His-Tag Question (pQE30/31/32 Qiagen Vector) - (Aug/01/2006 )
Has anybody had success w/ the pQE30/31/32 Qiagen vector?
What's the difference between the T5 promoter and the T7 promoter?
We are getting extremely low expression levels (~1% expression), and we are looking at different vectors and cell lines. Right now, we're getting maybe 1 ug of purified protein from ~100 ml of culture. We need about 1 mg of protein for our project.
We recently realized that multiple arginine residues in our desired proteins could be responsible for this, and we have considered switching to the Rosetta cell line. Does anybody have any other suggestions? The proteins that I am trying to expression are between 25-50 kDa, and are kinases and transcription factors. Will inducing at low temperatures overnight help?
Also, which vectors have people had success with? We're looking at the pRSET T7 vector from Invitrogen.
Thanks!
We use pcDNA 3.1 myc-His from invitrogen to clone our transgenes and use it for further protein expression and purification.
What's the difference between the T5 promoter and the T7 promoter?
We are getting extremely low expression levels (~1% expression), and we are looking at different vectors and cell lines. Right now, we're getting maybe 1 ug of purified protein from ~100 ml of culture. We need about 1 mg of protein for our project.
We recently realized that multiple arginine residues in our desired proteins could be responsible for this, and we have considered switching to the Rosetta cell line. Does anybody have any other suggestions? The proteins that I am trying to expression are between 25-50 kDa, and are kinases and transcription factors. Will inducing at low temperatures overnight help?
Also, which vectors have people had success with? We're looking at the pRSET T7 vector from Invitrogen.
Thanks!
Hi!
I use Rosetta 2 cell lines with pQE80's vectors and I get different expression, depending on the gene that I am using and its effect on the cells. I have found that if one vector doesn't give me great expression, sometimes switching the vector will help.
C.