On 25/09/2010 20:44, Cathal Garvey wrote: > > -mRNA detected (rtPCR?) but no translated product.
Correct. Formally, the primers used for the RT-PCR were designed to amplify the complete ORF of my gene, so they don't show that the ORF is in-frame with the tag. On the other hand, I've sequenced every individual plasmid construct with appropriate fwd/rev primers (T7 , M13 or EGFP depending on the construct) and shown that the ORF is in-frame with the tag.
Even if chaperones are necessary, surely you'd detect misfolded protein?
Yes and no. I only have the one peptide antibody to the ORF itself, which I'm not 100% sure is detecting the right thing, and in any case is directed near the C' terminus, so it won't detect prematurely terminated polypeptides. It should however detect misfolded full-length stuff. Most of my constructs have been C' fusions (since I wanted to purify full-length protein and not partial products), so anythin that isn't full-length might just not be seen.
When I use a C-terminal His tag in bacteria or in lysates, I get no signal at the expected size. When I use a C-terminal GFP tag in mammalian cell culture, I get a (low) signal at ~native GFP size, which may be internal initiation or contamination with uninserted vector.
The only N-terminal tag I've tried is a GST tag in bacterial culture. In this case, using anti-FP antibody I get a signal at ~native GST size (maybe fractionally larger). Either the translation is terminating early, or there's contamination with uninserted vector. Yes, I did pick a single colony, sequence it from both ends, etc.
Wht I can conclude (and I'm more than willing to be corrected!) is that if a chaperone is necessary, it's actually necessary for *production* of the full-length protein, not just for correct folding. Alternatively, codon usage is a strong possibility.
A 5' fusion with GFP might answer this question too: as GFP will be translated first, you'll surely see it being produced. If it's not attached to your protein, then it's probably a codon use issue and translation is stalling at an absent codon.
That's what I see with the N terminal GST fusion in bacteria. Bacteria != mammalian cells though. Time to make a mammalian constuct with N terminal tag, I guess...
Peter _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
