Fig 4. Production and characterization of selected Fn3 variants.

Engineered Fn3 clones 1.4.1 and 2.4.1 were expressed in bacteria with a C-terminal hexahistidine tag and a short peptide tag containing GKSK residues for later bioconjugation chemistry. (A) Fn3 protein 1.4.1 was purified by nickel affinity chromatography followed by SEC, demonstrating desired product with retention time of ~ 42 min. (B) Fn3 protein 2.4.1 was purified by nickel affinity chromatography followed by HPLC, demonstrating desired product with retention time of ~30 min. (C) Proteins were purified to high purity > 99% as analyzed by SDS-PAGE. Yields of Fn3 protein production were routinely ~ 10 mg/L.