CPP Tagged Adaptor Proteins for Intracellular Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for delivering proteins into cells are limited by the need for multiple tags, which can be hazardous and complicate purification, and often require advanced methods to distinguish membrane-associated constructs from translocated ones, leading to artifacts in studying cell penetration mechanisms.
Innovation Solution
The use of CPP tagged adaptor proteins, such as TAT-calmodulin, which facilitate the internalization of proteins through strong protein-protein interactions, allowing for the delivery of a broad range of payloads into cells using a unified strategy that simplifies purification and reduces hazards, and enables dissociation upon internalization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple tags are used to deliver proteins into cells, then delivery capability is improved, but purification complexity and hazards increase
Solution Approach 1:
The system divides the delivery function into two separate components: a cell-penetrating peptide (CPP) that handles membrane penetration and an adaptor protein that handles payload binding and internalization. This segmentation allows each component to be optimized independently and simplifies purification since only the adaptor needs to be purified, not the entire CPP-payload construct.
Solution Approach 2:
The adaptor protein serves as an intermediary between the CPP and the payload. The CPP binds to the adaptor, which in turn binds to the payload, creating a stepwise assembly that simplifies purification and reduces hazards compared to direct CPP-payload conjugation.
2Productivity
If cell membrane permeability is enhanced to deliver proteins into cells, then delivery efficiency is improved, but hazards and artifacts increase
Solution Approach 1:
The patent converts the potentially harmful effect of membrane permeability enhancement into a beneficial controlled process. By using CPP-adaptor complexes, the system achieves efficient delivery while maintaining control over the process, reducing artifacts in studying cell penetration mechanisms.
Solution Approach 2:
The adaptor protein acts as a temporary, disposable intermediary that facilitates delivery and then can be discarded or degraded, avoiding the need for permanent modification of the payload or cell membrane.
3Measurement precision
If advanced methods are used to distinguish membrane-associated constructs from translocated ones, then measurement precision is improved, but complexity increases
Solution Approach 1:
The patent employs fluorescent tags that enable visual distinction between membrane-associated and translocated forms of the construct. This allows real-time detection and differentiation of constructs at various stages of internalization without requiring complex analytical methods.
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 enables efficient and safe delivery of proteins into cells, allowing for real-time detection of internal components and manipulation of signaling pathways, while reducing the risks associated with cell membrane permeability enhancement and simplifying protein purification.
Implementation Method 1
Proteins tagged with a variety of cell penetrating peptides (CPPs) have been used to manipulate the interior of cells in culture and in situ for more than a decade
Implementation Method 2
The use of CPP tagged adaptor proteins, such as TAT-calmodulin, which facilitate the internalization of proteins through strong protein-protein interactions
Data Source
AI summary
Coupling proteins that make strong protein-protein interactions equipped with cell penetrating peptides (CPPs) provide a convenient and powerful method to perturb cell interiors; there are many potential payloads and a broad palette of selectively membrane permeable probes. In a preferred embodiment, the coupling protein will be calmodulin or a related calcium binding protein. In a preferred embodiment, the CPP will be TAT or another CPP. In a preferred embodiment, the coupling protein will release its payload after targeting to an interior compartment. Cargo proteins can be purified by affinity methods using the same tag that allows binding by the adaptor, enabling an integrated approach with ‘gains in both function and safety. Access to cell interior compartments has potential applications in research, diagnostics, and therapeutics.