One shot bl21 star

One Shot^®BL21 Star™ (DE3; Invitrogen) bacteria were transformed with plasmids encoding the scProtein of interest and plated on LB agar plates with appropriate antibiotic (kanamycin 50 µg/mL, Sigma). After overnight incubation at 37 °C, a medium sized, isolated colony was picked and inoculated into a 5 mL overnight seed culture of LB media. The seed culture was diluted 1:20 and grown in 2xYT media (Sigma) supplemented with 0.2 um filtered 40 mM MgSO4 (Sigma), 2.5 mM KCl (Sigma), and 20 mM glucose (Sigma) until obtaining OD600 ~0.1–0.3 and then, for protein expression, the bacteria were induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Sigma) and incubated overnight at 37 °C. Cultures were harvested by centrifugation at 3000 × g for 10 min and pellets stored at −80 °C or processed immediately.

PRL full length protein sequences fused to an N-terminal 6xHis-tag was cloned into pET28b bacterial expression vector. Proteins were expressed in One Shot BL21 Star bacteria (Invitrogen, Cat # C601003) by induction with 0.5 mM IPTG (Fisher Scientific, Cat # BP175510) for 16 h. Cells were resuspended in 10 ml of lysis buffer [300 mM NaCl (VWR Cat. No. BDH9286), 20 mM Tris pH 7.5, 10 mM Imidazole pH 8.0 (Sigma-Aldrich I2399), 1:1000 protease inhibitor cocktail (Sigma-Aldrich Cat. No. P8465)] per gram of cell pellet and lysed using a microfluidizer (Avestin, EmulsiFlex-C5). Protein was isolated using Ni–NTA Resin (VWR, Cat # 786–940) and eluted with 2 mL of elution buffer (300 mM NaCl, 20 mM Tris pH 7.5, and 250 mM Imidazole pH 8.0). Following cleavage with TEV protease, samples were reapplied to Ni–NTA column to remove uncleaved protein as well as TEV. Samples were further purified using an S200 column on a Superdex 10/300 in buffer containing 100 mM NaCl and 200 mM HEPES pH 7.5. Purified fractions were then run on 4–20% Mini-PROTEAN TGX Stain-Free (Bio-Rad). The purest fractions were pooled, concentrated together, flash frozen and stored at -80 °C.