Pmal expression system

RA3, TPP4 and TPP12 coding sequences were cloned into pMAL-c5x (New England Biolabs), and site-directed mutagenesis was performed on pMAL-c5x-TPP4 to introduce the various point mutations described in the Results section. All sequences were verified by Sanger sequencing before transformation into the Rosetta E. coli strain. Cultures were grown to an OD600 of 0.6 at 37°C, cooled to 16°C prior to addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM, and grown for an additional 12-16 hours at 16°C. Culture harvesting and purification of MBP-TPPs were performed with the pMAL expression system (New England Biolabs) according to the manufacturer’s instructions. Protein purity and concentration were assessed using SDS-PAGE gels. For in vitro assays, equal amounts of purified TPPs were incubated for 30 minutes at 28°C in buffer containing 10 mM Tris-HCl pH 7.6, 0.2 mM EDTA, 5 mM MgCl₂, 0.1 mg/mL BSA, and 0.5 mM T6P, S6P, G6P or F6P (Sigma-Aldrich). Phosphate release was measured using a colorimetric assay as OD₆₀₀ using the Serine/Threonine Phosphatase Assay System (Promega). Activity was expressed as a percentage of the activity measured with purified recombinant TPP4.