Hi, I found there are 1 ATG and 1 stop codon in front of our interested gene. Although we can still induce protein expression by Doxicycline in cells(in pSLIK-vector), is it possible that other unwanted transcript or fusion proteins are also expressed? Thanks for your time.
Hi Wen,
by the way you are describing it, it could be possible that you are producing a fusion protein or an unwanted peptide, there are however a few things to consider.
First of all, is the 5' ATG (the one in front of your gene of interest) in frame with the ATG of your gene of interest? If the answer is no, then there is no possibility of a fusion protein but you could have a small peptide.
Assuming that you are working with and eukaryotic system, does your gene of interest contain a kozac sequence? What about the 5' ATG, is it part of a kozac sequence? Ideally you would have one in your gene of interest but not in the 5' ATG as that would favor the transcription of your gene.
Regarding the Stop codon, is it in frame with the 5' ATG one, if so that could produce a polypeptide that most likely will be degraded as it probably will not be stable.
Finally how close is the 5'ATG from the promoter region, could it be that is actually part of the promoter? If you keep going 5' you will probably find more ATGs but that does not mean that they will be part of the mRNA.
Hope this helps,
Ezequiel
Hi Wen,
by the way you are describing it, it could be possible that you are producing a fusion protein or an unwanted peptide, there are however a few things to consider.
First of all, is the 5' ATG (the one in front of your gene of interest) in frame with the ATG of your gene of interest? If the answer is no, then there is no possibility of a fusion protein but you could have a small peptide.
Assuming that you are working with and eukaryotic system, does your gene of interest contain a kozac sequence? What about the 5' ATG, is it part of a kozac sequence? Ideally you would have one in your gene of interest but not in the 5' ATG as that would favor the transcription of your gene.
Regarding the Stop codon, is it in frame with the 5' ATG one, if so that could produce a polypeptide that most likely will be degraded as it probably will not be stable.
Finally how close is the 5'ATG from the promoter region, could it be that is actually part of the promoter? If you keep going 5' you will probably find more ATGs but that does not mean that they will be part of the mRNA.
Hope this helps,
Ezequiel
Hi Ezequiel,
The peptides are not stable sounds reasonable. Thank you. We express protein in mammalian cells. The ATG is not a part of the promoter and Kozak sequence as it is cloned by "cut and past" for convenience (by previous lab) during cloning, and it is in frame in front of our gene of interest (GOI). ATG are followed by 3 stop codon (between each there are about 20 amino acids). Sequences are looked like:
ATG-20 amino acids-stop-20 amino acids-stop-4 amino acids-stop-15 amino acid-GOI
Although we can still detect the protein by both tag and protein antibodies in western blot, I am still concerned about the unwanted protein/peptide. Thanks a lot for discussing this question~!
Best,
Wen An
Hi Wen,
from what yo said my guess is the you don't have to worry; (1) no kozac, (2) only 20 aa, (3) several stop codons.
Hope this helps,
Ezequiel
Hi Wen,
Depending on whether the ATGs and also stop codons are in frame with your gene of interest, there is a likelihood of expressing your protein and also some truncated short peptides. However if the yield of your protein is good enough you may not worry about the short peptides. You get lid of them during purificaton.
I hope this helps.Good luck
Lucy
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