I have cloned my genes of interest in pET22b and still not able to see the expression in BL21(De3)plys.
I see two possibilities :
- Your expression plasmid is incorrectly ligated, with no insert or an insert in the wrong position. You have to check your construct by thorough sequencing, from the T7 promoter to the T7 terminator.
- Your transformed strain does not express enough protein in the conditions that you applied. You should test several induction temperatures and time (37°C-3h, 30°C-3h, 24°-overnight, 18°C-overnight, for example) and run total lysates and soluble lysates over an SDS-PAGE. If you still see no expression, switch to a different strain such as BL21(DE3)-Gold or Rosetta2-pLysSRAR.
Thank you very much for the suggestions.....I have tried inducing at 37 C for 6 hrs and 16h with 1.5mM IPTG. Actually I have cloned 5 of my genes and only one is showing the induction that to very less. I will try to transform in different strain though..Thanking you
I would leave temperature screening as the last thing since it relates more to protein solubility (i.e. inclusion body formation) than to protein expression itself.
I haven't checked pEt22 features, but you should add a tag (like HisTag) that could help you to detect a very small amount of protein by western-blot analysis.
As suggested, codon usage could also be an issue.
Considering your gene has been cloned in right orientation and is in-frame, I'd suggest you to
1. replace ampicillin with carbenicillin.
2. Not to use pLysS unless your gene of interest is toxic to the host.
3. Use 0.1mM concentration of IPTG
Hope this helps, please update ur results...
Good luck :)
Thanks Shridhar
I will try out your suggestions and upload the results....at present I am trying Rosette pLys cells with all the conditions same. If I dont get the induction I will try your suggestions
Vikas
Yeah... sure... one more thing.. is your gene of interest is codon optimized? if not, then along with Rosetta, you may want to ty other hosts like BL21 (DE3) RIPL and RIL. Both of these are available from Stratagene.
You can make what called ( Cell free system ) that you don't need for the competent cell to ensure that your insert expressed or not.
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