PACE Protein Stability via Negative Selection
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Solution Overview
Problem
Proteins evolved using conventional methods often suffer from decreased stability and solubility issues, making them unsuitable for therapeutic or commercial use.
Innovation Solution
The use of phage-assisted continuous evolution (PACE) systems, including specific expression constructs and vectors, to enhance the stability and solubility of proteins by employing negative selection strategies, such as the E. coli sigma 32 reporter system and split T7 RNA polymerase complementation, and solubility-based selection systems like the Npu split intein and GCN4 tag systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional iterative evolution methods are used to evolve proteins, then protein diversity is generated, but the time and human effort required are excessive
Solution Approach 1:
The patent implements continuous evolution through a PACE system where a phage population continuously replicates and evolves in a lagoon over extended periods (months to years). The system maintains continuous mutation and selection pressure without interruption, allowing proteins to evolve continuously rather than through discrete iterative steps. This continuous action dramatically accelerates the evolution process compared to conventional batch methods.
2Adaptability or versatility
If proteins are evolved to have novel functionality, then protein diversity increases, but stability and solubility decrease
Solution Approach 1:
The patent implements feedback mechanisms through multiple selection systems that monitor and respond to protein stability and solubility. The sigma32 promoter system provides feedback by activating expression of dominant-negative pIII in response to misfolded proteins, while the Npu intein system provides feedback by preventing maturation of pIII when proteins are insoluble. These feedback loops continuously screen evolving proteins and eliminate unstable or insoluble variants, maintaining protein quality throughout the evolution process.
Solution Approach 2:
The patent uses intermediary systems to mediate between protein evolution and stability selection. The sigma32 promoter acts as an intermediary that translates protein folding status into gene expression signals. The Npu intein serves as an intermediary that translates protein solubility into maturation signals for pIII. These intermediaries enable indirect selection of stable and soluble proteins without directly measuring these properties.
3Reliability
If selection pressure is applied to enrich desired variants, then protein function is improved, but properly folded and soluble proteins are not adequately selected
Solution Approach 1:
The patent segments the selection process into multiple independent but complementary systems: (1) function-based selection through phage propagation, (2) stability-based selection through the sigma32 promoter system, and (3) solubility-based selection through the Npu intein system. Each segment addresses a specific aspect of protein quality, and together they provide comprehensive selection for properly folded, soluble, and functional proteins.
Solution Approach 2:
The patent merges multiple selection systems into a unified PACE framework. The sigma32 promoter system and Npu intein system are combined with traditional phage propagation selection to create an integrated selection apparatus. This merging allows simultaneous selection for multiple protein properties (function, stability, solubility) within a single continuous evolution experiment, rather than requiring separate experiments for each property.
Data Source
AI summary
Some aspects of this disclosure relate to systems, apparatuses, compositions (e.g., isolated nucleic acids and vectors), and methods for improving the stability and/or solubility of proteins evolved using phage-assisted continuous evolution (PACE). In some embodiments, vectors described herein comprise nucleic acids encoding selection systems (e.g., positive and/or negative selection systems) that link expression of genes required for production of infectious phage particles to a desirable physiochemical (e.g., stability or solubility) and/or desired function of an evolved protein.