Unexpressable protein

Peter Ellis via methods%40net.bio.net (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 
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



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