High pressure homogenizer

Proteins were expressed in vivo from BL21 (DE3) strains using IPTG induction. Briefly, 1 L cultures in terrific broth (Invitrogen) supplemented with ampicillin at 100 μg/mL were grown at 37 °C to OD ~1.5 and induced with IPTG at 100 μM. At this point, the temperature was reduced to 25 °C and proteins were expressed overnight. Cultures were harvested by centrifugation at 3000 × g and cells were lysed in a single pass through a high-pressure homogenizer (Avestin) at >18,000 PSI. Debris and insoluble material were removed by centrifugation at 23,000 × g. The soluble supernatant was purified on a streptactin column (IBA Life Sciences) according to the manufacturer’s protocol. After purification, elution fractions were pooled, concentrated ~30 X using a centrifugal filter device (Amicon Ultra, 10 kDa cutoff, Millipore), formulated in 20 % sucrose, flash frozen in liquid nitrogen, and stored at − 80 °C. Typical yields were 1 mg of purified protein from 3 (wet) grams of cells.

Eucalyptol was loaded into NLC by high-pressure homogenization technique using HPO, Lipoid S-100, olive oil, thimerosal, Tween-80, D-Sorbitol and eucalyptol (99%). The method used to prepare NLC-Eu was similar but slightly modified from previous study [26 (link), 27 (link)]. The formulation consisted of 2 phases; lipid phase and aqueous phase. The lipid phase consisting of 4 g of HPO, 1 mL of olive oil, and 1.77 g of Lipoid S-100 were mixed and heated in a beaker at 70 °C. Then, 500 μL of eucalyptol was added to the mixture with constant stirring at 1000 rpm for 5 min. On the other hand, the aqueous phase, which contained 0.005% (w/v) thimerosal, 4.75% (w/v) D-Sorbitol and 2% (w/v) Tween 80, were dissolved and heated at 70 °C. The aqueous phase was then added into the lipid phase under constant stirring for 5 min. The mixture was then further mixed using Ultra-Turrax® (IKA, Staufen, Germany) for 10 min at 13,000 rpm. The emulsion was then pressurized using a high-pressure homogenizer (Avestin, Ottawa, ON, Canada) for 15 cycles at 70 °C. The clear nano-emulsion was then allowed to cool down to room temperature at 25 °C and sealed for 24 h. The NLC-Blank was also formulated using the same method without the addition of eucalyptol during the preparation of lipid phase.

Expression of GST-tagged protein was done by transforming BL21(DE3) cells with the pGEX plasmids and then inducing the expression of the protein at 37 degrees for 3 h with 0.5 mM IPTG. Cells were lysed using a high-pressure homogenizer (Avestin) and centrifuged at 20,000g for 30 min. The lysate was incubated with glutathion beads (GE Healthcare) for 60 min and washed with 50 column volumes of 250 mM NaCl, 50 mM Tris pH 8.0, 5% glycerol, 5 mM beta-mercaptoethanol. GST protein bound to beads was stored at 4 degrees.
Strep-His-Bub1 and Bub1 Δ266–311 were expressed in HEK293 6E cell lines by transfection with 100 μg ml⁻¹ polyethylenimine ‘MAX'(PEI) (polysciences). After 3 days, the Bub1 protein was affinity purified using a Strep-tag/Strep-Tactin purification system (IBA) according to manufacturer's description. The protein was dialysed overnight into 150 mM NaCl, 25 mM Tris-HCl pH 8.0, 5% glycerol, 5 mM beta-mercaptoethanol and stored at 4 degrees.
His-BubR1 was expressed in insect cells using the pFastBac system (Invitrogen). High Five cells were infected with virus in plates and collected after 48 h. The cells were lysed using a nitrogen cavitation bomb and BubR1 purified using Ni-affinity column and eluted with imidazole-containing buffer.

B56α was expressed and purified as previously described (Kruse et al., 2013 (link)). The Plk1 polo box domain (residues 367–603) was expressed in BL21(DE3) cells overnight at 18°C. Cells were harvested by centrifugation and resuspended in buffer L [50 mM sodium phosphate, pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 0.5 mM TCEP, and 1× complete EDTA-free tablet (Roche)]. Lysis was done using a high-pressure homogenizer (Avestin) and lysate clarified by centrifugation. The clarified lysate was applied to a 5-ml HiTrap Nickle column and proteins eluted with a 10–500 mM imidazole gradient and peak fractions collected. Subsequently, TEV protease was added to remove tag and protein dialysed into buffer L lacking imidazole and loaded onto a 5-ml HiTrap Nickle column and unbound protein collected and pooled. The untagged Plk1 polo box domain was finally loaded on a superdex 200 16/60 column equilibrated with buffer G (50 mM sodium phosphate, 150 mM NaCl, 10% glycerol, and 0.5 mM TCEP) and peak fractions were collected.