Hi
I have cloned a 44.1 kDa protein (which exist as trimer in native state) in pET28b. It is expressing well in BL-21, Rosetta(DE3) as well as C41 strain. I have attached the visualization tool protter diagram for this sequence. Does anybody has experience with purifying such protein (which detergent, strain to use etc) (It has two transmembrane region only near N-terminal end, rest part is cytosolic). Total amino acid is 406.
Hi,
I have some questions for you before offering some suggestions.
- Is this proteing beeing expressed in bacterial cytoplasm or attached to cell mambrane?
- Have you ever tried purifying this protein?
Membrane proteins are usually insoluble. Thus you'll probably need to use refolding techniques.
How do you intend to purify you protein? (I mean, your purification tag).
It is being expressed in bacterial cell membrane. I plan to purify it by N terminal His tag (Ni NTA).
Hi Haaris Safdari,
If this protein is from bacterial origin or has a signal peptide recognized by bacterial transporters then it very likely to be located in the membrane (inner or outer membrane depending on the signal peptide). If that is the case, then you can probably confirm expression checks with western blotting to see if the His-tagged protein is being expressed. If it is expressed, then you can do an inner membrane/outer membrane extraction. These are usually done with various detergents. There are many articles on OM extraction of proteins from E. coli, so you can read up on several of them and then come up with your own 'recipe' based on available reagents: Article A Rapid Selective Extraction Procedure for the Outer Membran...
https://bio-protocol.org/e1287
Now if your protein is of non-bacterial origin it may be complicated. If your protein is expressed and folded in the bacterial membrane, then try the above. If not, it may be unfolded/aggregates in inclusion bodies. You may have to do a check to confirm if it is in the membrane or in inclusion bodies since inclusion bodies and membranes are both located in the cell pellet after cell lysis.
If it is in inclusion bodies, but you want it expressed in a membrane environment, you can try inserting an E. coli protein specific signal peptide on your construct to locate it to either the inner membrane or the outer membrane.
However, you can purify it from the inclusion body as well. You will have to use denaturing conditions such as 6-8M urea or 6M GdmCl to solubilize the inclusion body. Then you will have to try a refolding protocol with detergent or liposomes. Unless some literature is known about your protein or maybe a homolog, then you may have to screen several refolding conditions
Hope some of this is helpful to you...Cheers
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