Purification of Flag-tagged Sso2 protein

Strains were grown at 25°C overnight to an OD₆₀₀ around 1.0. Seventy OD₆₀₀ units of cells were collected from each strain. Cell lysates were prepared as described previously with modifications (Yue et al., 2017 (link); Liu et al., 2022 (link)). Briefly, cell pellets were washed once in cold water and resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 5 mM NaF, 1 mM sodium pyrophosphate, and 1 mM DTT) and a protease inhibitor cocktail (Roche). Cells suspensions were transferred to 2 ml screw cap tubes containing prewashed 2 mg zirconia/silica 0.5 mm beads. Cell were lysed using a bead beater; 1% (v/v) Triton X-100 was added to the cell lysates and incubated for 15 min at 4°C. The cell lysates were then spun at 20,000× g for 30 min and supernatants were incubated with 10 µl prewashed anti-Flag agarose beads (Sigma, A2220) at 4°C for 3 hr. Beads were washed five times with lysis buffer containing 0.1% (v/v) Triton X-100. Proteins bound on the beads were eluted with ×1 sample buffer. Proteins were detected with anti-Flag antibody (Sigma, F1804, monoclonal, 1:1000) or anti-Sso antibody (rabbit antiserum, 1:2000).
Coordinates and structure factors for the Sec3-Sso2 complex have been deposited in the Protein Data Bank with accession code 7Q83 (https://www.rcsb.org/structure/7Q83).