I am trying to overexpress a 13 kDa soluble protein in BL21-DE3. So far I am getting very poor yields (0.7 mg/Liter) and am needing tonnes of cultures to have enough protein for ITC and crystallography. I have tried autoinduction. Does anybody have any suggestions as to what else I could try? My protein is in the pHUE plasmid where it is expressed as an ubiquitynated protein.
Hello Bratati,
If you are using BL21(DE3), it is notoriously leaky. This could lead to loss of the production capacity of your cultures due to the presence of low- or non-producing segregants. I would use a host that affords a tighter control of expression in non-induced cells, such as BL21(DE3)(pLysS) or BL21(DE3). Also, did you look at the insoluble fraction ? It could be that a large fraction of your protein is misfolded and precipitates as inclusion bodies. In that case, you could try inducing at low temperature, e.g. 14 °C, or using strains that are suppose to be better at refolding recombinant proteins, such as SoluBL21(DE3) or strains harbouring plasmids overproducing chaperones.
Good luck
Pierre
You could also try experimenting with OD600 at induction and IPTG concentration used. You could also have a look to see if your protein contains lots of rare codons that E. coli might not like (you can get strains containing rare tRNAs)
did you optimize the codons for e.coli expression? Are you following an established protocol, or do you need to optimize the conditions?
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