All ER-PE24 ITs and anti-EGFR monobody affinity variants were solubly expressed at 20 °C for 48 h in E. coli strain SoluBL21(DE3) (Genlantis, San Diego, CA, USA) [15 (link)], purified using Ni-NTA resin (Qiagen, Hilden, Germany; 30210), and finally formulated in a phosphate-buffered saline (PBS) buffer (2.67 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4, pH 7.4) [34 (link)].
For the expression of EGFR-ECD-Fc protein, the corresponding plasmid was transiently transfected into HEK293F cell cultures in Freestyle 293F medium (Invitrogen, Waltham, MA, USA) according to the standard protocol [35 (link)]. Human EGFR-ECD-Fc protein was purified from the culture supernatants using protein-A agarose resin (Captiva PriMAB, Repligen, Waltham, MA, USA) and finally formulated in a PBS buffer. The concentration of purified proteins was determined using the Bicinchoninic Acid (BCA) assay.
For the expression of EGFR-ECD-Fc protein, the corresponding plasmid was transiently transfected into HEK293F cell cultures in Freestyle 293F medium (Invitrogen, Waltham, MA, USA) according to the standard protocol [35 (link)]. Human EGFR-ECD-Fc protein was purified from the culture supernatants using protein-A agarose resin (Captiva PriMAB, Repligen, Waltham, MA, USA) and finally formulated in a PBS buffer. The concentration of purified proteins was determined using the Bicinchoninic Acid (BCA) assay.