Target bacterial operon is reported to be co-transcribed and co-translated in wild type host. However, protein expression analysis (SDS PAGE) indicates translation of only first gene in frame with His tag.
Thankyou.
Hi Maryam,
Although BL21 strains do not possess a suppressor tRNA gene in their genome, it is possible to obtain TGA stop codon read-through mediated by a near-cognate recognition of tRNATrp. There is a mispairing on the third base of the codon (TGA-CCA vs TGA-TCA or TGG-CCA) but the misincorporation still occurs.
However, if I understand your question correctly, you have put your 2 gene operon into pET-28, and upon expression, you obtain a single protein which has a C-terminal his-tag. If you are suspecting stop codon suppression then I imagine that your tagged protein is the size of the predicted fusion between the two proteins? Are you using a native promoter or the T7 promoter that's on the plasmid?
@Ana thankyou for your input.
Yes, I have cloned a two gene operon in pET28a, which is in frame with N terminal His tag. Vector promoter T7 is used.
About my target proteins, literature describes they are co- transcribed and co- translated in wild type strain and exist as heterodimers. I can see two protein bands of my desired sizes 50 kDa and 10 kDa in 2M imidazole elution after nickel affinity chromatography. Does this mean both my genes are expressed and translated?? But why both proteins are seen as two distinct bands on SDS PAGE??
PS. I am new at protein expression analysis. Hope my question is making sense😊
Dear Maryam,
It would seem that your experiment is working just fine. Because your two proteins assemble into a heterodimer the complex is retained on Ni-NTA via the N-terminal His-tag on one of the subunits. When you load your eluted proteins on SDS-PAGE the heterodimer decomposes into subunits and you see a band for each of the proteins.
The only thing you might want to change is the imidazole concentration in your elution buffer - 0.25 M imidazole should be more than enough.
Good luck!
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