problem in expression of his tag protein - (Nov/06/2005 )

Hi All, I am trying to express on his tag protein (of 80kda) in BL 21 cells, which is cloned in pet 7.1. It is not at all indusing by IPTG. I am not seeing any difference between uninduced and induced bands on sds-page. Even I tried to bind the induced lysate with ni-nta, but nothing is binding (exept some non-specific junks ).Please any one can help me regarding this, what might be reason for this unexpresion of this protein.
thanks

have you check cell line? They may be lactose insensitive?

you might need BL21(DE3) instead of BL21

the difference is, the (DE3) version has a copy of the T7 RNA polymerase inducible with IPTG. if you use a T7 promoter expression system without the proper polymerase it is hard to get good induction

I learned this the hard way awhile back myself

Hi, thanks. But, I am using BL21 (DH3) comp.cells only. Even it is not expressing.Please help me regarding this.
thanks.

have you sequenced your clone? perhaps the insert is not properly in-frame, or you ended up with some sort of mutation in the start codon or RBS during cloning?

Thanks..I will sequence my clone

hi,

I am also trying to express his -tag 22-28 kda proteins (3 proteins). I am using Bl-21 De3 cells. And i dont see any difference in bewteen uninduced and induced sample, using IPTG as a an inducer.

The only thing which comes to my mind is that

"since the vector also has a start codon and my insert has also one...., will 2 start codons is playing any role???"

i have sequenced my insert...its complete sequence and inframe.

If anyone has any idea , whats going on, i will be happy.

thanks