[Protein-analysis] pET system expression

Julio Cortazar juliocortazar at o2.pl
Fri Jul 14 05:13:49 EST 2006


Hi there,

I'm trying to express and purify my protein in pET system. We decided
to try it as in pTrc plasmids and DH5alpha cells purification yields
were low for our purposes. What was really strange for me that even I
transformed BL21 (DE3)  and LysS cells by sequenced in MCS region
plasmid there was no induction (have monoclonal antibody for a fragment
of my protein and I see nothing on my blots no matter I grow my cultres
in 25 or 37 deg for 3 7 or 20h. Just nothing. There is in periplasmatic
space some protein (I see it on SDS-PAGE) which might be my (the same
mass) but it is unlikely for me thats it because there are no
difference in protein profile in induced and uninduced samples.
What it could be? I use pET 22b plasmid, I have exchanged original
signal sequence in my protein and ligated fragment of DNA coding mature
protein just after pelB sequence..,

Julio



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