Insoluble Protein - (Dec/08/2011 )
I got this protein and I really wanna produce a reasonable quantity for protein crystallization.
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?
Thanks!
Both lowering the growth temperature and reducing the IPTG concentration can help. What temperature, IPTG concentration and growth medium do you use?
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
TheBalrog on Fri Dec 9 22:39:13 2011 said:
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.
protolder on Mon Dec 12 07:28:02 2011 said:
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.
hola yes it would be enough to have the protein soluble, if it has S-S-bridges add 2mM of reducer to the lisys buffer and increase the volume of it, and try any tioredoxine strain as Origami family, buena suerte
I did an experiment once to compare the amount of soluble protein produced in the following media: LB, 2-YT, Super Broth and Terrific Broth. I then loaded equal volumes of the soluble fractions from each culture onto an SDS PAGE gel (rather than equal protein) and did a western blot to detect the construct. Only in Terrific Broth was my protein soluble. The formulae for these media can be found on this website:
http://www.exptec.com/Bacterial%20E.coli%20Growth%20Media.htm
However, the optimum growth medium should be tested for each construct.