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
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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DOI：
10.1007/s11307-018-1266-z
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发表时间：
2019-06-01
影响因子：
3.1
通讯作者：
Yanai, Kazuhiko
中科院分区：
文献类型：
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作者：
Ai Yanai;Harada, Ryuichi;Yanai, Kazuhiko
目的虽然已经报道了使用无细胞翻译系统和4-[F-18]氟-L-脯氨酸代替L-脯氨酸制备F-18标记蛋白的方法，但是其引入依赖于靶蛋白的氨基酸序列。本研究的目的是提出一种利用无细胞翻译系统添加来自詹氏甲烷球菌的工程正交氨酰-tRNA合成酶（pCNF-RS）/抑制性tRNA的F-18位点特异性标记方法（tRNA（CUA）（opt））对，O-2-[F-18]氟乙基-L-酪氨酸（[F-18]FET），步骤[F-18]从相应的前体制备FET，并确定[F-18] FET可以作为第21个氨基酸掺入人表皮生长因子受体2型（HER 2; Z（HER 2：342））的抗体分子中，与pCNF-RS-tRNA（CUA）（opt）对和模板DNA一起使用，在无细胞翻译系统中插入琥珀密码子。使用SKOV-3细胞，我们进行了[F-18]FET-Z（HER 2：342）的体外结合试验。结果通过使用市售的无细胞蛋白合成试剂，用pCNF-RS-tRNA（CUA）（opt）对和插入琥珀密码子的所需蛋白的模板DNA成功地将[F-18]FET掺入蛋白质中。[F-18]FET-Z（HER 2：342）的平均放射化学产率（非衰变校正）为6.54.1%。体外细胞结合试验显示，SKOV-3细胞结合的[F-18]FET-Z（HER 2：342）表达HER 2。体内PET显像结果显示，[F-18]FET-Z（HER 2：342）在SKOV-3移植瘤中有一定的蓄积。结论本研究建立的方法可用于制备含F-18的蛋白质，并可用于临床前的分子显像。
PurposeAlthough a preparation method for F-18-labeled proteins that used a cell-free translation system and 4-[F-18]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 (tRNA(CUA)(opt)) pair, O-2-[F-18]fluoroethyl-L-tyrosine ([F-18]FET), and template DNA inserted with an amber codon.Procedures[F-18]FET was prepared from the corresponding precursor and determined whether [F-18]FET could be incorporated into an affibody molecule for human epidermal growth factor receptor type 2 (HER2; Z(HER2:342)) as the 21st amino acid used with the pCNF-RS-tRNA(CUA)(opt) 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 [F-18]FET-Z(HER2:342). Furthermore, in vivo positron emission tomography (PET) imaging in SKOV-3 xenograft-bearing mice was performed after the intravenous administration of [F-18]FET-Z(HER2:342).Results[F-18]FET was successfully incorporated into proteins by using commercially available cell-free protein synthesis reagents with a pCNF-RS-tRNA(CUA)(opt) pair and template DNA of the desired proteins inserted with an amber codon. The mean radiochemical yield (non-decay-corrected) of [F-18]FET-Z(HER2:342) was 6.54.1%. An in vitro cell binding assay revealed that SKOV-3 cells-bound [F-18]FET-Z(HER2:342) expressed HER2. The in vivo PET imaging in SKOV-3 xenograft-bearing mice revealed that [F-18]FET-Z(HER2:342) accumulated in SKOV-3 xenografts.Conclusion The method proposed in this study might be useful for preparing proteins with F-18 and molecular imaging in the preclinical development.