Transformation of spMSP plasmids into BL21 cells.
1. Thaw 1 vial of BL21 STARTM (DE3) cells on ice.
2. Add 1 µL of spMSP plasmids (10-50 ng/ µL) into the vial and keep it on ice for 10 minutes.
3. Heat shock at 42 °C for 30 seconds and place the vial on ice for 2 minutes.
4. Add 500 µL LB broth into the vial and shake it at 37 °C for 1 hour.
5. Spread the cells on LB agar Kanamycin plates.
6. Incubate the plates overnight at 37 °C.
Expression of spMSPs in BL21 cells.
1. A single colony from the plate was picked into 10 ml LB media supplemented with 50 µg/ml kanamycin. Shake the culture at 37 °C overnight (200 rpm).
2. Transfer the 10 ml culture into 1 L LB supplemented with 50 µg/ml kanamycin. Incubate the 1L culture at 37 °C with shaking at 200 rpm until OD600 = 0.7.
3. Add IPTG (0.2 mM) into the culture and continue incubation with shaking at 120 rpm for 20h at 16 °C.
4. Collect the cells by centrifugation at 3450 x g for 20 minutes using 5920 R.
Purification of spMSPs.
1. Cells were resuspended in buffer A (50 mM Tris-HCl (pH 8),100 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol) plus one cOmplete tablet.
2. Cells were lysed on ice using a Branson cell disrupter (60% duty cycle, 45 secs).
3. Cell lysate was clarified by centrifugation at 12, 000 x g for 45 mins using JXN26.
4. The supernatant was loaded onto a 1 ml Ni2+-NTA column, followed by extensive wash (20 column volume) using buffer B (50 mM Tris-HCl (pH 8), 20 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol). Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), desalted in buffer A using PD MiDiTrap G-25 (GE Healthcare), and stored at -80 °C.
1. Purified spMSPs were incubated with PC lipids at different ratios in buffer A containing 0.05% DDM. Samples were kept on ice for 30 min, and detergents were slowly removed with BioBeads (1/3 volume) and gentle shaking (4 °C, overnight).
2. cNDs were fractionated by size exclusion chromatography (SEC) using the Superose 6 10/300 in buffer A. Fractions corresponding to cNDs were kept and subjected to analysis by negative stain electron microscopy.
Negative stain electron microscopy
1. Formvar/carbon-coated copper grids were glow discharged (15 mA, 25 secs) using PELCO easiGlowTM.
2. cNDs (10 µg/ml) were applied onto the grids for 30 secs, followed by staining with 0.75% uranyl formate for 1 minute.
3. Images were collected using a ThermoFisher Science Tecnai G2 TEM (100 kV) equipped with a Veleta CCD camera (Olympus).