assays, eliminating non-specific binding issues while maintaining high target expression concentration.
Solution Approach 2:
The patent creates recombinant viral particles (such as vaccinia virus or adenovirus) that express the integral membrane proteins on their surface. These viral particles serve as simplified models that display the target protein in a controlled manner, enabling high concentration target expression without the complexity of whole cells, thus resolving the contradiction between quantity and reliability.
Data Source
AI summary
This disclosure provides compositions and methods for expressing and displaying isolated integral membrane proteins (IMPs) or fragments thereof in a native conformation on poxvirus extracellular virions and methods for screening, selecting, and identifying antibodies or antibody-like molecules that bind to a target IMP of interest.