Metal Complex Labeling of His-Tagged Proteins Without Oxidation
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Solution Overview
Problem
Existing methods for attaching labels and/or carriers to target molecules, such as proteins, are inefficient and can interfere with the function and stability of the molecules due to the use of harsh oxidation steps and lack of site-specificity, leading to batch variability and protein destabilization.
Innovation Solution
A complex comprising a metal cation coordinating with a carbonate or nitrate ligand and a metal cation chelating domain is used to directly attach a label and/or carrier to a target molecule, bypassing oxidation steps and forming a kinetically inert linkage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional labeling methods (amino acid coupling, enzymatic conjugation, click chemistry) are used to attach labels to proteins, then labeling can be achieved, but the labels may detach under physiological conditions or require complex purification steps
Solution Approach 1:
The patent introduces a bivalent molecule as an intermediary that simultaneously binds to the target protein (via affinity to a binding site) and carries the label. This intermediary approach allows stable label attachment without requiring complex purification steps, as the bivalent molecule acts as a bridge between the protein and label, resolving the contradiction between reliability and complexity
Solution Approach 2:
The patent extracts the labeling function from complex multi-step conjugation processes and isolates it into a single-step binding event. By using bivalent molecules that pre-combine the binding functionality and labeling functionality, the method simplifies the overall process while maintaining stable attachment, addressing both reliability and complexity concerns
2Manufacturing precision
If site-specific conjugation methods are used to attach labels to proteins, then labeling precision is improved, but the methods are either kinetically inert (requiring activation) or lack versatility
Solution Approach 1:
The patent creates bivalent molecules that can universally bind to various protein targets through their affinity modules while simultaneously providing versatile labeling capabilities. The same bivalent molecule framework can be adapted to different protein targets and label types, achieving both site-specific conjugation and method versatility without requiring kinetic activation, resolving the contradiction between precision and adaptability
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
The method allows for fast and efficient attachment of labels and/or carriers to target molecules without affecting their function, reducing the need for cumbersome washing steps and ensuring stability, particularly suitable for oxidation-sensitive molecules like proteins.
Implementation Method 1
a bivalent molecule having a first binding site that binds to a target molecule
Implementation Method 2
conjugation of labels and/or carriers to target molecules such as his-tagged proteins via metal complex reagents
Data Source
Figure 1
Figure 2A~2C
Figure 2D
AI summary
The present invention relates to means and methods for conjugating/attaching target molecules such as proteins to a label and/or carrier. Specifically, the present invention provides a complex comprising a metal cation coordinating (i) a metal cation ligand being a carbonate selected from CO3 2- and HCO3- and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier. This complex can be used for attaching a label and/or a carrier to a target molecule, preferably a protein. The attachment of the label or carrier via the complex of the invention involves the replacement of the metal cation ligand with a coordinating group of the target molecule so that a product complex with the target molecule as primary ligand in the coordination sphere of the metal cation is formed. Accordingly, the present invention also provides for uses and methods involving the attachment of a label and/or carrier to a target molecule. Also provided are the products obtained by the labeling and or carrier-attaching methods of the invention and uses thereof. The invention further relates to methods for producing the complex of the invention and kits comprising the components for producing the complex of the invention.