Hi,
I am working on a protein which is known to anchor in the membrane post translation. I am recombinantly expressing it in E. coli and on lysis the protein is detected in pellet fraction and not the cytosolic fraction. On harsh sonication some protein can be detected in soluble cytosolic fraction which confrms previous reports and bioinformatics observations that the the protein is not integral membrane protein but membrane anchored protein. Is there any method other than use of detergent solublization to bring the protein in soluble fraction? Any experience or specific literature reference would be of great help.
I would refer you to:
There is another method indeed.
Detergents are the "traditional" method if you want. For a more up-to-date approach I would go with synthetic nanodiscs.
https://cube-biotech.com/what-are-nanodiscs
Dear Ameya
Do you really need to express also the membrane anchor domain?
Because in general for production of recombinant proteins carryng a single membrane anchor, you can remove this domain at the cloning stage and produce only the intracellular or extracellular functional domain.
For example in the past i did it to study different proteins as Human Sco1 and Sco2 or streptococcal sortases proteins.
you can find some guidelines for this on
https://www.researchgate.net/project/Optimizing-new-protocolls-for-recombinant-protein-production/update/5c9ce24e3843b0342433f828
and a video summmarizing some free tools avaialbes to perform this analisys is on my blog:
ProteoCool https://proteocool.blogspot.com/ at PAGE6
The production of the soluble dom,ain will facilitate a lot your work, because the presence of membrane region require the use of detergents (eg OG, DDM, DM) and is not obvious find a detergent able to solubilze the protein but not affect the folding of the soluble part.
If you need to express the entire protein, one alternative could be use a E.coli strain able to produce OMV (eg delta TOlR or Delta OmpA strains) and check if the protein is expressed on the vesicles. But in this case you can you just compare properties of empty vesicles vs vesicles carryng your protein, but protein purification from vesicles will be subjected to the same problems of protein purification from E.coli membranes.
good luck
Manuele
Hi,
I recommend you try nanodiscs technology. Phospholipid nanodiscs are 10 nm sized discoloidal protein-lipid particles which are consisting of around 150 phospholipids arranged in a central bilayer, stabilized by two amphipathic membrane scaffold proteins that wrap around the rim of the bilayer. Nanodiscs can shield the acyl chains of lipids from the outside aqueous environment, providing a soluble membrane mimetic into which membrane proteins can be incorporated.
Hope it helps!
- Badges
- Science topic