High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics.

Autor: Bruni R; Center on Membrane Protein Production and Analysis (COMPPÅ), New York Structural Biology Center, New York, New York, USA., Laguerre A; Center on Membrane Protein Production and Analysis (COMPPÅ), New York Structural Biology Center, New York, New York, USA., Kaminska AM; Center on Membrane Protein Production and Analysis (COMPPÅ), New York Structural Biology Center, New York, New York, USA., McSweeney S; NSLS-II, Brookhaven National Laboratory, Upton, New York, USA., Hendrickson WA; Center on Membrane Protein Production and Analysis (COMPPÅ), New York Structural Biology Center, New York, New York, USA.; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA., Liu Q; NSLS-II, Brookhaven National Laboratory, Upton, New York, USA.; Biology Department, Brookhaven National Laboratory, Upton, New York, USA.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2022 Mar; Vol. 31 (3), pp. 639-651. Date of Electronic Publication: 2021 Dec 23.
DOI: 10.1002/pro.4259
Abstrakt: Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and apply it to screen 61 eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functionally competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.
(© 2021 The Protein Society.)
Databáze: MEDLINE
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