soluble protein, low expression - (Apr/17/2007 )
I'm expressing a protein in BL21 DE3, and I have it soluble, but there is very low expression. There seem like a lot of things I could possibly change, but I'm not sure what is best to do in order to raise expression levels.
Right now I grow an overnight culture in LB, split 1 in 20 in the morning into 2xYT, grow for 1 hr at 30degrees, then induce expression with 0.5mM IPTG and grow for 4 hrs at 30 degrees.
I'm thinking more IPTG, lower temperature and a longer time for expression, but I've never done this before so really I have no idea.
Go for longer time expression first. I always think in order to get good expression, start from the time as it increases with the time until the cells die. Then lower the temperature. 25 C is pretty good for expression.
Try the overnight express instant TB medium from Novagen
A very stupid question here.
What is the difference by using terrific broth and LB broth? I mean... I can understand TB has much more nutrient. But does it really make a significant difference for expression? What are the other advantages to use TB over LB? Thanks. 
Hi
Induction works best when given at OD600nm of 0.6 to 0.8. Besides what u are planning try doing this also.
gud luk
Try to cultivate your bacteria in presence of glucose (0.4%) and induce later for a shorter time, your protein is mayby toxic for the bactéria. You can try to use strain like BL21 pLys too...
What is the difference by using terrific broth and LB broth? I mean... I can understand TB has much more nutrient. But does it really make a significant difference for expression? What are the other advantages to use TB over LB? Thanks.
Oh yes... If the protein is a bit toxic for the bacteria, their growth will be difficult in a poor medium. And less you have bacteria at the end of the culture, less your production would be good... Logical ;-)