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

Abstract

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 applied it to screen 61eukaryotic 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 functional 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.

Authors:
ORCiD logo [1];  [1];  [1];  [2];  [3];  [4]
+ Show Author Affiliations
  1. New York Structural Biology Center, New York, NY (United States). Center on Membrane Protein Production and Analysis (COMPPÅ)
  2. Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II)
  3. New York Structural Biology Center, New York, NY (United States). Center on Membrane Protein Production and Analysis (COMPPÅ); Columbia Univ., New York, NY (United States)
  4. Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II); Brookhaven National Lab. (BNL), Upton, NY (United States)
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
OSTI Identifier:
1837204
Report Number(s):
BNL-222489-2021-JAAM
Journal ID: ISSN 0961-8368
Grant/Contract Number:  
SC0012704; 5P41GM116799
Resource Type:
Accepted Manuscript
Journal Name:
Protein Science
Additional Journal Information:
Journal Volume: 31; Journal Issue: 3; Journal ID: ISSN 0961-8368
Publisher:
The Protein Society
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; cell-free expression; eukaryotic; membrane; proteins; high; throughput; lipidic

Citation Formats

Bruni, Renato, Laguerre, Aisha, Kaminska, Anna‐Maria, McSweeney, Sean, Hendrickson, Wayne A., and Liu, Qun. High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics. United States: N. p., 2021. Web. doi:10.1002/pro.4259.
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Bruni, Renato, Laguerre, Aisha, Kaminska, Anna‐Maria, McSweeney, Sean, Hendrickson, Wayne A., & Liu, Qun. High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics. United States. https://doi.org/10.1002/pro.4259
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Bruni, Renato, Laguerre, Aisha, Kaminska, Anna‐Maria, McSweeney, Sean, Hendrickson, Wayne A., and Liu, Qun. Wed . "High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics". United States. https://doi.org/10.1002/pro.4259. https://www.osti.gov/servlets/purl/1837204.
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@article{osti_1837204,
title = {High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics},
author = {Bruni, Renato and Laguerre, Aisha and Kaminska, Anna‐Maria and McSweeney, Sean and Hendrickson, Wayne A. and Liu, Qun},
abstractNote = {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 applied it to screen 61eukaryotic 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 functional 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.},
doi = {10.1002/pro.4259},
journal = {Protein Science},
number = 3,
volume = 31,
place = {United States},
year = {Wed Dec 15 00:00:00 EST 2021},
month = {Wed Dec 15 00:00:00 EST 2021}
}
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https://doi.org/10.1002/pro.4259
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