Ni nta column

To examine the expression of full-length His-tagged Cas9n in P. putida KT2440, a single colony of each, pCas9n or pPROBE-GT (empty vector), transformants was cultivated in LB medium. Cells were lysed in the SDS loading buffer, and then supernatant cell lysates were subjected to 12% gel SDS-PAGE separation. In addition, Cas9n protein was purified by Ni-NTA column (Takara) and separated by SDS-PAGE as previously described^{5 (link)}.

The cells were centrifuged at 5000 rpm for 15 min (4 °C), and the pellets were resuspended in lysis buffer containing 50 mM NaPi (pH 7.4), 10% glycerol, 500 mM NaCl, 5 mM β-mercaptoethanol, 0.5 mM phenylmethanesul fonylfluoride, and 10 mM imidazole^{20 (link)}. And then the cells were disrupted using a high pressure cell crusher (AH-1500, ATS, Canada) at 4 °C. Unbroken cells were removed by centrifugation at 15,000 rpm for 30 min. The hTop2β^core protein was isolated from the cell lysate supernatant using Ni-NTA column (Clontech, USA) and eluted with elution buffer containing 250 mM imidazole without NaCl. The resulting protein was loaded onto a HiPrep 16/10 Heparin FF column (GE Healthcare). The protein was eluted in a linear gradient over 10 column volumes with buffer A (30 mM Tris-HCl pH 7.5, 15 mM NaCl, 2 mM β-mercaptoethanol, and 1 mM EDTA) and buffer B (buffer A containing 1 M NaCl). The eluted fractions were pooled and purified on a gel filtration column (Superdex 200, GE Healthcare) in buffer C (buffer A containing 70 mM NaCl). The hTop2β^core protein (with molecular weight about 180 kDa) was collected and concentrated to 6.5 mg/ml for crystallization.

Synapsin (a.a. 1–705, human sequence) and Syph Ct (a.a. 219–308, mouse sequence) were expressed in Expi293 cells (Thermo Fisher Scientific) for three days following induction^{7 (link)}. Cells were harvested and lysed in a buffer that contained 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.5 mM TCEP (buffer A), and protease inhibitor (Complete EDTA-free, Roche). The lysates were centrifuged for 1 h at 17,000×g, followed by a two-step purification. The first step was affinity purification on a Ni-NTA column (Clontech) for 45 min (elution with 400 mM Imidazole in buffer A). The second step was gel filtration chromatography (Superdex 200, GE Healthcare) in 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM TCEP (buffer B). For imaging, the two proteins were desalted using PD-10 column (Sigma-Aldrich), resuspended in 25 mM Tris-HCl (pH 7.4), 0.5 mM TCEP and supplemented with NaCl of various concentrations (from 0 to 1 M). The mixture was pipetted on 35‐mm glass-bottom dishes (MatTek Corp). When formed, droplets of synapsin with Syph Ct adhered to the glass surface that was coated with poly-D-lysine (Sigma-Aldrich). Imaging was performed as described above for live imaging, but at RT.