I have successfully transformed E. coli BL21 (DE3) cells with a plasmid encoding my desired mammalian protein. The transformed cells were cultured in LB medium at 37 °C, and protein expression was induced with 0.5 mM IPTG once the culture reached an optical density at 600 nm (OD₆₀₀) of 0.6–0.8. Following induction, the culture was incubated for 5 hours at 37 °C. However, the resulting protein yield was very low. When I repeated the induction at 30 °C, a noticeable improvement in protein expression was observed. Unfortunately, due to limitations in available equipment, I am unable to maintain cultures at 30 °C. Therefore, I would appreciate it if you could recommend an optimized protocol for improving protein yield at 37 °C.
@Reeshma P V
Try switching the medium from LB to Terrific Broth or 2x YT for better yield. Else, you may further reduce the temperature to 20-25 ⁰C for enhanced protein folding and yield depending on your protein of interest.
Dear Reeshma P V
generally lower temperature are preferable since the protein stability is high, protease activity low and the bacteria growth slowly and therefore an higher fractioin of nutrients is dedicated to protein expression than biomass production.
If the problem is the availability of shakers to be setted up at 30°C, What about perform the expression at Room temperature? Do you have the possibility to test it?
best
Manuele
As Manuele has said lower temp is an excellent idea.
I've had great luck with Studier's "autoinduction media" at RT for a day or two!