Chimeric Protein Covalent Linking via Self-Processing Anhydride
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Solution Overview
Problem
Current methods for covalent protein conjugation face challenges in targeting endogenous proteins without modifying them, especially in therapeutic settings where immune responses need to be minimized, and often require complex post-translational modifications or unnatural amino acids, which are difficult to generalize and can be harmful due to issues like phototoxicity.
Innovation Solution
A system using chimeric polypeptides with a self-processing module that generates a reactive anhydride group upon calcium-induced autoproteolysis, allowing for specific covalent bonding with nucleophiles on other proteins under mild conditions, enabling efficient and specific conjugation of proteins without extensive modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If UV-induced photocrosslinking is used for protein conjugation, then conjugation efficiency is improved, but phototoxicity and limited tissue penetration occur making it unsuitable for cellular use
Solution Approach 1:
The patent replaces UV light-induced photocrosslinking with a chemical reaction system using a self-processing module that undergoes calcium-dependent autoproteolysis to generate a reactive anhydride group. This chemical substitution eliminates phototoxicity while maintaining conjugation efficiency, enabling in vivo and cellular applications.
Solution Approach 2:
The patent changes the activation parameter from UV light exposure to calcium ion concentration. The self-processing module is activated by calcium-dependent autoproteolysis, allowing control of the conjugation reaction through calcium concentration rather than UV irradiation, thereby avoiding phototoxic effects.
2Stability of the object's composition
If peptide tags are used for covalent conjugation, then stable covalent bonds are formed, but both proteins require modification and distinct strategies are needed for different protein types
Solution Approach 1:
The patent creates a universal self-processing module that can be fused to any protein of interest to enable covalent conjugation. This single module design works across different protein types and conjugation scenarios, eliminating the need for distinct strategies for different proteins while forming stable covalent bonds.
Solution Approach 2:
The self-processing module autonomously undergoes calcium-dependent autoproteolysis to generate the reactive anhydride group without requiring external enzymes or catalysts. This self-activating capability simplifies the overall process by eliminating the need for separate modification steps.
3Measurement precision
If small molecules with affinity for target protein are used for proximity-directed ligation, then specific targeting is achieved, but the approach is limited to proteins with deep and unique pockets and requires post-translational modification
Solution Approach 1:
The patent introduces a binding polypeptide as an intermediary that specifically binds to the target protein and presents the self-processing module in proximity to nucleophiles on the target. This intermediary approach enables specific targeting without requiring deep pockets, expanding applicability to a broader range of protein targets.
Solution Approach 2:
The patent divides the conjugation system into two separate components: a binding polypeptide that provides specificity through protein-protein interaction, and a self-processing module that provides the covalent bonding capability. This segmentation allows each component to be optimized independently and applied to diverse protein targets.
4Object-affected harmful factors
If constitutive weak electrophiles are used for proximity ligation, then cell-friendly conditions are maintained, but reaction rate is slow due to low reactivity
Solution Approach 1:
The patent creates a dynamic reactivity system where the electrophile remains latent until calcium-dependent autoproteolysis activates it. This dynamic control allows the system to maintain cell compatibility during handling while achieving fast reaction rates upon activation, resolving the trade-off between reactivity and biocompatibility.
Solution Approach 2:
The self-processing module is pre-designed with the latent anhydride group that is activated only upon calcium-dependent autoproteolysis. This preliminary preparation allows the reagent to be stable and cell-friendly during storage and handling, while enabling rapid reaction when activated by calcium ions in the desired location.
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 allows for broad and specific covalent conjugation of proteins under cell-friendly conditions, minimizing immune responses and avoiding the need for complex modifications, with the ability to form both intramolecular and intermolecular bonds efficiently and selectively.
Implementation Method 1
a self-processing module that undergoes autoproteolysis to generate a reactive anhydride group on a polypeptide of interest
Implementation Method 2
The self-processing module displays calcium-dependent autoproteolytic activity at an Asp-Pro bond
Implementation Method 3
generate an anhydride group on a polypeptide of interest that can be used to direct the formation of a covalent bond
Implementation Method 4
The reactive anhydride group is directed to react with an amine group... to produce an intermolecular isopeptide bond
Data Source
AI summary
The present invention relates to a system for generating intermolecular covalent bonds (e.g. amide, e.g. isopeptide bonds) between polypeptides. In particular, it provides the use of a chimeric protein to generate an anhydride group on a polypeptide for the formation of a covalent bond, wherein the chimeric protein comprises (i) a domain comprising the polypeptide and (ii) a domain comprising a self-processing module that contains an N-terminal dipeptide of aspartate or glutamate and proline (D/E-P), wherein (i) and (ii) are linked by a peptide bond between the aspartate or glutamate residue at the N-terminus of (ii) and the amino acid at the C-terminus of (i) and wherein the self-processing module cleaves the peptide bond between the proline residue and the aspartate or glutamate residue in the self-processing module to release the polypeptide and generate the anhydride group on the aspartate or glutamate residue.