Yeast Display System Adapter Molecule for Protein Folding
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Solution Overview
Problem
Current protein display methods, such as phage display and bacterial display, face challenges including denaturation of target proteins with high affinity, steric hindrance, and inability to provide post-translational modifications, limiting the identification of proteins with desired binding affinities and modifications.
Innovation Solution
A three-component system using yeast cells with a cell surface molecule, an adapter molecule with specific binding sites, and a display molecule, where the adapter molecule binds specifically to both the cell surface and the display molecule, allowing for proper folding and post-translational modifications, and efficient surface display of proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If phage display method is used to screen protein libraries, then high affinity binding proteins can be identified, but target proteins with very high affinity are not always identified because elution conditions denature the phage particle
Solution Approach 1:
The patent introduces a biotin-avidin system as an intermediary between the phage display library and the solid support. The biotinylated target protein binds to avidin immobilized on the solid support, and the phage display library binds to the biotinylated target. This intermediary system allows for stable binding without requiring harsh elution conditions that would denature the phage particle, thus maintaining both measurement precision and reliability.
Solution Approach 2:
The patent changes the binding parameters by using biotin-avidin interaction instead of direct protein-protein binding on the solid support. This parameter change allows for reversible binding under mild conditions, enabling the identification of high affinity binders without denaturing the phage particle through harsh chemical or physical treatments.
2Productivity
If target protein is immobilized on solid surface for screening, then library can be screened, but difficulties arise in determining the actual affinity of target protein for phage display protein
Solution Approach 1:
The biotin-avidin system serves as a mediator that separates the immobilization function from the binding measurement function. The avidin-biotin interaction provides stable immobilization for high-throughput screening, while the phage-display protein-target protein interaction remains in solution or on the surface in a manner that preserves native binding characteristics, allowing accurate affinity determination.
3Reliability
If protein library is displayed on bacterial cells, then many drawbacks of phage display are solved, but bacterial capsule causes steric hindrance to displayed proteins
Solution Approach 1:
The patent extracts the problematic bacterial capsule by using yeast cells instead of bacteria as the display host. Yeast cells lack the thick capsule that causes steric hindrance in bacterial display systems, while still providing eukaryotic cellular machinery for proper protein folding and post-translational modifications. This extraction of the harmful element (capsule) maintains the advantages of cellular display without the steric hindrance.
4Productivity
If eukaryotic proteins are expressed in bacteria, then library can be displayed, but bacteria cannot provide post-translational modifications like glycosylation and disulfide bonding
Solution Approach 1:
The patent copies the eukaryotic cellular environment by using yeast cells as the expression host instead of bacteria. Yeast provides the eukaryotic machinery necessary for proper folding, glycosylation, and disulfide bond formation, while maintaining high expression efficiency. This copying of the eukaryotic cellular context enables both high productivity and manufacturing precision for eukaryotic proteins.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This system enables specific and efficient binding of secreted protein libraries on eukaryotic cells, overcoming previous limitations by allowing for proper folding and post-translational modifications, and facilitating the identification of proteins with desired binding affinities.
Implementation Method 1
an adapter molecule with specific binding sites, and a display molecule, where the adapter molecule binds specifically to both the cell surface and the display molecule
Data Source
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AI summary
The present invention relates to the field of protein display libraries and library screening, In preferred embodiments, the present invention provides a three component system for display comprising a cell surface molecule, an adapter molecule and a display molecule.