Site-Selective Protein Modification Using Lysine-Histidine Acylation Tags
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Solution Overview
Problem
Existing methods for site-specific modification of proteins are not selective and lack protein-specificity, leading to heterogeneous mixtures and potential protein inactivation or aggregation, especially when targeting lysine residues.
Innovation Solution
A method involving the use of an acylation tag comprising a lysine residue and at least three histidine residues, which allows for site-specific modification of proteins by reacting efficiently with an acylating reagent, such as a phenyl ester, to introduce functional groups like biotin or fluorophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If labeling is performed via primary amino groups on lysine residues using NHS esters, then efficient labeling is achieved, but heterogeneous mixtures are produced due to lack of site-selectivity
Solution Approach 1:
The patent applies local quality by creating a specific local environment (acylation tag) with distinct chemical properties from the rest of the protein. The tag contains a lysine residue with modified pKa due to the microenvironment created by adjacent histidine residues and other structural features, making it uniquely reactive compared to other lysine residues in the protein. This allows selective modification at the tagged location while maintaining efficiency.
Solution Approach 2:
The patent changes the chemical parameter (pKa) of the lysine residue by modifying its microenvironment through the acylation tag structure. By altering the chemical surroundings of the lysine ε-amine, the pKa is shifted to a value that optimizes its reactivity toward the acylating reagent under physiological conditions, enabling selective labeling at the tag location.
2Manufacturing precision
If kinetically controlled labeling using substoichiometric reagent ratios is used, then some site-selectivity is achieved, but cumbersome optimization of conditions is required
Solution Approach 1:
The patent applies preliminary action by pre-installing the acylation tag on the protein before performing the labeling reaction. The tag is engineered into the protein sequence during expression, creating a pre-positioned reactive site with optimized local chemistry. This eliminates the need for post-reaction optimization of reagent ratios or conditions, as the selectivity is built into the tag structure itself.
3Manufacturing precision
If NTA carrying NHS ester is used to guide labeling of His-tagged protein, then proximity-based labeling is achieved, but protein inactivation and aggregation occur due to metal ion requirements
Solution Approach 1:
The patent extracts the metal ion dependency from the labeling system by replacing the NTA-metal ion complex mechanism with a direct chemical tag system. The acylation tag uses the lysine residue's amine group as the reactive site, eliminating the need for metal ions entirely. This removes the source of protein inactivation and aggregation while maintaining labeling specificity through the unique microenvironment of the tag.
4Measurement precision
If computational methods are used to predict residue pKa for selective labeling, then prediction of targeted lysine is attempted, but large error margins result in uncertain outcomes
Solution Approach 1:
The patent creates a simplified model system (the acylation tag) that copies the essential feature needed for selective labeling without the complexity of predicting the entire protein's pKa landscape. By focusing only on the local microenvironment of the tag and using principles of chemical microenvironmental effects, the system achieves reliable selectivity without requiring accurate computational predictions of the whole protein.
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
Enables site-specific conjugation of selected entities to proteins, maintaining protein functionality and stability, and facilitating selective modification even in complex mixtures.
Implementation Method 1
contacting the target protein or peptide from step (a) with an acylating reagent to form a modified target protein or peptide, wherein said acylation tag comprises a single lysine residue and at least three histidine residues, and wherein the target protein or peptide upon contact with the acylating reagent becomes modified at the ε-amine of the lysine residue of the acylation tag
Data Source
AI summary
The present invention concerns a method for site-selective modification of a target protein or peptide by use of an acylation tag comprising a single lysine residue and at least three histidine residues. Upon contact with an acylating reagent, the target protein or peptide becomes modified at the ε-amine of the lysine residue of the acylation tag.