Unexpressable protein

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Sat Sep 25 14:44:13 EST 2010


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" <dk from no.email.thankstospam.net> wrote:

In article <8g4cerFp61U1 from mid.individual.net>, Peter Ellis <pjie2 from cam.ac.uk>
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


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