The pGEX vector system does not include a termination signal sequence. The termination signal sequence has an important regulatory role in translation.
Does anyone have good experience with the use of pGEX vector?
Thank you for your answers.
Hi there,
Even if the downstream sequence of the 3 in frame stop codons is not actually documented in the pGEX serie leaflet, these are commercial expression vectors then one can expect them to be designed to work properly. If you are confronted with expression issues there are a lot of parameters to consider including the cloning strategy, expression strategy, nature of the protein of interest and also the nature of the plasmid itself.
I have had little experience with pGEX but had no expression issue.
Hi,
I have also used pGEX4T-1 quite a lot without any issues. Judging only from the picture you attached one cannot really say anything about the presence of a STOP codon, check the sequence for it. If the vector that you want to use doesn't have a STOP codon, simply add it to the end of your gene.
Good luck!
Thanks for all the answers!
We checked the sequence. The STOP codon is in place.
The question is: Does the tac promoter do not require a terminal signal sequence? The pGEX vector series does not contain terminal signal sequences. Is the presence of STOP codon sufficient to terminate mRNA synthesis?
Hi again,
I expect everything to be working fine in a commercial expression vector. However the insert you put in it may actually be responsible for expression issues (ie. starting with the same backbone you may observe a very nice expression for one specific insert and no expression at all for an other insert and also variation in optimal condition for expression according to the protein you intend to produce)...
And to answer your final question, stop codon itself has no effect on transcription termination but there are 2 ways to end transcription in bacteria : rho dependent and rho independent terminations. For the former, there is no known consensus sequence whereas there is for the latter.
I use pGEX vectors for heterologous protein expression. Is there any frameshift mistake in your construct?
Dear Abir,
Finally, production in the traditional IPTG expression system was unsuccessful for me. Using the same pGEX vector, in autoinduction broth at reduced temperature, the recombinant protein was successfully produced.
If you have problems with your production, I would recommend autoinduction broth testing based on my experiments.
good luck!!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA