Sortase-Mediated Protein Labeling for Rapid PET Tracer Synthesis
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Solution Overview
Problem
There is currently no facile and general method to efficiently and site-specifically modify proteins with short-lived radioisotopes like fluorine-18 for PET imaging, given their short half-life, which limits the rapid generation of radiolabeled peptides and proteins for diagnostic and therapeutic applications.
Innovation Solution
The use of sortagging technology, involving sortase-mediated transpeptidation with novel radiolabeled sortase substrates, allows for the efficient and site-specific labeling of proteins with radiolabels such as fluorine-18, enabling rapid generation of radiolabeled peptides and proteins for PET tracers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional labeling methods are used to label proteins with short-lived radioisotopes, then the labeling process can be performed with existing techniques, but the time required for labeling is too long to match the short half-life of the radioisotope
Solution Approach 1:
The protein is pre-modified with a sortase recognition motif and a reactive handle (such as a click chemistry group) before radioisotope introduction. This preliminary modification enables rapid labeling once the radioisotope is available, as the sortase enzyme and substrate are already in place to facilitate quick conjugation, thereby reducing the labeling time to match the short half-life of radioisotopes like fluorine-18
Solution Approach 2:
A sortase substrate peptide serves as an intermediary between the protein and the radioisotope. The substrate peptide contains a reactive handle that can rapidly conjugate with the radioisotope via click chemistry or other fast reactions, while the sortase recognition motif enables specific attachment to the protein. This intermediary system decouples the protein modification from the radioisotope attachment, allowing both steps to occur rapidly and sequentially
2Manufacturing precision
If site-specific labeling is achieved using conventional methods, then the labeling specificity is high, but the labeling time is extended beyond the acceptable window for short-lived radioisotopes
Solution Approach 1:
The sortase recognition motif (LPXTG sequence) is introduced at a specific local site on the protein where labeling is desired. This localized motif enables the sortase enzyme to specifically recognize and modify only that particular site, ensuring site-specific labeling. The reactive handle is also positioned at this same local site to enable rapid radioisotope attachment, combining specificity with speed
Solution Approach 2:
The sortase enzyme performs self-directed labeling by automatically recognizing its specific substrate sequence (LPXTG) on the protein and catalyzing the attachment of the radioisotope-labeled peptide. This self-service mechanism eliminates the need for complex external controls or multiple steps, achieving both high site specificity and rapid labeling in a single enzymatic reaction
3Productivity
If rapid labeling is performed to match radioisotope half-life, then the labeling speed is sufficient, but the labeling method lacks generality and applicability to different proteins
Solution Approach 1:
The sortase recognition motif (LPXTG) serves as a universal tag that can be introduced into any protein of interest through genetic engineering or chemical modification. The sortase enzyme universally recognizes this motif and catalyzes the same reaction mechanism regardless of which protein it is attached to. This universal system enables rapid, site-specific labeling of diverse proteins including antibodies, peptides, and therapeutic proteins, making the method broadly applicable across different protein types for PET imaging
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 method enables quick and efficient labeling of proteins, reducing the time required for tracer preparation and administration, while maintaining high specificity and purity, thus overcoming the limitations posed by the short half-life of commonly used radioisotopes.
Implementation Method 1
sortase-mediated transpeptidation
Implementation Method 2
click chemistry handle (e.g., tetrazine or trans-cyclooctene)
Data Source
AI summary
The present invention, in some aspects, provides methods, reagents, compositions, and kits for the radiolabeling of proteins, for example, of proteins useful for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (e.g., for diagnostic and therapeutic applications), using sortase-mediated transpeptidation reactions. Some aspects of this invention provide methods for the conjugation of an agent, for example, a radioactive agent or molecule to diagnostic or therapeutic peptides or proteins. Compositions comprising sortagged, radiolabeled proteins as well as reagents for generating radiolabeled proteins are also provided. Kits comprising reagents useful for the generation of radiolabeled proteins are provided, as are precursor proteins that comprise a sortase recognition motif.