Naptm 25 columns

Synthetic ligands (sequences published in [Mossner et al., 2020b (link)] and shown in Supplemental information) were stably expressed in CHO-K1 cells using neomycin resistance and single clone selection with 1.125 mg/ml G-418 (Genaxxon, Ulm, Germany). Transfected cells were cultivated with G-418 for two weeks and single clone selection was carried out with 0.5 cells/well. Single colonies were screened for protein expression. One clone was selected for protein expression in roller bottles (IBS Integra Biosciences, Zizers, Switzerland) with 10% low IgG fetal calf serum (GIBCO®, Life Technologies, Darmstadt, Germany) DMEM for two months. The supernatants were collected every 3-4 d and 1 L of supernatant was used for purification of Fc-tagged proteins using ProteinA MabSelect™ HiTrap™ columns (GE Healthcare, Chalfront St Giles, UK). Elution was carried out by pH shift using citrate buffer of pH 5.5 and 3.2. Buffer exchange to PBS was achieved using NAP™-25 columns (GE Healthcare, Chalfront St Giles, UK). GFP-mCherry fusion protein with Twin-Strep-tag was purified using StrepTrap™ HP column (GE Healthcare, Chalfront St Giles, UK). Elution was carried out by competitive displacement with 2.5 mM Desthiobiotin. Buffer exchange to PBS was achieved using NAPTM-25 columns (GE Healthcare, Chalfront St Giles, UK).