Expression of fusion proteins - (May/03/2010 )
Hi Everyone,
I'm having trouble with creating a GFP fusion protein. Originally I fused GFP to the C-terminal of my GOI, but could not get any fluorescence. Sequencing told me that everything was in-frame, I even tried multiple ~12a.a linker sequences. I figured this was a folding issue as GFP is a bulky protein. Next, I made an N-terminal GFP fusion, after which inserted a stop codon in the GFP. Theoretically, this should (and did) give me a dark GFP, but I should not see any expression of my GOI, fused at the C-terminal.
However, I do, in fact, see expression of my GOI, and all the colonies are dark. It seems like I'm getting read-through after my stop codon. My co-worker explained to me that when the polymerase hits a stop codon, it will scan up and downstream about 60nt to find another start, and will continue transcribing. There is an in frame ATG start about 8 aa upstream of the stop. Can anyone confirm if this is my problem?
Any help on GFP-specific fusions would be greatly appreciated, I've been at this for months!
Also, just for reference, this fusion is on a plasmid and is transformed into e.coli cells.
Cheers!
Jenn
ms.mutator on May 3 2010, 04:38 PM said:
I'm having trouble with creating a GFP fusion protein. Originally I fused GFP to the C-terminal of my GOI, but could not get any fluorescence. Sequencing told me that everything was in-frame, I even tried multiple ~12a.a linker sequences. I figured this was a folding issue as GFP is a bulky protein. Next, I made an N-terminal GFP fusion, after which inserted a stop codon in the GFP. Theoretically, this should (and did) give me a dark GFP, but I should not see any expression of my GOI, fused at the C-terminal.
However, I do, in fact, see expression of my GOI, and all the colonies are dark. It seems like I'm getting read-through after my stop codon. My co-worker explained to me that when the polymerase hits a stop codon, it will scan up and downstream about 60nt to find another start, and will continue transcribing. There is an in frame ATG start about 8 aa upstream of the stop. Can anyone confirm if this is my problem?
Any help on GFP-specific fusions would be greatly appreciated, I've been at this for months!
Also, just for reference, this fusion is on a plasmid and is transformed into e.coli cells.
Cheers!
Jenn
One correction, the polymerase doesn't look for a start site, the ribosome does. The RNA polymerase won't look for starts, it looks for promoters and falls off either due to the rho protein or an AT rich region at the end of a transcript. Sounds like a reasonable explanation, if the ribosome is meant instead of the polymerase. The ribosome is reading your fusion as an operon. I would suspect, however, that there may also need to be a ribosome binding site upstream of the start codon, something along the lines of AGGAGGA, but I'm not sure about it being necessary in that situation.