Protein Display Selection via Post-Expression Covalent Bonding
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Solution Overview
Problem
Current protein display selection methods, such as phage display, face challenges in efficiently selecting proteins with specific properties due to biases introduced by artificial fusion proteins and the need for laborious subcloning steps, especially in high-throughput applications.
Innovation Solution
A protein display selection method that uncouples the protein of interest library from the display system by forming covalent bonds post-expression using enzymatic ligation or spontaneous bond formation, allowing for direct selection and screening without artificial fusion, using tethering and capture sequences like SpyTag/SpyCatcher, enabling high-throughput selection and versatile use across different display systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If genetic fusion between POI and pIII is used for phage display, then the POI can be displayed on the phage surface, but biases are introduced in protein selection and subcloning steps are required
Solution Approach 1:
The invention divides the display system into separate components: the POI library remains in the phagemid without fusion to pIII, while a separate capture reagent contains the pIII binding domain. This segmentation eliminates the need for genetic fusion between POI and pIII, thereby removing selection biases while maintaining display functionality.
Solution Approach 2:
The invention introduces a capture reagent as an intermediary component that contains the pIII binding domain. This capture reagent mediates the interaction between the phage display system and the POI, allowing selection without direct genetic fusion between POI and pIII, thus eliminating selection bias.
2Measurement precision
If subcloning is performed to express POI without pIII for screening, then POI can be expressed for screening, but the process becomes laborious and time-consuming
Solution Approach 1:
The invention performs preliminary action by maintaining the POI library in the phagemid in a ready-to-use state without requiring subcloning. The capture reagent is designed to work directly with the POI-pIII fusion expressed from the phagemid, eliminating the time-consuming subcloning step while preserving screening accuracy.
3Productivity
If conventional pH shift elution is used to elute POI, then elution can be achieved, but low-affinity POI are preferentially eluted causing selection bias
Solution Approach 1:
The invention changes the elution parameter from pH shift to disulfide bond reduction. By using a reducing agent to cleave the disulfide bond between pIII and the POI, the elution mechanism is fundamentally altered to be independent of POI affinity, thereby eliminating selection bias while maintaining efficient elution.
4Quantity of substance
If hyperphage is used to achieve multivalent display, then all 5 copies of pIII originate from the phagemid, but the system complexity increases
Solution Approach 1:
The invention extracts the pIII binding domain from the phagemid system and places it in a separate capture reagent. This allows the POI library to remain simple in the phagemid while achieving multivalent display through the capture reagent, thereby reducing system complexity while maintaining multivalency.
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 approach avoids biases in protein selection, reduces the need for subcloning, and allows for high display rates and efficient selection of proteins with specific properties, facilitating high-throughput applications and versatility across various display systems.
Implementation Method 1
forming a covalent bond between the POI and the anchor protein post expression either by enzymatic protein ligation (e.g. SpyLigase, SnoopLigase, sortase, butelase, peptiligase etc.) or by spontaneous covalent bond formation (e.g. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, etc.)
Data Source
AI summary
Described herein is a protein display selection method which uncouples a protein of interest (POI) library from the display selection system. Display of the POI can be achieved by forming a covalent bond between the POI and the anchor protein post expression either by enzymatic protein ligation (e.g. SpyLigase, SnoopLigase, sortase, butelase, peptiligase etc.) or by spontaneous covalent bond formation (e.g. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, etc.). The POI library is fused to a tethering sequence, for example SpyTag, at the C-terminus of the POI which then forms a covalent bond to a capture sequence found on an anchor protein, for example, the SpyCatcher-fused anchor protein, e.g., a SpyCatcher-geneIII protein (SpyCatcher-pIII) fusion, for the most common form of phage display. Nucleic acid constructs, host cell systems and methods of producing the protein display systems are also provided.