Recombinant Feather Keratin Purification

Recombinant feather keratins were prepared as described previously (Jin et al., 2017 (link)) and used as native substrates. Briefly, E. coli cells expressing recombinant feather keratins were resuspended in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, pH 8.0) and disrupted by sonication. After centrifugation at 10,000 × g for 30 min, expressed keratins in the form of inclusion bodies were resuspended in lysis buffer containing 8 M urea and 1 mM PMSF, incubated on ice for 1 h, and centrifuged at 16,000 × g for 30 min. Supernatants were filtered through a 0.45 μm membrane, then applied to a 10 mL Ni-NTA agarose resin column (Qiagen, Germany) equilibrated with lysis buffer containing 8 M urea. Fractions containing unfolded keratins were eluted with 250 mM imidazole, concentrated using an Amicon Ultra-3K device (Millipore, USA), and buffer-exchanged by step-wise dialysis against 50 mM Tris-HCl (pH 8.0) at 4°C. Dialyzed samples were centrifuged at 10,000 × g for 30 min to remove insoluble material, and the resulting supernatants containing refolded keratins were concentrated using an Amicon Ultra-3K device (Millipore).