suggestions to express protein in e coli - (Jan/24/2007 )

Ok, so.. i finally changed the tag of my protein, put it in a pGEX vector , bought rosetta strains and now looking forward to purify it via GST.
I still dont have the beads, i am not sure which one to buy or should I buy columns. Nevertheless I was advised to first induce the expression of my protein and then think of purifying it.. Thats what I will do and I will appreciate any suggestions you might have regarding the conditiopns to test first as for example, temperature, IPTG concentration, scale, parameters of sonication.. etc. (and of course if suthink should I buy any special kit or beads .
I was thinking starting with a volume of 10 ml, at 37 and 30 for 2 and 4 hr of induction hoiw thats sound?

thanks!

not too sure about pGEX though. But i think you need thrombin to cleave away your GST.

Kit or Assay from Amersham Biosciences is pretty good. Check out their range of assays. You might find something useful for you.

10ml sounds a lil bit too much for me. I used about 5ml. The temperatures and induction hours sound ok with me. Good luck.

Thanks timjim, i just inoculated a colony into 2ml.. grow till od 0.5 and then will add IPTG

hope to return with good news tonight

my colleague use pRAR system. It's a plasmid coding tRNA for rare codons in bacterias, which help him on production.
He's now culturing his bacterias at 37, and cool the medium to 15' to express his proteins. This may not be the first adjustement you will follow, but that's for pointing out that slowering the culture tempmay help the bacteria and reduce protein degradation.