protein expression - I can not express in E.coli (Apr/25/2003 )

[COLOR=blue]Hi!
Now ,I want to express a protein in E.coli to study its function.It may be a regulator and has large size---about 100kD.
I have cloned it in pET30a(+) in frame and try to express it in BL21(DE3).The induction temprature(20, 25 37) and IPTG concentration(0.1, 1) have been tryed.
But when SDS-PAGE,no protein I expect is expressed.
could you give me some advice.
Thanks!!!

Hi!
I had that kind of problem before. I have 2 ideas in mind. First, ur protein may be toxic for E. coli. You should try to check for the integrity of the clone u take for ur overproduction. Also, u may want to use BL21 (DE3) pLysS or pLysE to get rid of the basal expression that is there even without overproduction. Second, I don't know from which organism is the gene u cloned, but this organism may have a different codon usage than E. coli. U could try with BL21(DE3)codonplus, a strain that has more tRNAs for the minor tRNA present in E. coli, permitting the overproduction of protein from eukaryotes, for example. Hope it helps...
dbernard