Cell-free protein expression under macromolecular crowding conditions.

<h4>Background</h4>Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the effic...

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Autores principales:	Xumeng Ge, Dan Luo, Jianfeng Xu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/eb3a7f8b31914a8aa9dfc8751490d9ad
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spelling	oai:doaj.org-article:eb3a7f8b31914a8aa9dfc8751490d9ad2021-11-18T07:32:43ZCell-free protein expression under macromolecular crowding conditions.1932-620310.1371/journal.pone.0028707https://doaj.org/article/eb3a7f8b31914a8aa9dfc8751490d9ad2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22174874/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.<h4>Methodology/principal findings</h4>Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.<h4>Conclusions/significance</h4>Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.Xumeng GeDan LuoJianfeng XuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 12, p e28707 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xumeng Ge Dan Luo Jianfeng Xu Cell-free protein expression under macromolecular crowding conditions.
description	<h4>Background</h4>Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.<h4>Methodology/principal findings</h4>Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.<h4>Conclusions/significance</h4>Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.
format	article
author	Xumeng Ge Dan Luo Jianfeng Xu
author_facet	Xumeng Ge Dan Luo Jianfeng Xu
author_sort	Xumeng Ge
title	Cell-free protein expression under macromolecular crowding conditions.
title_short	Cell-free protein expression under macromolecular crowding conditions.
title_full	Cell-free protein expression under macromolecular crowding conditions.
title_fullStr	Cell-free protein expression under macromolecular crowding conditions.
title_full_unstemmed	Cell-free protein expression under macromolecular crowding conditions.
title_sort	cell-free protein expression under macromolecular crowding conditions.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/eb3a7f8b31914a8aa9dfc8751490d9ad
work_keys_str_mv	AT xumengge cellfreeproteinexpressionundermacromolecularcrowdingconditions AT danluo cellfreeproteinexpressionundermacromolecularcrowdingconditions AT jianfengxu cellfreeproteinexpressionundermacromolecularcrowdingconditions
_version_	1718423321490489344

Cell-free protein expression under macromolecular crowding conditions.

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