The coding sequences of rabbit GM64–93, GM64–237, and GM2–237 were subcloned into a pET-M30-MBP vector containing an N-terminal His6-tag followed by maltose binding protein (MBP) and a tobacco etch virus (TEV) protease cleavage site. GMCBM21 (residues 102 to 237) was subcloned into pRP1B containing an N-terminal His6-tag followed by a TEV protease cleavage site. Escherichia coli strain BL21-Codon-Plus (DE3)-RIL (Agilent) cells were transformed with the GM expression vectors. Freshly transformed cells were grown at 37°C in LB medium containing selective antibiotics until they reached an OD600 (optical density at 600 nm) of 0.8 to 1.0. Protein expression was induced by addition of 1 mM β-d-thiogalactopyranoside to the culture medium, and cultures were allowed to grow overnight (18 to 20 hours) at 18°C. Cells were harvested by centrifugation (6000g, 15 min, 4°C) and stored at −80°C until purification.

Expression of uniformly 13C- and/or 15N-labeled protein was carried out by growing freshly transformed cells in M9 minimal media containing [13C]-d-glucose (4 g/liter) and/or 15NH4Cl (1 g/liter) (Cambridge Isotopes Laboratories) as the sole carbon and nitrogen sources, respectively. Expression of uniformly [2H,15N]-labeled GM64–237 was achieved by growing cells in D2O-based M9 minimal media containing 15NH4Cl (1 g/liter) as the sole nitrogen source. Multiple rounds (0, 30, 50, 70, and 100%) of D2O adaptation were necessary for high-yield expression. Cloning, expression, and purification of PP1α7–300, PP1α7–330, PP1β6–327, PP1γ7–308, and PP1γ7–323 were performed as previously described (18); human and rabbit PP1 are 100% identical. GM64–93, GM49–86, and GM64–105 peptides were purchased from Bio-Synthesis Inc. GM64–237 N228A was generated by site-directed mutagenesis and expressed using the same methods as those used for wt-GM64–237.

The plasmid of human glycogen synthase (pFastBacDual GST-GYG1+GYS1) was a gift from E. Zeqiraj, University of Leeds. Expression and purification were carried out according to previously published methods (30). Recombinant bacmid was generated in DH10Bac cells (Thermo Fisher Scientific) and purified using the PureLink HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific). Sf9 cells were cultured as suspension in Sf-900 III SFM medium (Thermo Fisher Scientific; 110 rpm at 26°C). P1 baculorvirus was produced in monolayer cultures by transfecting the recombinant bacmid using Cellfectin II Reagent (Thermo Fisher Scientific). P2 virus was generated by infecting Sf9 cultures (1.6× 106 cells/ml) with P1 virus at 400 μl per 100 ml of cells. The supernatant from P2 was harvested 3 days after infection, and 3 ml was used to infect 600 ml of Sf9 suspension culture to produce the P3 culture. P3 was used at a 1:10 ratio to infect 3 liters of Sf9 cell culture at 2.0 × 106 cells/ml for protein production. Cells were grown in suspension for 3 days, and the cell pellets were washed in phosphate-buffered saline before they were frozen and stored at −80°C until used.

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