problems with protein expression

[Carmen P Pena Diaz]

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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