Expression using pTrc/99A vector

vldnaseq at hkucc.hku.hk vldnaseq at hkucc.hku.hk
Mon Feb 13 00:16:08 EST 1995


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




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