I'm having trouble synthesizing a mollusk 173 aminoacids protein of unknown function in a E.coli cell-free protein expression system. The positive control works well and the plasmid has been sequenced and shows no problem, the sequence of the gene has been optimized for codon usage in this context, but the protein is not produced (no distinct opacity at the bottom as in the positive control) and is not detected in a subsequent Western blot at all. We used a pIVEX2.3 plasmid from Roche with a T7 promoter.
We had trouble obtaining the plasmid with the proper gene as well, we suspect it may be toxic to bacteria. Has any of you had the same issue?
Elisabetta Punzi ,
What kind of problems exactly? In what plasmid (i.e. under what promoter, if you're using coupled transcription/translation) is the gene cloned? What organism is the gene from?
Alejandro Martin , I edited the question with the required information.
Elisabetta Punzi ,
And the cell-free extract, does it have T7 RNA polymerase? The positive control, is it cloned in pIVEX2.3 as well?
Alejandro Martin yes, we even replaced the stock T7 to be sure. The positive control was cloned in pIVEX2.3 and this plasmid is commonly used in the lab for cell-free expression.
Elisabetta Punzi then honestly, I don't know. One possibility is that the 5' of the gene is involved in the formation of a secondary structure sequestering the RBS (you could check that in Salis' RBS calculator at https://www.denovodna.com/software/predict_rbs_calculator), but it would have to be really, really stable to prevent translation to such an extent that you cannot detect the target polypeptide by Western blotting.
Here's another possibility: if the specific plasmid prep you sequenced is different from the prep you are using for the TnT system (say, you sent the original miniprep for sequencing and then transformed and repurified from there to have material for the cell-free system), and the gene product is really toxic, there is a chance you are using a mutated, non-expressing version of the original clone.
By the way, you may skip the cloning step by amplifying the gene by PCR with oligonucleotides directly incorporating the sequence of the T7 promoter. Voila! Toxicity problems be gone.
Hope it helps!
I agree with Alejandro Martin last answer, if your plasmid is toxic in E. coli you may be generating mutants as you grow the stock. PCR amplifying the template gets around that. Worth a try!
Even I suspect gene toxicity. To make sure that recombinant protein is toxic, cell growth should be monitored before induction. If the growth rate of the recombinant strain is slower compared to an empty-vector bearing strain then you can draw a conclusion
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