problem in expressing A DNA binding protein - (Feb/16/2012 )

hi,
i have exprienced some problem in expressing a DNA binding protein in Bl21(DE3) cells.

i have checked my gene sequence in the plasmid after ligation by sequencing.it is correct n in correct reading frame.

i have tried many things. given IPTG induction at different O.D.s like 0.6, 0.8, 1.0 even. induced the protein for different time periods after induction i.e. for 4 hrs, 6 hrs. tried expressing at 37 degree and 25 degree(overnight).

even after trying all i get only 1mg of my protein from 1litre of the culture.

a wierd thing that i have noticed is that my Bl21 cells transformed with plasmid carrying my gene of interest reach the O.D. of 0.6 only after about 4 hours which i think is about double the time taken by Bl21 cells without the plasmid.

so i have two questions:
1) how can i increase the expression of my protein.
2) why are the bl21 cells growing so slowly after transformation with my plasmid.

What is your expression system? pET? pGEX? I used the pGEX and met the same problems. Furthermore, I have another band besides the recombinant protein and the GST band.

i had used pET expresion system,,

so what else did u try zhengming??

Well, I know some people who would be lucky to get 1mg protein from 1 L. You can grow e.g. 3 x 1L and after purification you can concentrate your protein sample.

If your protein is going into the soluble fraction then you can try for auto induction media. You need not monitor O. D. of the culture, just inoculate and leave it at 18/25 OR 37 whichever is suitable/optimum for your protein for 24-30hrs. then check for protein on SDS-PAGE.

DNA binding protein could mean that the protein might be toxic ...or cause any other things the bugs don't really like ...therefore they will try to get rid of the expression at any cost. You could try the walker strains for the expression of toxic proteins (E. coli C41/C43 ...distributed by e.g. Lucigen).

Regards,
p

for best protein expression, always use colony from fresh transformed plate, no glycerol stocks or week old plates. I've seen crappy expression of a construct when old plate used, but decent expression with fresh plate. Fresh is best.
Also, try baffled erlenmeyer flasks- the dents in the flask does a better job aerating cultures and cells grow and express better. I've seen big difference between cultures grown in regular and baffled flasks.
I've heard good things about the Lucigen high comp cells- good for toxic proteins that kill your culture