Human-Derived Split Protein Actuators for Immune-Compatible Control
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Solution Overview
Problem
Existing chemically inducible dimerization systems face limitations due to undesirable characteristics of ligands and non-human protein components, leading to immune recognition and limited diversity, which hampers their clinical application and simultaneous regulation of multiple processes in human cells.
Innovation Solution
Designing chemically inducible split protein actuators (CISPAs) using human-derived ligand-binding proteins split into two fragments that reassemble only in the presence of a cognate ligand, with one fragment being significantly smaller than the other, allowing for precise regulation of cellular processes and therapeutic applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If non-human protein components are used in chemically inducible dimerization systems, then dimerization function is achieved, but immune recognition and elimination occur
Solution Approach 1:
The patent changes the fundamental parameter of protein origin from non-human to human-derived. By selecting ligand-binding proteins from human sources (such as human FKBP12, human FRB, or other human protein domains), the system maintains functional dimerization capability while eliminating immune recognition issues. This parameter change directly resolves the contradiction between achieving reliable dimerization and avoiding immune responses.
2Ease of operation
If ligand-binding proteins are split into two fragments, then precise regulation of cellular processes is achieved, but system complexity increases
Solution Approach 1:
The patent applies segmentation by dividing ligand-binding proteins into two separate fragments (first fragment and second fragment). Each fragment can be independently expressed and controlled. When the ligand is present, the fragments dimerize to form the functional complex. This segmentation enables precise spatial and temporal regulation of cellular processes, as the system remains inactive until both fragments are present and the ligand triggers their association.
Solution Approach 2:
The patent introduces a ligand as an intermediary molecule that mediates the interaction between the two protein fragments. The ligand binds to specific sites on each fragment, inducing a conformational change that promotes dimerization. This intermediary mechanism provides precise control over when and where the protein complex forms, enabling regulated activation of downstream effects without direct complexity in the protein-protein interaction interface.
3Adaptability or versatility
If diverse ligands are used for simultaneous regulation of multiple processes, then versatility is improved, but system design complexity increases
Solution Approach 1:
The patent employs universal human-derived protein domains (such as FKBP12, FRB, or other ligand-binding domains) that can be paired with different effector proteins. The same ligand-binding module can regulate multiple different cellular processes by simply changing which effector protein it is fused to. This universality allows simultaneous or sequential regulation of multiple processes using the same basic architecture, reducing design complexity compared to creating entirely separate systems for each function.
Solution Approach 2:
The modular segmentation of the system into separate ligand-binding domain, dimerization interface, and effector domain allows for easy recombination. Different effector proteins can be paired with standardized ligand-binding modules to create multiple orthogonal regulation systems. This segmentation enables the design of versatile systems where the same ligand-binding technology can control multiple distinct biological processes without increasing overall system complexity.
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
CISPAs provide a human-compatible system for precise regulation of cellular processes and therapeutic applications, avoiding immune response and enabling simultaneous control of multiple processes using diverse ligands.
Implementation Method 1
selecting a ligand-binding protein or protein domain, wherein the ligand-binding protein or protein domain is capable of binding a ligand
Data Source
AI summary
The present invention relates to chemically inducible split protein actuators (CISPA), which utilize ligand-binding proteins or protein domains originating from humans or other organisms, which are rationally split into two fragments that reassemble only in the presence of a cognate ligand. In particular, the invention relates to their design, manufacture, structure, and uses. The designed CISPAs can be used to regulate cellular processes such as gene expression, conditionally reconstitute of the function of a protein such as enzyme activity, as biological sensors, or for other applications.