The pFUSE-Fc plasmid (400 mg) was supplied by the Department of Pathology, Sapporo Medical University School of Medicine. The retroviral reprogramming plasmids pFUSE-hIgG1-Fc2 (InvivoGen, CA, U.S.A.) have been previously described (21 (link)). pFUSE-Fc2 (IL2ss) plasmids facilitate the secretion of Fc-Fusion proteins from pFUSE-Fc-transfected cells. To obtain single-chain Fc fragments of IgH and IgK, we followed the principle of PCR assembly (22 (link)), and the information of the inserted synthesized representative cDNA and the amplified primers are shown in Supplementary Table 1. Briefly, the plasmid was digested with EcoRV and NcoI (Takara Bio, Shiga, Japan), and the targeted IgH and IgK domains were obtained using a two-step overlapping PCR. The PCR product was subcloned into the digested plasmid using In-Fusion HD Cloning kits (Takara Bio); the In-Fusion reaction mixture was transformed into competent cells, and individual isolated colonies were picked from the culture plate. Plasmid DNA was isolated using a Plasmid DNA purification kit (Macherey-Nagel GmbH& Co.), and 2.5 μg of the DNA transfected into 293 T cells using Lipofectamine 3000 (Thermo Fisher Scientific, MS, U.S.A.). Subsequently, the 293 T cells were grown in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution with zeocin (300 μg/mL, InvivoGen), and the supernatant was collected from the culture well on day 2 and days 7–10. IgGs from the supernatant were collected from 3–5 culture dishes and purified using a spin column-based antibody purification kit (Protein G; Cosmo Bio, Tokyo, Japan). The density of IgG was measured using the NanoDrop One (Thermo Fisher Scientific).
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