Protein expression and purification
Growing expression Cultures
Growth of standard expression cultures (100 ml)
- Inoculate 10 ml culture medium containing relevant antibiotics in a 50 ml flask. Grow the cultures overnight at 37°C.
- Inoculate 100 ml prewarmed medium (with antibiotics) with 5 ml of the overnight culture and grow at 37°C with vigorous shaking until an OD600 of 0.6 is reached (30–60 min).
Tip: Take a 1 ml sample immediately before induction. This sample is the noninduced control. Pellet cells and resuspend them in 50 µl 5x SDS-PAGE sample buffer (see table 5x SDS-PAGE sample buffer). Store at –20°C until SDS-PAGE analysis - Induce expression (e.g., by adding IPTG to a final concentration of 1 mM).
- Incubate the cultures for an additional 4–5 h.
Tip: Collect a second 1 ml sample. This sample is the induced control. Pellet cells and resuspend them in 100 µl 5x SDS-PAGE sample buffer. Freeze and store the sample at –20°C until SDS-PAGE analysis.
Tip: If expressing a protein for the first time, take a 1 ml sample every hour and treat as above to produce a time-course of expression - Harvest the cells by centrifugation at 4000 x g for 20 min.
- Freeze the cells in dry ice–ethanol or liquid nitrogen, or store cell pellet overnight at –20°C
5x SDS-PAGE sample buffer
| Composition of working solution | Component | Amount per liter |
|---|---|---|
| 0.225 M Tris·Cl (pH 6.8) | 1 M Tris·Cl, pH 6.8 | 2.25 ml |
| 50% glycerol | Glycerol | 5 ml |
| 5% SDS | SDS | 0.5 g |
| 0.05% bromophenol blue | Bromophenol blue | 5 mg |
| 0.25 M dithiothreitol (DTT)* | 1 M DTT | 2.5 ml |
Culture growth for preparative production (1 liter)
- Inoculate 20 ml culture medium containing the relevant antibiotics. Grow overnight at 37°C with vigorous shaking.
- Inoculate a 1 liter culture 1:50 with the noninduced overnight culture. Grow at 37°C with vigorous shaking until an OD600 of 0.6 is reached.
Tip: Take a 1 ml sample immediately before induction. This sample is the noninduced control. Pellet cells and resuspend in 50 µl 5x SDS-PAGE sample buffer (see table 5x SDS-PAGE sample buffer). Store at –20°C until SDS-PAGE analysis.
- Induce expression (e.g., by adding IPTG to a final concentration of 1 mM).
- Incubate the culture for an additional 4–5 h.
Tip: Collect a second 1 ml sample. This is the induced control. Pellet cells in a microcentrifuge tube and resuspend in 100 µl 5x SDS-PAGE sample buffer. Store at –20°C until SDS-PAGE analysis.
- Harvest the cells by centrifugation at 4000 x g for 20 min. Freeze the cells in dry ice–ethanol or liquid nitrogen, or store cell pellet overnight at –20°C.