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Wason, Irvinder Singh

University of British Columbia

Purification and characterization of membrane proteins is often limited by the ability to reconstitute membrane proteins into a soluble state with a membrane mimetic, while ensuring the protein is in a native state. Here we present a method to stabilize the entire E. coli membrane proteome into a soluble state using the peptidisc library, simultaneously purify a target protein, as well as verify native conformation of the target utilizing a native ligand of the target membrane protein. We purify two β-barrels, the ferrichrome receptor FhuA, and the vitamin B-12 receptor BtuB, as well as three α-helical protein complexes, the inner membrane translocon SecYEG, and the expanded holo-translocon (HTL), and a novel expanded holo-translocon we term the HMD complex. We purify these proteins with native ligands and antibodies in a highly pure and soluble state, as well as identify native interactors via mass spectrometry analysis. This is the first time any of these proteins have been purified via their native ligands, in a soluble state in the absence of detergent, as well as the first time the HMD complex has been isolated. This offers a rapid methodology to fish for target membrane proteins in soluble, native states.

Text

2022-07-26

Attribution-ShareAlike 4.0 International

http://hdl.handle.net/2429/82201

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-sa/4.0/