Luria bertani media

Escherichia coli O157:H7 EDL 933 (ECO157) was transformed with the plasmid pGFPuv (Clontech, Mountain View, CA, USA) with an additional kanamycin resistance cassette as previously described (McGaughey and Nayduch, 2009 (link)). Stock cultures of GFP-expressing E. coli O157:H7 (GFP-ECO157) were maintained on Luria-Bertani media (Fisher Scientific, Atlanta, GA, USA) with 100 μg/ml (w/v) of ampicillin sodium and 50 μg/ml (w/v) kanamycin sulfate (LBAK agar or broth). Prior to fly feeding, bacteria were cultured in 50 ml LBAK broth for 8–9 h while shaking at 37°C, and 1 ml was sub-cultured in 25 ml LBAK broth until an OD₆₀₀ of 1.00–1.20 (± 0.05) was reached.

Buffer constituents, Luria-Bertani (LB) media, restriction endonucleases and antibiotics (ampicillin, kanamycin) were purchased from ThermoFisher Scientific (Pittsburgh, PA). BSK-II media containing 6% rabbit serum, the substrates (MTA, SAH, 5’dADO), nucleosides (adenosine, 5’-chloroadenosine) and nucleotide (5’-adenosine monophosphate) were obtained from Sigma-Aldrich (St. Louis, MO). Nucleoside analogs (5’-ethylthioadenosine [ETA], 5’-propylthioadenosine [PTA], 5’-isopropylthioadenosine [iPTA], 5’-butylthioadenosine [BTA]) were the kind gift of Dr. Michael Riscoe (Portland VA Medical Center, Portland, OR). The early stage transition state analogs, 1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol (MT-ImmA), 1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-homocysteinyl-D-ribitol (HCY-ImmA), 1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-deoxyethyl-D-ribitol (5’-dEt-ImmA) were the kind gift of Dr. YS Babu (BioCryst Pharmaceuticals, Birmingham, AL). The late stage transition state analogs, 1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-(methylthiomethyl) pyrrolidine (MT-DADMe-ImmA), and 1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(butylthiomethyl)pyrrolidine (BuT-DADMe-ImmA) were graciously provided by Dr. Vern Schramm (Albert Einstein College of Medicine, Bronx, NY).

Rosetta2 cells were transformed with WT or p.Arg808His encoding construct of PTPN4. Overnight starter cultures were prepared by inoculating 100 mL of Luria-Bertani (LB) media (Thermo Fisher Scientific) with several colonies and grown overnight at 37°C. Large-scale cultures were grown in 2× TY media at 37°C until optical density (OD) reached ~0.6 and then induced with 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown at 18°C overnight. The cells were harvested by centrifugation and lysed in buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 10% glycerol, 0.25 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP,Sigma-Aldrich), 5 mM MgCl₂, and protease inhibitor cocktail. PTPN4s were purified using Roche cOmplete Ni-NTA resin using manufacturer’s instructions. Proteins were further purified using Superdex 75 (GE Healthcare) size exclusion column in the final formulation buffer (50 mM HEPES [pH 7.5], 10% glycerol, 500 mM NaCl, 0.25 mM TCEP).