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

Bruni, Renato; Laguerre, Aisha; Kaminska, Anna‐Maria; McSweeney, Sean; Hendrickson, Wayne A.; Liu, Qun

doi:10.1002/pro.4259

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

Journal Article · 14 December 2021 · Protein Science

DOI: https://doi.org/10.1002/pro.4259 · OSTI ID:1837204

^[1]; Laguerre, Aisha ^[1]; Kaminska, Anna‐Maria ^[1]; McSweeney, Sean ^[2]; Hendrickson, Wayne A. ^[3]; Liu, Qun ^[4]

New York Structural Biology Center, New York, NY (United States). Center on Membrane Protein Production and Analysis (COMPPÅ)
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II)
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)
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II); Brookhaven National Lab. (BNL), Upton, NY (United States)

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.

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Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0012704

OSTI ID:: 1837204

Report Number(s):: BNL--222489-2021-JAAM

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 3 Vol. 31; ISSN 0961-8368

Publisher:: The Protein SocietyCopyright Statement

Country of Publication:: United States

Language:: English

References (66)

High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli Bruni, Renato; Kloss, Brian Current Protocols in Protein Science, Vol. 74, Issue 1 https://doi.org/10.1002/0471140864.ps2906s74	journal	November 2013
A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins Chae, Pil Seok; Rasmussen, Søren G. F.; Rana, Rohini R. Chemistry - A European Journal, Vol. 18, Issue 31 https://doi.org/10.1002/chem.201200069	journal	June 2012
Hydrophobic supplements in cell-free systems: Designing artificial environments for membrane proteins Hein, Christopher; Henrich, Erik; Orbán, Erika Engineering in Life Sciences, Vol. 14, Issue 4 https://doi.org/10.1002/elsc.201300050	journal	March 2014
Membrane proteins and membrane proteomics Tan, Sandra; Tan, Hwee Tong; Chung, Maxey C. M. PROTEOMICS, Vol. 8, Issue 19 https://doi.org/10.1002/pmic.200800597	journal	October 2008
How bilayer properties influence membrane protein folding Corin, Karolina; Bowie, James U. Protein Science, Vol. 29, Issue 12 https://doi.org/10.1002/pro.3973	journal	October 2020
Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms Wallin, Erik; Heijne, Gunnar Von Protein Science, Vol. 7, Issue 4 https://doi.org/10.1002/pro.5560070420	journal	April 1998
Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents: Polymer-Based Cell-Free Expression of GPCRs Klammt, Christian; Perrin, Marilyn H.; Maslennikov, Innokentiy Protein Science, Vol. 20, Issue 6 https://doi.org/10.1002/pro.636	journal	May 2011
Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels Miroux, Bruno; Walker, John E. Journal of Molecular Biology, Vol. 260, Issue 3 https://doi.org/10.1006/jmbi.1996.0399	journal	July 1996
Reconstitution and Imaging of a Membrane Protein in a Nanometer-Size Phospholipid Bilayer Bayburt, Timothy H.; Carlson, Joseph W.; Sligar, Stephen G. Journal of Structural Biology, Vol. 123, Issue 1 https://doi.org/10.1006/jsbi.1998.4007	journal	September 1998
Nanodiscs as a New Tool to Examine Lipid–Protein Interactions Schuler, Mary A.; Denisov, Ilia G.; Sligar, Stephen G. Methods in Molecular Biology https://doi.org/10.1007/978-1-62703-275-9_18	book	December 2012
Structural genomics for membrane proteins Lundstrom, K. Cellular and Molecular Life Sciences, Vol. 63, Issue 22 https://doi.org/10.1007/s00018-006-6252-y	journal	September 2006
Engineering of the baculovirus expression system for optimized protein production Martínez-Solís, María; Herrero, Salvador; Targovnik, Alexandra M. Applied Microbiology and Biotechnology, Vol. 103, Issue 1 https://doi.org/10.1007/s00253-018-9474-7	journal	October 2018
Lipid-Assisted Membrane Protein Folding and Topogenesis Dowhan, William; Vitrac, Heidi; Bogdanov, Mikhail The Protein Journal, Vol. 38, Issue 3 https://doi.org/10.1007/s10930-019-09826-7	journal	April 2019
The Center for Eukaryotic Structural Genomics Markley, John L.; Aceti, David J.; Bingman, Craig A. Journal of Structural and Functional Genomics, Vol. 10, Issue 2 https://doi.org/10.1007/s10969-008-9057-4	journal	January 2009
Structural genomics target selection for the New York consortium on membrane protein structure Punta, Marco; Love, James; Handelman, Samuel Journal of Structural and Functional Genomics, Vol. 10, Issue 4 https://doi.org/10.1007/s10969-009-9071-1	journal	October 2009
Specific lipid requirements of membrane proteins—a putative bottleneck in heterologous expression Opekarová, M.; Tanner, W. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1610, Issue 1 https://doi.org/10.1016/S0005-2736(02)00708-3	journal	February 2003
Lipid–protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles, bicelles and liposomes Lyukmanova, E. N.; Shenkarev, Z. O.; Khabibullina, N. F. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1818, Issue 3 https://doi.org/10.1016/j.bbamem.2011.10.020	journal	March 2012
Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant Blesneac, Iulia; Ravaud, Stéphanie; Juillan-Binard, Céline Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1818, Issue 3 https://doi.org/10.1016/j.bbamem.2011.12.016	journal	March 2012
Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs Periasamy, Agalya; Shadiac, Nadim; Amalraj, Amritha Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1828, Issue 2 https://doi.org/10.1016/j.bbamem.2012.10.003	journal	February 2013
Cell-free production and characterisation of human uncoupling protein 1–3 Rebuffet, Etienne; Frick, Anna; Järvå, Michael Biochemistry and Biophysics Reports, Vol. 10 https://doi.org/10.1016/j.bbrep.2017.04.003	journal	July 2017
Breaking the barriers in membrane protein crystallography Kang, Hae Joo; Lee, Chiara; Drew, David The International Journal of Biochemistry & Cell Biology, Vol. 45, Issue 3 https://doi.org/10.1016/j.biocel.2012.12.018	journal	March 2013
Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins Sonoda, Yo; Cameron, Alex; Newstead, Simon FEBS Letters, Vol. 584, Issue 12 https://doi.org/10.1016/j.febslet.2010.04.015	journal	April 2010
Membrane protein production in Escherichia coli cell-free lysates Henrich, Erik; Hein, Christopher; Dötsch, Volker FEBS Letters, Vol. 589, Issue 15 https://doi.org/10.1016/j.febslet.2015.04.045	journal	May 2015
Crystal Structure of the Eukaryotic Light-Driven Proton-Pumping Rhodopsin, Acetabularia Rhodopsin II, from Marine Alga Wada, Takashi; Shimono, Kazumi; Kikukawa, Takashi Journal of Molecular Biology, Vol. 411, Issue 5 https://doi.org/10.1016/j.jmb.2011.06.028	journal	September 2011
Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: A case study of the human endothelin A receptor Junge, Friederike; Luh, Laura M.; Proverbio, Davide Journal of Structural Biology, Vol. 172, Issue 1 https://doi.org/10.1016/j.jsb.2010.05.004	journal	October 2010
Expression screening of membrane proteins with cell-free protein synthesis Isaksson, Linnéa; Enberg, Johan; Neutze, Richard Protein Expression and Purification, Vol. 82, Issue 1 https://doi.org/10.1016/j.pep.2012.01.003	journal	March 2012
A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems Haberstock, Stefan; Roos, Christian; Hoevels, Yvette Protein Expression and Purification, Vol. 82, Issue 2 https://doi.org/10.1016/j.pep.2012.01.018	journal	April 2012
Structural genomics plucks high-hanging membrane proteins Kloppmann, Edda; Punta, Marco; Rost, Burkhard Current Opinion in Structural Biology, Vol. 22, Issue 3 https://doi.org/10.1016/j.sbi.2012.05.002	journal	June 2012
Protein production from the structural genomics perspective: achievements and future needs Almo, Steven C.; Garforth, Scott J.; Hillerich, Brandan S. Current Opinion in Structural Biology, Vol. 23, Issue 3 https://doi.org/10.1016/j.sbi.2013.02.014	journal	June 2013
Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures Sonoda, Yo; Newstead, Simon; Hu, Nien-Jen Structure, Vol. 19, Issue 1 https://doi.org/10.1016/j.str.2010.12.001	journal	January 2011
Rationalizing membrane protein overexpression Wagner, Samuel; Bader, Mirjam Lerch; Drew, David Trends in Biotechnology, Vol. 24, Issue 8 https://doi.org/10.1016/j.tibtech.2006.06.008	journal	August 2006
Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli Skretas, Georgios; Makino, Tomohiro; Varadarajan, Navin Metabolic Engineering, Vol. 14, Issue 5 https://doi.org/10.1016/j.ymben.2012.05.001	journal	September 2012
Preparative scale cell-free expression systems: New tools for the large scale preparation of integral membrane proteins for functional and structural studies Schwarz, Daniel; Klammt, Christian; Koglin, Alexander Methods, Vol. 41, Issue 4, p. 355-369 https://doi.org/10.1016/j.ymeth.2006.07.001	journal	April 2007
High-throughput protein purification and quality assessment for crystallization Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert Methods, Vol. 55, Issue 1 https://doi.org/10.1016/j.ymeth.2011.07.010	journal	September 2011
A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability Seneviratne, Rashmi; Khan, Sanobar; Moscrop, Ellen Methods, Vol. 147 https://doi.org/10.1016/j.ymeth.2018.01.021	journal	September 2018
Expression of recombinant proteins in insect and mammalian cells McKenzie, Edward A.; Abbott, W. Mark Methods, Vol. 147 https://doi.org/10.1016/j.ymeth.2018.05.013	journal	September 2018
The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology Garamella, Jonathan; Marshall, Ryan; Rustad, Mark ACS Synthetic Biology, Vol. 5, Issue 4 https://doi.org/10.1021/acssynbio.5b00296	journal	February 2016
Optimization of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR Michou, Myrsini; Kapsalis, Charalampos; Pliotas, Christos ACS Synthetic Biology, Vol. 8, Issue 7 https://doi.org/10.1021/acssynbio.9b00120	journal	June 2019
Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance Bazzacco, Paola; Billon-Denis, Emmanuelle; Sharma, K. Shivaji Biochemistry, Vol. 51, Issue 7 https://doi.org/10.1021/bi201862v	journal	February 2012
Drug—target network Yıldırım, Muhammed A.; Goh, Kwang-Il; Cusick, Michael E. Nature Biotechnology, Vol. 25, Issue 10 https://doi.org/10.1038/nbt1338	journal	October 2007
Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems Schwarz, Daniel; Junge, Friederike; Durst, Florian Nature Protocols, Vol. 2, Issue 11 https://doi.org/10.1038/nprot.2007.426	journal	November 2007
A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins Angius, Federica; Ilioaia, Oana; Amrani, Amira Scientific Reports, Vol. 8, Issue 1 https://doi.org/10.1038/s41598-018-26668-y	journal	June 2018
Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system Niwa, Tatsuya; Sasaki, Yoshihiro; Uemura, Eri Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep18025	journal	December 2015
Cell-free methods to produce structurally intact mammalian membrane proteins Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep30442	journal	July 2016
Mitochondrial pyruvate transport: a historical perspective and future research directions McCommis, Kyle S.; Finck, Brian N. Biochemical Journal, Vol. 466, Issue 3 https://doi.org/10.1042/BJ20141171	journal	March 2015
Durable vesicles for reconstitution of membrane proteins in biotechnology Beales, Paul A.; Khan, Sanobar; Muench, Stephen P. Biochemical Society Transactions, Vol. 45, Issue 1 https://doi.org/10.1042/BST20160019	journal	February 2017
Liposomes as models for membrane integrity Routledge, Sarah J.; Linney, John A.; Goddard, Alan D. Biochemical Society Transactions, Vol. 47, Issue 3 https://doi.org/10.1042/BST20190123	journal	May 2019
Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression Jacobs, Miranda L.; Boyd, Margrethe A.; Kamat, Neha P. Proceedings of the National Academy of Sciences, Vol. 116, Issue 10 https://doi.org/10.1073/pnas.1814775116	journal	February 2019
Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems Rath, Parthasarathi; Demange, Pascal; Saurel, Olivier Journal of Biological Chemistry, Vol. 286, Issue 37 https://doi.org/10.1074/jbc.M111.276956	journal	September 2011
A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity Gray, Lawrence R.; Rauckhorst, Adam J.; Taylor, Eric B. Journal of Biological Chemistry, Vol. 291, Issue 14 https://doi.org/10.1074/jbc.M115.711663	journal	April 2016
Consequences of Membrane Protein Overexpression in Escherichia coli Wagner, Samuel; Baars, Louise; Ytterberg, A. Jimmy Molecular & Cellular Proteomics, Vol. 6, Issue 9 https://doi.org/10.1074/mcp.M600431-MCP200	journal	April 2007
Molecular Interaction Studies Using Microscale Thermophoresis Jerabek-Willemsen, Moran; Wienken, Chistoph J.; Braun, Dieter ASSAY and Drug Development Technologies, Vol. 9, Issue 4 https://doi.org/10.1089/adt.2011.0380	journal	August 2011
Properties and identification of human protein drug targets Bakheet, Tala M.; Doig, Andrew J. Bioinformatics, Vol. 25, Issue 4 https://doi.org/10.1093/bioinformatics/btp002	journal	January 2009
Shaping Escherichia coli for recombinant membrane protein production Karyolaimos, Alexandros; Ampah-Korsah, Henry; Zhang, Zhe FEMS Microbiology Letters, Vol. 365, Issue 15 https://doi.org/10.1093/femsle/fny152	journal	June 2018
Mitochondrial Pyruvate Carriers Prevent Cadmium Toxicity by Sustaining the TCA Cycle and Glutathione Synthesis He, Lilong; Jing, Ying; Shen, Jianlin Plant Physiology, Vol. 180, Issue 1 https://doi.org/10.1104/pp.18.01610	journal	February 2019
The JCSG high-throughput structural biology pipeline Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 66, Issue 10 https://doi.org/10.1107/S1744309110038212	journal	September 2010
Cell-free complements in vivo expression of the E. coli membrane proteome Savage, David F.; Anderson, Corey L.; Robles-Colmenares, Yaneth Protein Science, Vol. 16, Issue 5 https://doi.org/10.1110/ps.062696307	journal	May 2007
Circularized and solubility‐enhanced MSP s facilitate simple and high‐yield production of stable nanodiscs for studies of membrane proteins in solution Johansen, Nicolai Tidemand; Tidemand, Frederik Grønbæk; Nguyen, Tam T. T. N. The FEBS Journal, Vol. 286, Issue 9 https://doi.org/10.1111/febs.14766	journal	February 2019
Evaluation of detergents for the soluble expression of α-helical and β-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system Klammt, Christian; Schwarz, Daniel; Fendler, Klaus FEBS Journal, Vol. 272, Issue 23, p. 6024-6038 https://doi.org/10.1111/j.1742-4658.2005.05002.x	journal	November 2005
A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans Bricker, Daniel K.; Taylor, Eric B.; Schell, John C. Science, Vol. 337, Issue 6090 https://doi.org/10.1126/science.1218099	journal	May 2012
Identification and Functional Expression of the Mitochondrial Pyruvate Carrier Herzig, Sébastien; Raemy, Etienne; Montessuit, Sylvie Science, Vol. 337, Issue 6090 https://doi.org/10.1126/science.1218530	journal	May 2012
Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli Skretas, G.; Georgiou, G. Applied and Environmental Microbiology, Vol. 76, Issue 17 https://doi.org/10.1128/AEM.00963-10	journal	July 2010
A systematic approach for testing expression of human full-length proteins in cell-free expression systems Langlais, Claudia; Guilleaume, Birgit; Wermke, Nadja BMC Biotechnology, Vol. 7, Issue 1 https://doi.org/10.1186/1472-6750-7-64	journal	October 2007
Mitochondrial pyruvate carrier 1 mediates abscisic acid-regulated stomatal closure and the drought response by affecting cellular pyruvate content in Arabidopsis thaliana Shen, Jian-Lin; Li, Chun-Long; Wang, Mei BMC Plant Biology, Vol. 17, Issue 1 https://doi.org/10.1186/s12870-017-1175-3	journal	November 2017
Acyl-Lipid Metabolism Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred The Arabidopsis Book, Vol. 11 https://doi.org/10.1199/tab.0161	journal	January 2013
Preparation of amino acid mixtures for cell-free expression systems Caschera, Filippo; Noireaux, Vincent BioTechniques, Vol. 58, Issue 1 https://doi.org/10.2144/000114249	journal	January 2015

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59 BASIC BIOLOGICAL SCIENCES
cell-free expression
eukaryotic
high
lipidic
membrane
proteins
throughput

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

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