I have cloned a cDNA coding for 7 kDa cysteine-less protein into pET20b which has leader sequence. The recombinant vector was sequenced using T7 promoter and the reading frame of the protein was correct. However, the protein was not expressed. I used E. coli BL21(DE3)pLysS for the expression of the protein. What could be the reason that the protein is not expressing? How should I troubleshoot this?
If you have already exhausted changing expression temperature, amount of IPTG ..., I would suggest to re-transform fresh competent cells. Describing your expression conditions (temperature and amount of IPTG used) and how you tested expression (SDS-PAGE, antibody …) will, however, earn you a more targeted suggestion. Good luck
I tested the expression on SDS-PAGE gel. Re-transformation is a good idea. I will do it and do the expression trial. Thank you for the suggestion.
Is not expressing or is expressing at low levels (e.g. detectable by Western blotting)?
You can try transformation into a pLysS-minus host, e.g. plain BL21(DE3). The presence of T7 lysozyme can decrease expression levels.
Are your cells growing well? Did you check for the transcription of your gene of interest? Maybe your gene is correctly transcribed into mRNA but translation is not working. Do you use antibodies against your gene or do you use the c-terminal His-Tag of pET20b? You could include a n-terminal tag to check if translation stops premature.
Recently some collegues used the pCold expression system and boosted their protein expression. Maybe it helps.
It might seem like a very small thing, but I would recommend going back to your initial cloning design to check the orientation of the gene insert with respect to the promoter you used. Also, do a quick check to ensure that the open reading frame of the inserted gene is maintained. A frame-shift due to an improper insertion of the gene into the vector, might target it for degradation, leading to no detectable expression what so ever.
We have sequenced the plasmid and the insert is in the correct frame and orientation. We have also cloned the gene into pET32a and it is expressing well with the fusion partner.
without knowing more specifics of your method for detecting recombinant protein expression - not all protein engineered for periplasmic expression are translocated across the membrane (for a variety of reasons). Assuming your constructs are in the right orientation and sequence verified, are you basing your findings on looking at the cell supernatants only? Best to look at the whole cells, cell pellets post lysis, cell sups and media to sort this out.
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