You’ve got the right construct in the right host – now how you induce expression can make all the difference. A common mistake is blasting cells with a high concentration of IPTG and incubating at 37 °C, only to get mountains of misfolded protein.
In reality, less is often more in expression optimization. Try inducing at a lower temperature (like 18 °C or 20 °C overnight); cooler temps give proteins more time to fold properly, often boosting soluble yield. Adjusting IPTG concentration can help too – sometimes a little (e.g. 0.1 mM instead of 1 mM) is enough to trigger expression without stressing the cells. Also consider the cell density at induction: inducing at mid-log (OD₆₀₀ ~0.6) versus late-log can impact yields and protein quality.
Another powerful approach is auto-induction media, which slowly induce expression as the culture grows to high density – great for obtaining high yields without constant supervision.
Finally, don’t overlook shaking speed and aeration; sample oxygenation can improve protein production in bacteria. Tuning these parameters requires a bit of trial and error, but the payoff is big: higher yields and properly folded protein.