Hello everyone,
I have a protocol to express a enzyme (human 17B-HSD1), but it doesn't list the shaker speed, just the temp (18C for 12-18 hours). I obviously don't want inclusion bodies but I do want to maximize the protein I get. Is 250-300rpm too much?
Most likely, shaker speed won't affect the formation of inclusion bodies. The main effect will be on aeration, but at 18 °C, the growth rate will be slower and oxygen solubility will be higher than at 37 °C, so aeration is probably not a limiting factor. You may consider choosing a lower speed, though, to reduce the mechanical wear and tear of your shaker: 150 rpm should be OK for media such as LB or 2YT. You should also take care of choosing a culture vessel big enough so that the depth of liquid doesn't exceed 4-5 cm to get a good surface:volume ratio for optimal aeration. 1-L Fernbach flasks are a good option for culture volumes up to 700-800 ml.
it should be fine, as Pierre has stated, with other refs using 0.25 mM IPTG and 13C ON to keep this protein soluble. Good luck!
Shaker speed is not likely to affect inclusion bodies, but these factors can help:
- Lowering the temperature to between 20°C and 30°C
- Inducing at lower cell densities (A600 = 0.5)
- Inducing for a shorter period of time
- Inducing using a lower concentration of the inducing agent
https://lab.plygenind.com/how-to-prevent-inclusion-bodies-formation-in-protein-expression
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