With the bacterial vectors I am using, convenient cloning sites would insert the gene and also a 5' bacterial IRES into pFastbac1. Will this cause problems downstream in trying to make a baculovirus and trying to express protein in Sf9 cells?
Hi Gabby,
You can find some info in the following link; https://www.ncbi.nlm.nih.gov/books/NBK143992/
Do you plan to use the IRES to express 2 proteins at the same time? I am a bit confused about that part since IRES used for bi-cistronic gene expression are usually of viral origin and not bacterial.
I did work with IRES and I can tell you that an IRES sequence directly after a promoter will still lead to protein production. However, if the IRES contains an ATG this might cause some issues with the translation and a lower product yield.
In general I have noticed that using IRES sequences to express 2 genes of interest always results in reduced protein expression of both genes. I guess that is because a single, long mRNA is much more unstable and harder to process than 2 short ones.
Thanks for your reply! The IRES I am referring to is an artifact from the original construct, which is bacterial in origin.
I see. As long as the IRES artifact is not too long it should not interfere with protein expression. After formation of the ribosome it moves along the DNA until it finds the start codon ATG.
Have you considered amplifying your construct using PCR? When I had trouble with some DNA artifact I did not want in my final construct I used primers which directly annealed to the start and end of the cDNA. I then added restriction sites to my primers and amplified my whole construct using a high fidelity polymerase. I isolated the PCR product an cut it with the restriction enzymes. The great advantage is you can add any restriction site you like with overhang primers and since your PCR product is not methylated you can even use DAM sensitive enzymes.
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