Some of this may have been discussed offlist, but: -mRNA detected (rtPCR?) but no translated product. -Codon use has several very poor codons (any references out there support the possibility that different tissues have different codon use profiles?)
Gene synthesis would answer things pretty quickly, especially if it's biobricked for ease of fusion to GFP (be sure to use a protein-fusion compatible format, and request a compatible super-folder GFP from the registry if you're academic). However, the cost might be a bit much if it emerges that this isn't the problem. Even if chaperones are necessary, surely you'd detect misfolded protein? 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. --- Twitter: @onetruecathal Sent from my beloved Android phone. On 25 Sep 2010 20:26, "DK" <[email protected]> wrote: In article <[email protected]>, Peter Ellis <[email protected]> wrote: >Hiya, > >The pro... >pEGFP-N1 vector (CMV promoter, N terminal GFP fusion). No soluble or >insoluble expression in two... Peter, sorry to be skeptical but - Occam's razor - I find it hard to believe. More details perhaps? How do you detect expression of this protein? I hope by Western using some really specific primaries. No signal at all? Not even against GFP? What you say is that you have a gene that cannot be transcribed or its mRNA translated without testis-specific factors. That is really extraordinary claim, right? (Particularly considering that you start with cDNA). If true, it's a significant discovery on its own and warrants separate investigation. In that vein, have you tried in vitro transcription/mRNA detection? Curious, DK _______________________________________________ Methods mailing list [email protected] http://ww... _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
