problems with protein expression

[David-Paul Minde]

Dear Shen,
... your protein might be constantly degraded (and therefore be "invisible"
even though it is actually highly "expressed"). Try low-temperature
expression (e.g. 18°C or 15 °C[maybe w/o antibiotics]) or even 10°C if you
have arcticexpress strains.
best wishes,
David
PS
... if it's that small, you might think of simply redesigning of your gene
and just order the (e.g. E. coli) codon optimized cDNA.


2008/9/18 Qingwu Shen <shenq from purdue.edu>

> 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. Could
> anybody
> help me to figure out this problems? I just started to express protein
> using
> E.coli. Any help is highly appreciated.
>
> Shen
>
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-- 
David Minde MSc (TUM)

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