Natarajan Arutselvan, Abou-Elkacem Lotfi
Department of Radiology, James H. Clark Center, Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2019;2033:301-313. doi: 10.1007/978-1-4939-9654-4_20.
Bioconjugation of biologically useful proteins is in great demand (e.g., conjugation to biotins, metal chelators, and drug carriers to target specific tissues for both in vitro and in vivo use). These conjugates provide widespread opportunities for various biological and biomedical applications. Evolving state-of-the-art protein conjugation strategies have led to the development of many affinity ligands, including for cancer imaging and diagnosis. However, to achieve the desirable protein conjugates, there are many challenges that remain to be addressed in order to obtain a reproducible procedure for all proteins and ligands. These include a control over the protein modification and the efficiency of the conjugation while retaining the original biological protein affinity postmodification. Here we present detailed conjugation methods for the human fibronectin tenth type III domain (FN3) protein scaffold for use in preclinical PET imaging. More specifically, this chapter provides detailed methods to produce a FN3 and a FN3-chelator-conjugate, its labeling with the radionuclide 64-Cu, and its use for noninvasive PET imaging in mice.
生物活性蛋白的生物共轭需求巨大（例如，与生物素、金属螯合剂和药物载体共轭，以靶向特定组织用于体外和体内应用）。这些共轭物为各种生物学和生物医学应用提供了广泛的机会。不断发展的先进蛋白质共轭策略已促成了许多亲和配体的开发，包括用于癌症成像和诊断的配体。然而，为了获得理想的蛋白质共轭物，仍有许多挑战有待解决，以便为所有蛋白质和配体获得可重复的程序。这些挑战包括控制蛋白质修饰和共轭效率，同时在修饰后保留原始生物蛋白质亲和力。在此，我们展示了用于临床前PET成像的人纤连蛋白第十个III型结构域（FN3）蛋白质支架的详细共轭方法。更具体地说，本章提供了制备FN3和FN3 - 螯合剂共轭物、用放射性核素64 - Cu对其进行标记以及将其用于小鼠无创PET成像的详细方法。
