Hi...
Concerning your protein expression issue, i should add that the rossetta strain growth rate is very low compared to others. So you should induce for longer times to obtain a better yield. Also, did you check the protein through western blot? Probably it is there and the yield is so low is not detectable on an SDS-PAGE compared to the negative control.
P.
-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu on behalf of DK
Sent: Fri 19/09/2008 01:42
To: methods from magpie.bio.indiana.edu
Subject: Re: problems with protein expression
In article <mailman.45.1221756825.29717.methods from net.bio.net>, Qingwu Shen <shenq from purdue.edu> wrote:
>Hi everybody,
>>I'm trying to expression some rabbit skeletal TnI using E.coli. The plasmid I'm
>>using came from some other lab and has been stored in freezer for many years.
> I'm
>using Rossetta (DE3) PlysS to express protein and the yield is very low. Even
>worse after I changed two Cys to Ser in the protein using PCR mutagenesis, I
> got
>no protein (mutant TnI) expression although there was a induction band on SDS -
>PAGE gel when compared the induced sample to non-induced control.
That would say that there was expression, wouldn't it?
Did you mean to say "soluble expression"? Well, some proteins don't
fold well in E.coli, that's a fact of life. Troponin I is small enough and,
supposedly, simple enough in its structure, that refolding might do the
job nicely.
Also, some proteins don't *express* well in E.coli. May be a codon
usage thing or something more complicated. I.e., we've never been
able to express large amounts of yeast calmodulin in E.coli - even
though bovine expresses ridiculouslywell. And that is in a strain
that provides all the tRNAs that are rare in E.coli.
DK
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