Purification of Fn3 Variants for Binding Studies

MSLN-binding Fn3 variant 3.4.4 and negative control protein Fn3 RDG, in which the RGD integrin-binding motif has been mutated to RDG (plasmid DNA provided by B. Hackel, University of Minnesota), were prepared as previously described (Sirois et al., 2018 (link)). Briefly, Fn3 genes were cloned into a pET vector with a C-terminal hexahistadine tag (plasmid provided by B. Hackel, University of Minnesota) and expressed in BL21(DE3) E. coli. Cultures were grown in LB and induced overnight at 20°C with 0.5 mM Isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were resuspended in lysis buffer (35 mM Na₂HPO₄·dibasic, 15 mM NaH₂PO₄·monobasic, 500 mM NaCl, 5 mM CHAPS, 25 mM imidazole, 5% glycerol) supplemented with an EDTA-free protease inhibitor (Pierce), and lysed by repeated freezing and thawing. Soluble fractions were isolated by centrifugation. Fn3 variant 3.4.4 was purified by cobalt affinity chromatography with HisPur cobalt resin (Thermo Fisher) while Fn3 RDG was purified by nickel affinity chromatography with Ni-NTA agarose resin (Thermo Fisher) followed by size exclusion chromatography (SEC) on a Superdex 75 10/300 column (GE Healthcare Life Sciences). Protein samples were analyzed for purity by SDS-PAGE on a BioRad ChemiDoc MP imaging system.