(by pjie2 from cam.ac.uk)
Sat Sep 25 20:47:08 EST 2010
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
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.
> 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
>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).
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