Hi! Could anybody tell me if it is possible to clone a GFP tag with my trasngene of interest without being a fusion protein, just a separated elements? Thank you.
Lately I have been using the 2A peptide (P2A to be precise). WE used this approach to generate recombinant Reporter Viruses. SO we put the marker Gene (eGFP) under the IE protein (immediate early promoter). So and when the cell gets to the cell and start expression the gene then the reporter would give GFP signal also. This technique on paper is better in a couple of ways then conventional techs like fuse product or internal IRES. This minimally affects your protein of interest i.e. if the P2A is sitting on the N terminus of your protein of interest then only one amino acid is added to your protein. and the expression of your protein and reporter is equimolar which is not there in the internal IRES site.
But what we observed is that the GFP signal is rather a little weak than the fused product under the same promoter. But then again this is a viral promoter that would be regulated in a number of ways. But I think if you just want to use your protein and marker tag in a vector under a strong promoter like CMV then this weak signal should not be a problem. I have personally not used 2A peptides in vector and used them in full length virus (256K genome). Hope this would be helpful, If you have further questions regarding this I would be happy to help. And regarding generation of these clones you can use seamless cloning so there wont be any scars left in your final product.
Yes. You have to use a bicistronic IRES plasmid (IRES=internal ribosomal entry site). In these you clone GFP and your favorite cDNA in the two cloning sites that are sepparated by the IRES. The only problem is that in the first generation plasmids translation from the second ORF was very low. Hope that was fixed....
Good luck
Estanis
Hi, If I understood correctly, you would like to express the two proteins as separate proteins in the same cell using 1 plasmid. there are many methods but how about just taking a GFP containing plasmid and adding your protein of interest (if it is not too long) after GFP using long primers. in the forward primer, add an end codon (to end your gfp) and then start codons to start your protein of interest. keep some base pairs in between and also make sure there is no frameshift, you can also add a R.E site! so your construct looks like--> start codon-GFP-end codon- spacer with R.E. site-start codon-protein of interest-end codon. and then during expression, they will be separate proteins. otherwise you have techniques like above :)
Do you mean by adding a Restriction site between the GFP and my protein? I am not sure about that...I guess that by others techniques you mean for example by bicistronic promoters or directly cloning my protein into a GFP containing vector. The point is that I have to use an specific vector which is already bicistronic, so I could not take advantage for this to express the reporter. That is why I was asking for a possibility of cloning my protein together with GFP in the same fragment but not as a fusion protein...I was pretty sure that this is not possible o difficult to achieve..but...I had to try!. Thank you anyway!
Try 2A viral peptides approach. They induce "ribosomal-skip" or "STOP-and-GO". I don't know about the nature of your protein, if it's a trans-membrane protein then there are some tricks, otherwise should not be a problem.
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