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bioRxiv · 10.1101/2021.09.08.459519

Semisynthesis of functional transmembrane proteins in GUVs

Podolsky, K. A.·Masubuchi, T.·Debelouchina, G. T.·Hui, E.·Devaraj, N. K.
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Abstract

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

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Podolsky, K. A., Masubuchi, T., Debelouchina, G. T., Hui, E., Devaraj, N. K.. 2021-09-08. Semisynthesis of functional transmembrane proteins in GUVs. https://doi.org/10.1101/2021.09.08.459519

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