Hi everyone,
I am trying to express and purify a protein in BL21 (DE3) and arctic express RIL cells, which has a membrane binding region. I am using Triton X100 and CHAPS as detergents for its successful purification. However, most of the protein goes into the pellet. Could you please let me know what else could be added into the lysis buffer for the enhanced solubility of the protein?
Thanks
This is likely soluble expression, correct? Is there any evidence for inclusion body formation (phase contrast microscopy) even though you employ the Arctic Express strain? The cold-adapted heterologous chaperonins may not necessarily guarantee solubility and correct folding. Is there AT richness due to codon optimization? When you mention a pellet, is this a pellet of inclusion bodies with a membrane layer/cell debris on top at around 10K x g or a membrane fraction only? Often the over-expression of foreign receptors into the inner membrane/periplasm of E coli will result in crowding and precipitation with mis-folding which can pellet out upon centrifugation (even though you're trying to prevent this with the chaperonin system). Is this C-terminally His tagged?
How is the cell disruption done; are you using only the detergents without any mechanical or enzymatic intervention? Does the lysis procedure involve digestion of the peptidoglycan follwed by a freeze/thaw and a clarifying centrifugation? Is there a reason why you avoided the maltoside-based detergents such as 1% DDM (dodecyl beta maltoside) which is a commonly used surfactant for membrane proteins, IMP's for example? Subsequent downstream processing would then involve ultracentrifugation and perhaps Dounce homogenization of the pellet and SEC purification. This type of procedure has worked not only for the isolation of membrane bound proteins but also membrane intercalated organic molecules.
Hi Grant Shimamoto,
Thanks for your reply.
The protein is cytosolic however it can go and bind to membrane. Therefore, i want to purify it using detergents and different additives. I am using sonication for cell lysis. I do not specialize in membrane protein purification. i would appreciate if you could suggest something for its successful purification.
Regards
Sunil
Have you considered the isoelectric point of your protein and than used the right buffer and pH? Sarcosyl (max 0,3%, N-LAUROYL SARCOSINE SODIUM SALT) is also used, there are lots of detergents useful.
Orwick-Rydmark M, Arnold T, Linke D. The Use of Detergents to Purify Membrane Proteins. Curr Protoc Protein Sci. 2016 Apr 1;84:4.8.1-4.8.35.
In my experience in protein expression when you express one membrane protein in E.coli, if the protein is not toxic it forms inclusion bodies that are the only way to store high amount of protein expressed. Regards
The literature is plethoric with methods do do what you want to accomplish and it appears you already have a procedure since you seem committed to triton and chaps. The cell disruption method may also be from a literature procedure you are following. Again, check for inclusion bodies, check for complete cell homogenization, and be certain that enhancing solubility during lysis is not the same as tryng to solubilize misfolded protein to prevent them from forming a non-ultracentrifugal pellet.
- Badges
- Science method