Cellular lipid membranes are embedded with transmembrane proteins crucial to cell function. Elucidating membrane proteins’ diverse structures and biophysical mechanisms is increasingly necessary due to their growing prevalence as a therapeutic target and sheer ubiquity in cells. Most biophysical characterization strategies of transmembrane proteins rely on the tedious overexpression and isolation of recombinant proteins and their reconstitution in model phospholipid bilayers. Unfortunately, membrane protein reconstitution depends on the use of denaturing and unnatural detergents that can interfere with protein structure and function. We have developed a detergent-free method to reconstitute transmembrane proteins in model phospholipid vesicles. By utilizing split intein ligations, we are able to semisynthesize single pass transmembrane proteins on liposomes. Split inteins are natural or engineered protein trans-splicing domains. By leveraging the biorthogonality and chemoselectivity of split inteins, overexpressed soluble domains are ligated to synthetic transmembrane peptides to build semisynthetic membrane proteins directly on phospholipid vesicles. This one-pot method bypasses the painstaking expression of recombinantly expressed integral membrane proteins and the multistep process of detergent-based protein reconstitution, making it easier to study these important biomolecules in an isolated system.

Detergent-free reconstitution of functional transmembrane proteins into liposomes

ACS Fall 2021

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