Hi everyone!
I am trying to express a bacterial protein which I have cloned in pet28a+ vector with NcoI site and XhoI site with C-terminal his-tag. The bacterial host I am using is BL21DE3 plysS. I have used following conditions.
1) IPTG cont. - 0.5mM to 3mM
2) Induction temp - 37 deg Celcius for 4hrs as well as for 18 hrs.
I did not see any expression.
Any suggestions where I might be going wrong?
Bacterial gene expression
In my view, before protein induction with IPTG (0.5mM to 3mM) and Induction temp – 37°c for 4hrs as well as for 18 hrs, you should analyze the recombinant vector pet28a+/gene vector and validate the gene insertion by PCR using the recombinant vector extracted from the recombinant bacteria with a negative control using the initial non recombinant vector.
You also have to verify the codon frequency usage of your Heterologous gene in E Coli may be you have a rare codon. that could limit or block translation.
May be you have also to verify the gene and its ORF sequence in your recombinant vector. Sometimes the gene cloning could introduce changes in the ORF especially if your gene sequence is very G/C rich, thus some changes could introduce nonsense codon. Yet a higy rich G/C sequence may limitgene transcription in BL21DE3 E coli.
Hi Mohamed Nejib Marzouki , thank you for the suggestions, I have confirmed the clone by
1) colony PCR
2) by double digestion of construct using restriction enzymes for fragment release and
3) DNA sequencing,the results when I checked in expasy showed my gene sequence.
Hi Mangal
I have a question, how to check whether gene is in frame or not?
Gene frame verification
You should sequence in both directions the insert gene of your recombinant vector and verify the frame sequence ORF and codons lecture.
However sometimes and especially when you prepare your gene fragment by PCR you could introduce nucleotide changes by the taq polymerase. Yet it depends of the quality of the taq enzyme.
Best regards
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