Purification of recombinant RAC and Ssb1

Recombinant RAC was purified as described in our previous work³⁷. Coding fragment of Ssb1 was amplified from S. cerevisiae genomic DNA (S288C), and cloned into the vector pET28a. The plasmid was transformed into Escherichia coli BL21 (DE3) cells for overexpression. Cells were induced at 18 °C with 0.4 mM IPTG for 12 h, harvested, resuspended in buffer A (20 mM HEPES-KOH pH 7.5, 500 mM KCl, 10 mM MgCl₂, 1 mM PMSF, and 20 mM imidazole) and subjected to ultrasonic lysis. The cell lysates were then clarified by centrifugation at 30,970 x g for 30 min at 4 °C in a JA 25.50 motor (Beckman Coulter). Supernatants were loaded onto a Ni-NTA column (GE Healthcare) and eluted with buffer B (20 mM HEPES-KOH pH 7.5, 120 mM KCl, 10 mM MgCl₂, and 250 mM imidazole). Eluates were further purified with a Resource Q column (1 ml, GE Healthcare). The Ssb1-containing fractions were pooled, concentrated and loaded onto a pre-equilibrated Superdex 200 column (10/300 GL, GE Healthcare) with buffer C (20 mM HEPES-KOH pH 7.5, 100 mM KCl, 10 mM MgCl₂).