Posted 08 December 2011 - 04:23 AM
I grow the cells 37C then induce it with 400uM IPTG. Harvest, lyse and run a gel but I don't see any band at all in the lysate. Band is clearly on the pellet.
I checked the sequence. The GRAVY index is 0.14, which I don't think is really terrible, since I got some constructs which have index higher than that (0.38) and produce very well as soluble protein.
Any suggestions? I don't really wanna go to the route of mutagenesis as much as possible.
Would lowering down growth temperature and lower IPTG do the trick?
Posted 09 December 2011 - 02:39 PM
Posted 11 December 2011 - 11:28 PM
Posted 15 December 2011 - 12:25 AM
Both lowering the growth temperature and reducing the IPTG concentration can help. What temperature, IPTG concentration and growth medium do you use?
LB media. I use 37C then induce at 16C with 400uM IPTG.
Hola, depending of the host, some protein is expressed in a quasi constitutive way, so check time 0 of induction for it woudn´t be necessary induce. Yes lowering incubation/induction temperature helps you to have soluble protein, and more important, check the isoelectric point of it because if it´s near of your lisys buffer pH, solubility is low and you can solve working with a buffer with a pH enought far of isoelectric point to facilitate solubility. Buena suerte
Isoelectric point for the protein is 7.14. I used Tris buffer at pH 8.5.
Posted 15 December 2011 - 11:03 PM
Posted 17 December 2011 - 08:17 AM
However, the optimum growth medium should be tested for each construct.