All buffers and solutions described herein were prepared in MilliQ water from a Barnstead GenPure water filtration system (Thermo Scientific) with a resistivity of at least 18.2 MFor all proteins, thawed cell pellets were resuspended in 5 mL wash buffer/g of cells (wash buffer = 50 mM HEPES (Sigma: Biocertified), 300 mM NaCl (Sigma: BioUltra), 5 mM imidazole (Sigma: ReagentPlus), pH = 7.5). Resuspension was supplemented with Pierce™ Protease Inhibitor, EDTA-free tablets (Thermo Scientific). After resuspension, cells were lysed by sonication and centrifuged to remove any non-soluble cell debris (20,000 RPM, 20 min, 4°C). Supernatant was filtered with 0.43 micron syringe filters and loaded onto a wash-buffer equilibrated 5 mL HisTrapFF column (Cytiva, 2 mL/min binding rate) using an AKTA Start protein purification system (Cytiva). After sample application, the column was washed with 15 CV of wash buffer to elute any non-specific binding proteins. Elution occurred using a linear gradient from 0% to 100% elution buffer (elution buffer = 50 mM HEPES, 100 mM NaCl, 250 mM imidazole, pH = 7.5). For TycF, an isocratic elution was performed by washing the column with 10 CV of 30% elution buffer then 10 CV of 100% elution buffer. Relevant protein fractions, as determined by SDS-PAGE analysis, were pooled. Sfp and TycF were dialyzed against RXN buffer (20 mM HEPES, pH =7.5) overnight, filtered, concentrated using Amicon™ Ultra 10 kDa MWCO centrifugal filter units (Millipore Sigma), flash frozen in liquid nitrogen, and stored at -80°C.