Protein expression problems - (Feb/15/2005 )

Hi,
I've been having trouble getting any expression of my gene(2Kb) when inserted into pET14b vector using NdeI and BamHI Restriction enzymes. This is odd because I got great expression in a pET32b vector. I've verified that my gene is inserted in the vector by bacterial PCR. I've check my primers to see if they may have caused frame shifting. What else should I be checking?

KK

well, this is going to be of no practical help for you, sorry...
but, for the morale I can tell that what you experience is not that unusual...same thing happen to me two times. a gene that got expressed well enough in one vector and a e.coli strain wasn't expressed at all when inserted in another vector, though that other vector worked perfectly with an other gene.... checked everything, no help... sometimes things just won't work the way we want them!

mike

jadefalcon is right on about this. That said, you shoud try as many reasonable modifications as possible.
Are you using Bl21(DE3) or Bl21(DE3) pLysS? The pLysS may take more IPTG to induce, and often have lower expression levels. How much IPTG are you using? At what temp are you growing the cells?

good luck

Thanks for the reply, I am using BL21 DE3 cells and a 1mM final concentration in a 1 L culture and inducing for 14-16 hrs. I've tried two inducing temps.....16 and 21 degrees. They both gave the same result. Any suggestions?

Hi,

Did you try to express at 37 or 30 degree for 2 hours? If you can get the protein at those temp. and can not get at lower degree so you got the same problem with me.
Since the AmpR plasmids are easy to be thrown out of the cell. My case is pET21a, hic.

Try another vector is the best way I think.