Protein Expression with T7 Strains
T7 Protein Expression
- Transform expression plasmid into T7 Express, T7 Express Iq, T7 Express lysY or T7 Express lysY/Iq (Iq and lysY hosts allow for more stringent control of protein expression). Plate out on antibiotic selection plates and incubate overnight at 37°C (24 hours at 30°C for SHuffle® strains).
- Resuspend a single colony in 10 ml liquid culture with antibiotic.
- Incubate at 37°C until OD600 reaches 0.4-0.6.
- Induce with 40 µl of a 100 mM stock of IPTG (final conc. = 0.4 mM) and induce for 2 hours at 37°C (4 hours at 30°C or 16°C overnight for SHuffle strains).
- Look for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction alone.
- For large scale, inoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 37°C (30°C for SHuffle strains) until OD600 reaches 0.4-0.6. Add IPTG to 0.4 mM. Induce 2 hours at 37°C or 15°C overnight (4 hours at 30°C or 16°C overnight for SHuffle strains).
No colonies or no growth in liquid culture
- Even though T7 expression is tightly regulated, there may be a low level of basal expression in the T7 Express host. If toxicity of the expressed protein is likely, transformation of the expression plasmid should be carried out in a more tightly controlled expression strain:
- In Iq strains over-expression of the LacIq repressor reduces basal expression of the T7 RNA polymerase
- In lysY strains mutant T7 lysozyme is produced which binds to T7 RNA polymerase, reducing basal expression of the target protein. Upon induction, newly made T7 RNA polymerase titrates out the lysozyme and results in expression of the target protein
- Incubation at 30°C or room temperature may also alleviate toxicity issues.
- Check antibiotic concentration (test with control plasmid).
No protein visible on gel or no activity
- Check for toxicity - the cells may have eliminated or deleted elements in the expression plasmid. If this is the case, test Iqand/or lysY strains to reduce basal expression.
- Culture cells for protein induction. Just before induction, plate a sample on duplicate plates with and without antibiotic selection. If toxicity is an issue, significant fewer colonies will be seen on plates containing antibiotic (indicating that the plasmid has been lost) compared to plates without antibiotic.
Induced Protein is insoluble
T7 expression often leads to very high production of protein that can result in the target protein becoming insoluble. In this case:
- Induce at lower temperatures (12 -15°C overnight)
- Reduce IPTG concentration to 0.01 mM - 0.1 mM
- Induce for less time (as little as 15 minutes)
- Induce earlier in growth (OD600 = 0.3 or 0.4)