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Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells

Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, ha...

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Autores principales: Umbach, Simon, Levin, Roman, Neumann, Sebastian, Steinmetzer, Torsten, Dötsch, Volker, Bernhard, Frank
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9355040/
https://www.ncbi.nlm.nih.gov/pubmed/35935506
http://dx.doi.org/10.3389/fbioe.2022.906295
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author Umbach, Simon
Levin, Roman
Neumann, Sebastian
Steinmetzer, Torsten
Dötsch, Volker
Bernhard, Frank
author_facet Umbach, Simon
Levin, Roman
Neumann, Sebastian
Steinmetzer, Torsten
Dötsch, Volker
Bernhard, Frank
author_sort Umbach, Simon
collection PubMed
description Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes.
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spelling pubmed-93550402022-08-06 Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells Umbach, Simon Levin, Roman Neumann, Sebastian Steinmetzer, Torsten Dötsch, Volker Bernhard, Frank Front Bioeng Biotechnol Bioengineering and Biotechnology Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes. Frontiers Media S.A. 2022-07-22 /pmc/articles/PMC9355040/ /pubmed/35935506 http://dx.doi.org/10.3389/fbioe.2022.906295 Text en Copyright © 2022 Umbach, Levin, Neumann, Steinmetzer, Dötsch and Bernhard. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Umbach, Simon
Levin, Roman
Neumann, Sebastian
Steinmetzer, Torsten
Dötsch, Volker
Bernhard, Frank
Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
title Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
title_full Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
title_fullStr Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
title_full_unstemmed Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
title_short Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
title_sort transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9355040/
https://www.ncbi.nlm.nih.gov/pubmed/35935506
http://dx.doi.org/10.3389/fbioe.2022.906295
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