Integral Membrane Protein Display on Poxvirus Virions
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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 discovery, particularly due to difficulties in correctly assembling antigen binding regions in bacterial cells and the complexity of whole cells as antigen mixtures.
Innovation Solution
The method involves expressing IMPs as fusion proteins with the vaccinia virus F13L protein on extracellular enveloped virions (EEVs), allowing for the display of IMPs in a native conformation, which facilitates the selection of antibodies binding to these proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If integral membrane proteins are expressed in bacterial cells, then antibody libraries can be constructed, but the antigen binding regions cannot be correctly assembled and folded
Solution Approach 1:
The patent uses a eukaryotic expression system (insect cells or mammalian cells) as an intermediary to properly fold and assemble the antigen binding regions of integral membrane proteins before presenting them on the virus surface. This intermediary system provides the necessary cellular machinery for correct protein folding that bacterial cells lack.
Solution Approach 2:
The patent changes the biological system parameter from bacterial to eukaryotic expression, which fundamentally alters the folding and assembly capabilities. This parameter change enables proper formation of complex antigen binding regions that require eukaryotic post-translational modifications and chaperone systems.
2Quantity of substance
If whole cells are used for antibody discovery, then integral membrane proteins can be displayed, but the cells become complex antigen mixtures with low target expression
Solution Approach 1:
The patent extracts the integral membrane protein of interest from the complex whole cell environment and displays it on the surface of a simplified virus particle. This extraction removes the complexity of other cellular antigens while maintaining the target protein's native conformation and accessibility.
Solution Approach 2:
The patent segments the antigen presentation system by separating the target IMP from the complex cellular background and displaying it on a distinct viral platform. This segmentation allows for focused antibody selection against the specific target without interference from other cellular proteins.
3Productivity
If retroviral gag fusion is used to display IMPs on VLPs, then antibodies can be screened, but the display concentration is insufficient and competition from cell proteins persists
Solution Approach 1:
The patent employs a virus particle that serves multiple functions: it provides a simplified antigen presentation surface, achieves high-level target protein expression, enables concentrated display on the viral surface, and facilitates efficient antibody screening. The viral system universally addresses all these requirements simultaneously.
Solution Approach 2:
The patent creates a composite structure by fusing the integral membrane protein with viral envelope proteins to generate a chimeric viral particle. This composite virus combines the target antigen with viral structural components, enabling stable display at high concentrations on the viral surface.
Data Source
Figure 1A~1B
Figure 1C
Figure 2
AI summary
This disclosure provides compositions and methods for expressing and displaying isolated integral membrane proteins (IMPs) or fragments thereof in a native conformation for use in the screening, selecting, and identifying of antibodies or antibody-like molecules that bind to a target IMP of interest.