Surface Display Fusion Protein Reducing Steric Hindrance
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current surface display methods for proteins on eukaryotic cells are limited by the need for multiple gene expression and steric accessibility issues, making it difficult to achieve monocistronic expression and adjustable protein accessibility.
Innovation Solution
The method involves fusing a protein of interest with a signal polypeptide, a stalk polypeptide, and a surface anchor polypeptide to create a surface-accessible fusion protein, which can be introduced into eukaryotic cells using recombinant expression vectors, allowing for adjustable accessibility and binding measurements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple genes are used for surface display, then protein expression on cell surface is achieved, but system complexity and manufacturing difficulty increase
Solution Approach 1:
The patent combines multiple gene functions into a single fusion construct. The fusion protein integrates the protein of interest with the cell surface anchor (such as GPI anchor or transmembrane domain) in one continuous polypeptide chain, eliminating the need for separate gene expression systems while maintaining reliable surface display capability.
Solution Approach 2:
The invention creates a universal surface display system using a standardized fusion format. The通用 anchor sequences (GPI anchor or transmembrane domain) can be paired with any protein of interest, providing a multi-functional platform that works across different protein types without requiring protein-specific engineering of the anchoring mechanism.
2Reliability
If proteins are displayed on cell surface, then binding measurements can be performed, but steric accessibility is limited
Solution Approach 1:
The fusion protein is segmented into distinct functional domains: the protein of interest region and the anchor region (GPI anchor or transmembrane domain). This segmentation allows the protein of interest to be positioned optimally for binding accessibility while the anchor domain handles the cell surface attachment function, reducing steric hindrance between these functional elements.
Solution Approach 2:
The anchor domain acts as an intermediary between the protein of interest and the cell membrane. By positioning the anchor at the C-terminus or N-terminus depending on the configuration, it mediates the attachment to the cell surface while leaving the protein of interest exposed and accessible for binding interactions with ligands or antibodies.
Data Source
AI summary
Methods and compositions are provided for displaying a protein of interest (POI) on the surface of a eukaryotic cell by fusing the POI to a signal polypeptide, a stalk polypeptide, and a surface anchor polypeptide to generate a surface accessible fusion protein. Nucleic acids are provided that include nucleotide sequences encoding a signal polypeptide, a stalk polypeptide, and a surface anchor polypeptide. In some cases, a subject nucleic acid includes and insertion site for the insertion of a POI. In some cases, a subject nucleic acid includes a nucleotide sequence that encodes a POI. In some cases a stalk polypeptide is a synthetic stalk polypeptide and various example synthetic stalk polypeptides are disclosed. In some cases, a surface anchor polypeptide is a glycosylphosphatidylinisotol (GPI) anchor domain, which can be synthetic. Kits are also provided for practicing the subject methods.