Cloning, Expression and Purification of Mouse GS

The gene sequence encoding mouse GS (2–373, Uniprot assession #{"type":"entrez-protein","attrs":{"text":"P15105","term_id":"145559476","term_text":"P15105"}}P15105) was synthesized with an N-terminal 6×His-AVI-TEV tag (MHHHHHHGLNDIFEAQKIEWHEENLYFQG) and cloned into pET-24a(+) to yield pNG189 (Blue Sky Bioservices, Worcester, MA, USA). GS mutants T301A and S343A were synthesized and cloned similarly (numbering refers to the native mouse sequence). All sequences were codon optimized for expression in Escherichia coli. GS mutants T301E and T301V were generated with the QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) using pNG189 as a template according to the manufacturer’s protocol.

For expression of GS, pNG189 was transformed into BL21 (DE3) cells (New England Biolabs, Ipswich, MA, USA). Cultures were grown in LB medium with 25 μg/mL kanamycin at 37°C with shaking until they reached an OD of 0.6, at which point they were induced by addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 500 μM. After induction, cultures were incubated at 16°C overnight with shaking. Cells were harvested by centrifugation and stored at −20°C until further use. For protein purification, all steps were performed at 4°C. Two liters worth of cells were thawed and resuspended in lysis buffer containing 50 mM sodium phosphate pH 7.5, 500 mM NaCl, 10% glycerol, 500 μM TCEP, 10 mM imidazole, and 1× Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Scientific, Waltham, MA, USA). Cells were lysed by passage through a French press. Lysates were cleared by high-speed centrifugation and loaded onto a HisTrap HP column (GE Healthcare, Chicago, IL, USA) pre-equilibrated with 50 mM sodium phosphate pH 7.5, 500 mM NaCl, 10% glycerol, 500 μM TCEP, and 10 mM imidazole. The column was washed with the same buffer before eluting with a gradient of 1 M imidazole. Fractions containing GS were identified by SDS-PAGE, pooled, and concentrated to ~2 mL. Crude GS was then loaded onto a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, Chicago, IL, USA) pre-equilibrated with 50 mM sodium phosphate pH 7.5, 500 mM NaCl, 10% glycerol, and 500 μM TCEP. GS was eluted with the same buffer at a flow rate of 1 mL/min. Fractions containing GS were identified by SDS-PAGE, pooled, supplemented with 1 mM MgCl₂ and ATP, and concentrated. Protein concentration was determined by the Bradford method (Bio-Rad; #500–006) using bovine serum albumin as a standard (Thermo Scientific, Waltham, MA, USA; #23209). GS was stored at −80°C. GS mutants were expressed and purified similarly.