School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
PLoS One. 2013 Nov 18;8(11):e81350. doi: 10.1371/journal.pone.0081350. eCollection 2013.
Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
对新生儿 Fc 受体（FcRn）的研究揭示了其在哺乳动物中的多种重要功能，包括保护 IgG 和血清白蛋白（SA）免受溶酶体降解。因此，治疗性抗体、含有 IgG-Fc 和 SA 的药物的药代动力学行为受到其与 FcRn 相互作用的影响。如果可以在体外研究此类药物与 FcRn 的相互作用，则可以促进其临床前开发。出于这个原因，我们利用基于慢病毒的基因传递系统，开发了一种生产人 FcRn 可溶性细胞外结构域和全长受体与绿色荧光蛋白融合的稳健系统，该系统可轻松快速地生成稳定过表达细胞。在多层培养瓶中生产细胞外结构域，然后使用固定化 IgG 进行亲和纯化，可从每升细胞培养物中捕获毫克量的可溶性受体，并保留 IgG 结合。该受体进一步通过 SDS-PAGE、western blot、圆二色性光谱、ELISA、表面等离子体共振和温度稳定性测定进行了表征，结果表明该蛋白具有高纯度的功能性和稳定性。全长受体以膜结合形式成功过表达，并保留 pH 依赖性 IgG 和 SA 结合。
