Expression using pTrc/99A vector

Peter Alliger u7q11aa at sunmail.lrz-muenchen.de
Mon Feb 13 04:29:03 EST 1995


In article <1995Feb13.131608.1 at hkucc.hku.hk>
vldnaseq at hkucc.hku.hk writes:

> Hi, everybody.  Did anyone use the expression vector pTrc/99A from Pharmacia
> I've used it for the expression of eukaryotic protein which is nontoxic, in 
> E.Coli.  However, after purification of the protein (30X purification), only
> 500ug of protein/ L of culture, was obtained.  Besides, I didn't find any 
> significance difference  of the desired protein between the noninduced and
> the induced culture.
> What I've done to induce the protein was as follow:
> Bacteria culture grew until O.D.=0.6.  IPTG was added to a final concentration
> 4mM.  Culture was continuously incubated for 12 hr and harvest.  The cells were
> lysed by sonication.  Difference concentration of IPTG had been tried, 0.1mM,
> 0.4mM, 1mM, 2mM, 4mM. But, no difference of the expressed protein had been found
> 
> Would you please suggest any methods you've experienced?  Thank you.
> 
> Shannon
> 
I had good experience with this vector. A time coarse showed increasing
expression up to 2 to 3 hours with 1mM IPTG. Perhaps your induction is
to long or your construct is not ok (did you check for the correct
reading frame?). One more try would be to use a different strain of
E.coli (for example JM109).

good luck  PIT

===============================================================
Peter Alliger, Ph.D.                   
Institute for Immunology                 
University of Munich                    
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