Glutathione resin

GST-fusion proteins were produced and purified from bacteria lysates with glutathione resin (GE Healthcare) as described in the Protein Purification section. The glutathione beads coupled to GST-fusion proteins were equilibrated with TNET buffer and then incubated with appropriate amount of cell lysates for indicated time at 4 °C. Pull-down samples were collected and washed four times with TNET buffer. Samples were subjected to SDS–PAGE and western blot analysis.

PrgE pull-down experiments were performed in 20 mM Hepes, pH 7.5, and 200 mM NaCl by mixing either 2 nmol GST-PcfF or PcfG-His (baits) with 4 nmol PrgE without tag (prey) and 100 μl of the resin (glutathione resin [GE Healthcare] when using PcfF and Ni-NTA [Protino] for PcfG). The proteins were incubated for 15 min at 4°C before collecting the flow-through and washing with 5 × 5 CV wash buffer and eluting with 2 × 5 CV elution buffer. For GST-PcfF pull-downs, 20 mM Hepes, pH 7.5, and 200 mM NaCl were used as wash buffer and 20 mM Hepes, pH 7.5, 200 mM NaCl, and 30 mM glutathione as elution buffer. For His-PcfG pull-downs, wash buffer contained 20 mM Hepes, pH 7.5, 200 mM NaCl, 30 mM imidazole, and elution buffer, 20 mM Hepes, pH 7.5, 200 mM NaCl, 500 mM imidazole. The samples were analyzed on SDS–PAGE and stained with Coomassie Brilliant Blue.

CEP135^F2 was PCR-amplified from a FL CEP135 cDNA and subcloned into pMAL-c2x (NEB) to generate N-terminal maltose binding protein (MBP)-tagged constructs. The Plk4^F2 PB1-PB2 domain was PCR-amplified from a Plk4 cDNA and subcloned into pGEX-6p2 (GE Life Sciences). BL21 DE3 Escherichia coli were grown at 37 °C to an OD₆₀₀ of 0.6, induced with 0.1 mM isopropyl-b-D-thiogalactoside and cells were shifted to 16 °C for 18 h. Cells were centrifuged for 10 min at 2,100g, and then the pellets stored in Buffer A (PBS, 10 mM imidazole, 0.1% β-mercaptoethanol) at −80 °C. Cells were lysed in Buffer A by either sonication or using a cell disruptor (Avestin), centrifuged at 23,000g for 20 min at 4 °C and the supernatant was mix with amylose-resin (NEB) or glutathione resin (GE Life Sciences). Resins were washed in Buffer A and eluted with Buffer A+10 mM glutathione or 10 mM maltose. Protein-containing fractions were pooled, dialysed overnight in Buffer B (25 mM HEPES pH 7.4, 150 mM NaCl and 1 mM DTT) for glutathione S-transferase (GST) pull down assays. Purified proteins were also concentrated using Amicon10K Ultra Spin Concentrators (Millipore). GST-PB1-PB2 was immobilized on glutathione, mixed with MBP-CEP135^F2, rocked at 25 °C for 35 min and pelleted at 500g for 1 min. Inputs and pellets were analysed using SDS–PAGE.