Purification and Biotinylation of Talin R3-IVVI Domain

We used the mechanically stable talin R3-IVVI domain as a model substrate, as previously reported in force spectroscopic studies^{21 (link),22 (link)}. For purification, the protein construct was transformed into the E. coli BL21 (DE3 Invitrogen) competent cells. Cells were grown in Luria broth at 37 °C with carbenicillin, till the O.D. becomes 0.6–0.8 at 600 nm. The cultures were then induced with 1 mM Isopropyl β-d-thiogalactopyranoside (IPTG, Sigma Aldrich) overnight at 25 °C. The cells were pelleted and re-suspended in 50 mM sodium phosphate buffer pH 7.4, containing 300 mM NaCl and 10% glycerol. Phenylmethylsulfonyl (PMSF) was used as a protease inhibitor followed by lysozyme for membrane lysis. After incubating the solution for 20 min at 4 °C, the dissolved pellet was treated with Triton-X 100 (Sigma Aldrich), DNase, RNase (Invitrogen), and 10 mM MgCl₂ and kept at 4 °C in the rocking platform. The cells were disrupted in French press and cell lysate was centrifuged at 11,000 rpm for 1 h. The protein was purified from the lysate using Ni²⁺-NTA column of ӒKTA Pure (GE healthcare). For in vitro biotinylation of the polyprotein, and Avidity biotinylation kit was used and the biotinylated polypeptide was purified by Superdex-200 increase 10/300 GL gel filtration column in presence of Na-P buffer with 150 mM NaCl⁷⁸ .