Constructs bearing the catalytic domains of PTP1B, SHP1, SHP2, HePTP, TCPTP, and CD148 were prepared by over-expression in E. coli (see Supporting Information for protein sequences). All of these constructs had an N-terminal His6-tag followed by a TEV protease cleavage site. For CD45, our construct contained the tandem phosphatase and pseudophosphatase domains and an N-terminal His6-SUMO tag, and for YopH, a tagless version of the full-length protein was expressed. BL21(DE3) cells transformed with a phosphatase-encoding plasmid were grown at 37 °C in 2 L of terrific broth (TB), supplemented with the appropriate antibiotic. After cells reached an optical density at 600 nm of 0.5, isopropyl-β-D-1thiogalactopyranoside (IPTG) was added for a final concentration of 0.5 mM to induce the expression of proteins, and the cultures were incubated at 18 °C overnight. Cells were harvested by centrifugation (7800 rcf at 4 °C for 30 min), resuspended in lysis buffer (pH 8, 50 mM Tris, 300 mM NaCl, 20 mM imidazole, 2 mM 2-mercaptoethanol (BME), 10 % glycerol), and lysed by sonication on ice. After separation of insoluble material by centrifugation (33,000 rcf at 4 °C for 45 minutes), the supernatant was applied to a 5 mL HisTrap Ni-NTA column (Cytiva). The resin was washed with lysis buffer and wash buffer (50 mM Tris, pH 8.5, 50 mM NaCl, 10 mM imidazole, 2 mM BME, 10 % glycerol). The protein was eluted with pH 8.5 buffer (50 mM Tris, 50 mM NaCl, 500 mM imidazole, 2 mM BME, and 10% glycerol) and passed through a 5 mL HiTrap Q anion exchange column. The His6-tag of the collected fractions was cleaved by incubating with TEV protease or Ulp1 (SUMO protease) overnight. The reaction mixture was subsequently flowed through 2 mL of Ni-NTA resin (ThermoFisher). The cleaved protein was collected in the flow-through and washes. Proteins were further purified using a Superdex 75 or 200 16/600 gel filtration column (Cytiva) in 10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP, 10 % glycerol. Pure fractions were pooled then concentrated with centrifugal filters (Millipore). The resulting solution was aliquoted, and flash frozen in liquid N2 for long-term storage at -80 °C. For YopH, which was tagless, the protein was purified by cation exchange of the soluble fraction of the cell lysate over a 5 mL HiTrap SP column (Cytiva), followed by size-exclusion chromatography as described for the other phosphatases.