S75 10 300 gl

Both variants of PrgU, with either N- or C-terminal 10×His tag and a 3C protease site, were produced in E. coli BL21(DE3) and grown in terrific broth (TB) medium in a LEX bioreactor (Epiphyte). The cells were grown at 37°C to an OD₆₀₀ of 1.0 to 1.5, and the temperature was then lowered to 18°C before induction with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The cells were incubated for 18 h at 18°C before being harvested via centrifugation and lysed in lysis buffer (50 mM KPi [pH 7.8], 150 mM NaCl, 15 mM imidazole [pH 7.8], and 10% glycerol) using a Constant cell disruptor (Constant Systems) with a pressure of 25 kilopounds/in². Cell lysates were run over an immobilized metal-ion affinity chromatograph (IMAC) with Ni-nitrilotriacetic acid (NTA) beads using gravity flow and washed with 20 column volumes (CV) of wash buffer (50 mM KPi [pH 7.8], 500 mM NaCl, 50 mM imidazole [pH 7.8]) followed by 5 CV of wash buffer containing 2 M LiCl followed again by 20 CV of wash buffer. The proteins were then eluted with elution buffer (50 mM KPi [pH 7.0], 500 mM NaCl, and 500 mM imidazole [pH 7.0]). Eluted material was run over a size exclusion chromatograph (SEC) in 20 mM KPi (pH 7.0), 150 mM NaCl using either an S200 10/300 GL Increase (GE Healthcare) or an S75 10/300 GL (GE Healthcare) column.

30 µM of TrpA monomer, TrpB monomer, or 30 µM TrpA mixed with 30 µM TrpB were subjected to a S75 10/300 GL (GE Healthcare, Chicago, IL, USA) column pre-equilibrated in 50 mM Tris·HCl pH 7.5. Likewise, 65 µM TrpA monomer, TrpB monomer, or 65 µM TrpA mixed with 65 µM TrpB were subjected to the same column pre-equilibrated in 50 mM Tris·HCl pH 7.5, 100 mM NaCl, 5 mM serine. Samples were eluted in the same buffer, and protein peaks were detected at 280 nm.