TAT-GFP expression from pRSET vector - (Jul/10/2008 )

Hello there - here we go, long post!

I'm trying to express TAT-GFP contained in a parent vector which is pRSET, it is a pET vector, driven by T7 etc. (i.e. I know this should work in bacteria!). Just to clarify, TAT is a small peptide (11 amino acids) which allows for transduction of proteins fused to it, into target cells. (here is some more info for the parent vector http://www.addgene.org/pgvec1?f=c&cmd=...;vectorid=5899). As far as I can tell the sequence of my plasmids are correct, though I've never sequenced starting upstream of my insert.

I have tried a huge number of combinations of temperature and length of expression, BL21's from invitrogen, novagen, LB+IPTG, or MagicMedia from invitrogen, amongst other things, and I just can't get my protein expressed.

I have attempted to verify expression through coomassie, western blotting, growing transformants on selective plates with IPTG in search of green colonies, or just through looking at the bacterial pellet since I've been told you can see the green color without doing anything to the bacteria after expression of GFP. I've tried exposing my bacterial pellets to light from a microscope and the pellets do not glow green.

Further, I've tried streaking transformed colonies of BL21 onto plates with different amounts of IPTG, from 31.25 micromolar, doubling in concentration up to 500 micromolar, and I did not notice any 'green' on the plates, but I did notice that starting around 125 micromolar the bacteria grow a lot less readily, to the point where at 500 micromolar the plates are practically blank. I don't think this concentration of IPTG is toxic to bacteria, so does that mean that my fusion protein is cytotoxic? This is very strange, since other groups have made fusions of this type many times in the literature.

I have a number of questions:

1. Should I reclone my fusion protein into another magical vector that is more likely to yield expression? which vector?
2. should I use some other medium?? I know LB is supposed to work, but is there something better? I've read about 2xYT, or Terrific Broth.
3. should my bacterial pellet be visibly green in normal light, as I've been told (in which case screening would be super easy), since I'm looking for GFP?
4. do I need to sequence starting upstream of my insert, in case one of my restriction enzymes somehow damaged the promoter region? That doesn't sound likely, but I'm all out of ideas.

Thanks for any help,

H