I'm trying to over express an active protease in BL21 (DE3) following the setup below:
1. Inoculate starter culture at a 1:100 dilution into LB broth
2. Incubate at 37°C with shaking until OD600 reaches 0.6 ,induce with 1 mM IPTG and express protein for 3 hours at 37°C.
However no difference between nu-induced and induced was observed (after 3h, OD600 was 0.9)
Did you test the cell lysates for the expression of the protease using SDS-PAGE/Western blot?
Hi Syed,
I've done it with an anti-His tag antibody, I even did an un-induced culture as a control but the problem is that the expression pattern of this one was higher than the induced one, but still the protein (un-induced ) does not express well. I've even increased the protein conc. from 50 µg to 200 µg but no success...
Dear Antonio,
Thank you so much, please find attached.
Just wondering if the induced culture has inclusion bodies ?
Hello Aicha,
Your observation that more recombinant protein was present in uninduced cells compared to cells induced with 1 mM IPTG is suggestive of toxicity of this protease towards the bacteria. I ll suggest you to give BL21(DE3) pLysS a try. You can also try to express this protease at lower incubation temperatures (30C for 6-8 h or 22C for 12 h) and adding 1-2% glucose in seed cultures to suppress leaky expression. Please add less IPTG; something like 50 uM. What protease is this?
Hi Aicha,
Devil is in the details, so it would be really helpful to troubleshoot if you could upload gel image and give details of the sample making. Because if protein is insoluble then it will be in the pellet and then you should take care of loading the whole cell lysate onto SDS-PAGE.
Second thing I can think of is what Syed sir has suggested, if the protein is toxic you can try pLysS cells or you can try inducing at slightly higher OD (say 0.8) with lesser IPTG (say 100mM). Also, make sure that your IPTG is not too old or degraded.
Also, as Antonio sir has indicated, you should check whether your clone is perfect.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques