Bio scale mini nuvia imac cartridge

The protein expression plasmids with the target cloned in pET30a(+), pMAL-c5X-His, and pGEX-4T3 were transformed into BL21/DE3 pLysS E.coli bacteria (#L1195, Promega, Madison, WI). The recombinant His-, MBP-, Strep-, and GST-tagged proteins were purified as previously described [23 (link),49 (link),87 (link),88 (link)] or purified with Bio-Rad NGC Chromatography System (Bio-Rad Laboratories) using Bio-Scale Mini Nuvia IMAC Cartridges (#7800811, Bio-Rad, CA). The His-tagged NS1-70 was further purified by running through a HiLoad Superdex 200 pg column (#28989335, Cytiva) with Bio-Rad NGC Chromatography System at a flow rate of 0.5 ml/min as previously described [49 (link)].

The E. coli host Shuffle^® T7 (New England Biolabs, Inc., Ipswich, MA, USA), was transformed with a cloned expression vector. The cells were cultured in 500 mL of LB medium containing 50 μg/mL of ampicillin at 30 °C until the optical density (OD) at 600 nm was 0.5. Proteins were overexpressed by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) treatment at 20 °C. After 5 h of reactions, cells were harvested by centrifugation (3000× g for 20 min). Harvested cells were lysed via sonification in a lysis buffer of 50 mM Tris-HCl, 200 or 300 mM NaCl, and 10% glycerol at pH 7.0. The cell lysates were filtered through a polyether-sulfone syringe filter with a 0.45 μm pore size (Hyundai Micro Co., Ltd., Seoul, Republic of Korea) after centrifugation at 16,000× g for 10 min and purification via Ni-affinity chromatography (Bio-Scale™ Mini Nuvia™ IMAC Cartridges; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The purified samples were concentrated with Centriprep™ (Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). Protein samples were further separated by their size through size exclusion chromatography (SEC) with a Superdex™ 200 Increase 10/300 GL column (GE Healthcare, Uppsala, Sweden). After SEC, eluted fractions of the expected peak were pooled and concentrated with Centriprep™ (Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland).

The supernatant after cell disruption containing recombinant His-tagged protein was supplemented with 40 mM Imidazole and subsequently loaded onto a Ni²⁺-loaded HiTrap 5 ml chelating HP column (GE Healthcare) or Ni²⁺-loaded Bio-Scale mini Nuvia IMAC cartridge (BioRad) pre-equilibrated in buffer A (20 mM HEPES pH 7.5, 500 mM NaCl, 1 mM MgCl₂, 1 mM ß-Me for CbFic2 versions and FRET control constructs; 20 mM HEPES pH 7.5, 500 mM NaCl, 1 mM ß-Me for TS-H3 peptides and GST-H3 peptides). The column was washed with 40 mM imidazole and His-tagged protein was eluted using a 5 ml fractional gradient of 40–350 mM imidazole over 20 column volumes. Protein-containing fractions were analyzed by SDS PAGE and pooled for TEV protease cleavage, if applicable. For GST-H3 peptides, protein-containing fractions were analyzed by SDS PAGE, combined, and concentrated as well as buffer exchanged to 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT using Amicon Ultra centrifugal filter units (Merck Millipore), before being subjected to TEV cleavage.