I miss two Proteins from my overexpression in BL21 DE3 - (Nov/28/2006 )
I try to express a T4 Protein in BL21 DE3 RIL codon Plus via a pET28 vector. Here we go:
1. Transfection is very poor, down to 1-3 colonies per plate, which appear ~24-72h after transfection
2. I give them an antibiotic stress of 50ug/m Chloramphenicol and 100 ug/ml of Kanamycin, the pACYC Plasmid (chloramphenicol resistance) and my vector (pET28 with kanamycin marker) should be present. In liquid culture the cells grow up to OD 2.8 (haven't tried to grow them more) overnight.
3. After inducing expression with IPTG at OD 0.6 they grow slowly (~0.4 OD in 3h)
4. SDS Gel of the samples before and during expression shows no significant overexpression.
and last but not least:
5. The chloramphenicol acetyl transferase band at 25 kDA is missing !?!
How can that be? There is enough fresh cloramphenicol inside the culture to keep up the stress.
And where did my protein go? I mean, it is a T4 protein, E.coli should have no problem to express it in abundance.
1. Transfection is very poor, down to 1-3 colonies per plate, which appear ~24-72h after transfection
2. I give them an antibiotic stress of 50ug/m Chloramphenicol and 100 ug/ml of Kanamycin, the pACYC Plasmid (chloramphenicol resistance) and my vector (pET28 with kanamycin marker) should be present. In liquid culture the cells grow up to OD 2.8 (haven't tried to grow them more) overnight.
3. After inducing expression with IPTG at OD 0.6 they grow slowly (~0.4 OD in 3h)
4. SDS Gel of the samples before and during expression shows no significant overexpression.
and last but not least:
5. The chloramphenicol acetyl transferase band at 25 kDA is missing !?!
How can that be? There is enough fresh cloramphenicol inside the culture to keep up the stress.
And where did my protein go? I mean, it is a T4 protein, E.coli should have no problem to express it in abundance.
It sounds like you have a toxic protein. One way to get a reasonable yield if this is the case, is to grow the cells to a relatively high density, say 0.8 to 1.0, then induce for a short time.
In order to cross out other potential issues, like poor codon bias, try putting the sequence into this site: http://gcua.schoedl.de/ .
Good luck.