On 26/09/2010 02:13, DK wrote:
If I'm right about the soluble/insoluble forms, we can hypothesise that there is a specific chaperone protein in round spermatids that keeps the monomers in a soluble form until they're ready for use.
>
Now I'm confused. All the folding-related things apply to when the protein is *made*. In theory it is possible that a protein is so unstable w/o chaperone(s) that it gets chewed to tiny pieces immediately but in practice it is next to imposible that exact same thing would be happening in all expression systems.
My thought was that there might be a strong mutimerisation / polymerisation signal at the N terminus of the protein. In that case, the polypeptide chains might start aggregating together in the absence of <some factor> required to maintain them in solution.
In that case, translating the message in vitro / in culture in the absence of <some factor> would mean that the nascent polypeptides aggregate together, drop out of solution and are not able to continue translation.
That might explain why the N-terminal GST tag gave a band marginally larger than free GST, while C-terminal His- and GFP-tags gave nothing much.
> How
did that "crud" look on a gel - huge band/blob somewhere about or below right size or not?
The "crud" was seen in a bacterial culture, from a vector that should give a C terminal His-tag fusion. Anti-his gave a smear across all sizes, going up well beyond the expected size. Anti-(my protein) gave nothing much. Maybe some non-specific signal at higher AB concentrations. Note that anti-(my protein) is directed at the C terminus of the ORF.
That says to me that whatever the insoluble stuff was, it didn't contain the C-terminus of my protein and was unlikely to contain the C-terminal His tag.
>
If no huge band that it's just incomplete lysis with BugBuster.
... or that, of course. For what it's worth, I did process several constructs in parallel, and this one gave a larger pellet than the others (which had soluble, correct fusion protein expression).
Peter _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
