Site-Specific Labeling of F-18 Proteins Using a Supplemented Cell-Free Protein Synthesis System and O-2-[18F]Fluoroethyl-L-Tyrosine: [18F]FET-HER2 Affibody Molecule
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Although a preparation method for F-18-labeled proteins that used a cell-free translation system and 4-[18F]fluoro-L-proline instead of L-proline has been reported, its introduction depends on amino acid sequences of target proteins. The purpose of the study was to propose site-specific labeling method of F-18 by using cell-free translation systems supplemented with an engineered orthogonal aminoacyl-tRNA synthetase derived from Methanocaldococcus jannaschii (pCNF-RS)/suppressor tRNA (tRNACUAopt) pair, O-2-[18F]fluoroethyl-L-tyrosine ([18F]FET), and template DNA inserted with an amber codon.
[18F]FET was prepared from the corresponding precursor and determined whether [18F]FET could be incorporated into an affibody molecule for human epidermal growth factor receptor type 2 (HER2; ZHER2:342) as the 21st amino acid used with the pCNF-RS-tRNACUAopt pair and template DNA inserted with an amber codon in a cell-free translation system. Using SKOV-3 cells, we performed an in vitro binding assay of [18F]FET-ZHER2:342. Furthermore, in vivo positron emission tomography (PET) imaging in SKOV-3 xenograft-bearing mice was performed after the intravenous administration of [18F]FET-ZHER2:342.
[18F]FET was successfully incorporated into proteins by using commercially available cell-free protein synthesis reagents with a pCNF-RS-tRNACUAopt pair and template DNA of the desired proteins inserted with an amber codon. The mean radiochemical yield (non-decay-corrected) of [18F]FET-ZHER2:342 was 6.5 ± 4.1 %. An in vitro cell binding assay revealed that SKOV-3 cells–bound [18F]FET-ZHER2:342 expressed HER2. The in vivo PET imaging in SKOV-3 xenograft-bearing mice revealed that [18F]FET-ZHER2:342 accumulated in SKOV-3 xenografts.
The method proposed in this study might be useful for preparing proteins with F-18 and molecular imaging in the preclinical development.
Key wordsCell-free protein synthesis Engineered orthogonal aminoacyl-tRNA synthetase [18F]FET Affibody
This study was supported by Grants-in-Aid for Scientific Research (16 K15314) from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We wish to acknowledge the contribution of Mr. Takahiro Morito for technical support and the staff at the Cyclotron and Radioisotope Center of Tohoku University for the HM-12 cyclotron operation. We acknowledge the support of the Biomedical Research Core of Tohoku University Graduate School of Medicine.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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