Allosteric Protein Design via Directed Evolution
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Solution Overview
Problem
Rational protein design methods are often unsuccessful due to complex, non-intuitive interactions governing protein structure and function, making it challenging to engineer mutant proteins effectively.
Innovation Solution
The development of methods and compositions for designing allosteric proteins that bind to target small molecules, inducing conformational changes, which can be used to create sensor proteins and inducible gene expression systems, including orthogonal and inducible systems for mammalian cell culture and large-scale fermentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If rational mutagenesis methods are used to engineer protein mutants, then detailed knowledge of structure and function can be utilized, but the methods are generally unsuccessful due to complex non-intuitive interactions
Solution Approach 1:
The patent employs directed evolution to systematically change protein parameters through random mutagenesis and selection, bypassing the need to understand complex interaction mechanisms. This approach transforms the unreliable rational design process into a reliable evolutionary selection process where successful mutants are identified empirically rather than predicted theoretically
Solution Approach 2:
The patent utilizes the protein's own evolutionary capability by implementing in vitro evolution systems where proteins undergo self-mutation and self-selection. The system allows proteins to serve themselves by generating diversity through mutagenesis and selecting functional variants based on their inherent properties, eliminating the need for external rational design intervention
2Adaptability or versatility
If existing gene expression systems like Tet-On or IPTG-based systems are used, then gene expression can be regulated, but these systems lack orthogonality and applicability to different cell types and scales
Solution Approach 1:
The patent creates universal allosteric protein regulators that can function across different cell types and scales through directed evolution. The evolved proteins possess universal binding capabilities for small molecule effectors while maintaining orthogonality, allowing the same regulatory mechanism to be applied from mammalian cells to large-scale fermentation systems without cross-interference
Solution Approach 2:
The patent introduces small molecule effectors as intermediaries that mediate between the allosteric protein regulators and gene expression control. These effector molecules serve as universal signals that can be detected and responded to by engineered proteins in different biological systems, enabling cross-system compatibility while maintaining regulatory orthogonality
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
These methods enable the design of allosteric proteins that respond specifically to effector molecules, allowing for precise regulation of gene expression and biosynthetic pathways, overcoming limitations of existing systems like Tet-On and IPTG-based methods.
Implementation Method 1
an allosteric protein of the present invention binds an effector molecule and undergoes a conformational change, causing an increase or a decrease in one or more activities of the protein
Data Source
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AI summary
Methods and compositions for making and isolating allosteric DNA binding proteins that bind to one or more allosteric effectors to induce a conformation change in the proteins are provided.