Hi Navjyoti,

You can get the recombinant protein to the soluble phase by lowering the induction temperature and induce the protein with less amount of inducer. Lowering the temperature and the inducer concentration can allow the bacteria to grow slowly and can give time for your protein get folded properly and come to the soluble phase. Changing strains can also help you to get your protein to soluble fraction.

I have not worked on membrane proteins before but here is an article which looks like a promising option if you have not tried already. Please see Wagner et al. (2008) Proc. Natl. Sci. USA 105:14371–14376 and

http://kb.psi-structuralgenomics.org/update/2008/10/full/th_psisgkb.2008.9.html. on Tuning membrane protein overexpression.

Please post the solution at the end to help others. Good luck.

Hi Navjyoti,

You can get the recombinant protein to the soluble phase by lowering the induction temperature and induce the protein with less amount of inducer. Lowering the temperature and the inducer concentration can allow the bacteria to grow slowly and can give time for your protein get folded properly and come to the soluble phase. Changing strains can also help you to get your protein to soluble fraction.

I suggest to transform into C43 or C41 since they are known to perform well on membrane proteins. In my experience addition of a solvent e.g. 2% tritonX-100 before disrupting the cells helps to disconnect the protein from the membrane therby allowing you to purify it from the soluable fraction.

Could we have some details about your "membrane protein", intrinsic or peripheral ?, beta barrel or alpha helices ?, and how many helices ? It seems reasonable that an eucaryotic membrane protein would go into inclusion bodies in a normal E. coli strain. Then you need some detergent; for instance have a look at Methods Mol Biol. 2010;601:205-17. As an alternative, you may think about cell-free synthesis which is increasingly successful with membrane proteins.

To Chandrasekhar, could you explain how changing growth conditions can make a membrane protein to "get folded properly and come to the soluble phase" ? It seems that the proper folding for a membrane protein is precisely to make it embedded in a lipid membrane, and not be soluble in aqueous media.

It might help to make use of protein fusions, like maltose binding protein on the N-terminus and thioredoxin C-terminally, as was used for some GPCR by Grisshammer & Tate.

The pGEX vector you used does this give a GST-fusion ? If so these a prone to go o inclusion bodies. Look at the issue of Meth Mol Biol as Pierre proposed.

you can try dissolving the insoluble protein in 8 M Urea or 6 M GdHCl and dialyse it, against buffer, the protein will refold to its native form. Or if dialysis does not work. FIrst Dilute the the protein dissolved in urea to 50-200 times than dialyse it.

I am working on membrane proteins and i had this problem also. If your protein is mainly located in the inclusion bodies, i suggest you to try the protocol proposed by Feroz . But if it is located in the insoluble fraction (pellet = membrane and inclusion bodies) u could resuspend your pellet in a organic solvent (HFIP, ACN..) and try to purify you protein by RP-HPLC.

Good luck!

thanks a lot everyone. I am trying a new strain for now. will let you know

Dear all,

I have retransformed into C43 and tried to express the protein but can't get any expression with different temperatures and IPTG concentration.

Guess I have to purify from inclusion bodies. Can anyone please email the detailed protocol.

thanks

Hi,

Resuspend the pellet in the lysis buffer(20mMPhosphate + 5mM DTT + MgCl2 + Protease inhibitor + DNaseI + lysozyme) and ultrasound, centrifuge.

centrifugate will be loaded to IMAC and purify the membrane protein

goodluck

Hi Dear,

First thing to do your protein is check the properties like transmembrane, signal sequence, GPI-modification site, secondary structure etc. by different available online bioinfo tools so that at least you know your protein specially membrane protein very closely. Then start your wet work in right direction. As you earlier said your protein was going into pellet in BL21(DE3) strain, upto how much lowest IPTG conc. you are checked now with minimum temperature and also with induction time? If you try to solubilize your protein from pellet then the next problem will arise of functionality which will be more hectic, so try to make your protein in soluble form...

Good Luck for your experiment....

Radhey

Navjyoti,

I have had a lot of luck using SDS solubilization and on-column detergent exchange following the Sander's Lab protocol from Vanderbilt University. The major problems I have encountered using SDS is that it decrease binding affinity for nickel columns making it more difficult to get maximal yield form inclusion bodies. I have also read that mild denaturing conditions like SDS are much better than using 8M urea or other harsh denaturing conditions. I hope this helps! I am attaching the Sander's Lab protocol, but it is also available on their website for free.

Good Luck!

Jacob McMillan