5 replies to this topic

[Xerophytes01]

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!

[TheBalrog]

Both lowering the growth temperature and reducing the IPTG concentration can help. What temperature, IPTG concentration and growth medium do you use?

[protolder]

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

[Xerophytes01]

TheBalrog, on 09 December 2011 - 02:39 PM, 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 11 December 2011 - 11:28 PM, 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.

[protolder]

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

[TheBalrog]

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.co...wth%20Media.htm
However, the optimum growth medium should be tested for each construct.