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

• Published in: Protein science Vol. 31; no. 3; pp. 639 - 651
• Main Authors: Bruni, Renato, Laguerre, Aisha, Kaminska, Anna‐Maria, McSweeney, Sean, Hendrickson, Wayne A., Liu, Qun
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01-03-2022; Wiley Subscription Services, Inc; The Protein Society
• Subjects: BASIC BIOLOGICAL SCIENCES; cell-free expression; Cell-Free System - metabolism; Cellular structure; Complex formation; complexes; Detergents; Detergents - chemistry; Eukaryota; eukaryotic; eukaryotic membrane proteins; Full‐Length Paper; Full‐Length Papers; high; high throughput; High-Throughput Screening Assays; lipidic; lipidic mimetics; Lipids; Liposomes; membrane; Membrane proteins; Membrane Proteins - chemistry; Membranes; Membranes - metabolism; Metabolism; Mitochondria; Protein biosynthesis; Protein synthesis; Proteins; Pyruvic acid; Screening; Substrates; throughput; Workflow; cell-free expression; lipidic mimetics; eukaryotic membrane proteins; high throughput; complexes
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.4259

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.