pRSET expression system troubles

Hans Wessels hansw at sci.kun.nl
Sun Aug 25 06:35:00 EST 2002


"Vayuputra" <vayuputra369 at hotmail.com> wrote in message news:<ajruqi$or6$1 at charm.magnus.acs.ohio-state.edu>...
> I've done some work with the pRSET vectors and had decent success.
> 
> One of my observations was that in small cultures (3-5 ml) I couldn't detect
> the protein.  But when a large culture is grown (500 ml - 1 litre) I saw an
> obvious overexpression.
> 
> OD values seem to be specific to individual fusion proteins - try various
> conditions (induce the cells in increasing cell densities : 0.4, 0.6, 0.8
> and 1.0 OD600 values).
> 
> Try 0.1mM, 0.5mM IPTG concentrations to induce the protein.
> 
> (These can only be done after following another suggestion to your query -
> sequence the clones to verify if your protein is in frame with the tag.
> Some clones get induced better than others.  Once I identified cell clones
> expressing well I made glycerol stocks and have been using those cells
> eversince).
> 
> Hope this helps.
> Vayuputra


Hi,

i have had some problems using the pGEX 4T1 system in combination with
E. coli BL21 RiL codon plus. It turned out that adding glucose to a
final concentration of 0,5% did the job. It could also be that your
clone is unstable. This case you have a problem resolving in
transforming them right before use.



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