Condensation of an additive-free cell extract to mimic the conditions of live cells.

The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified t...

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Autores principales:	Kei Fujiwara, Shin-ichiro M Nomura
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b562b93482b448648e8e49b9b80c6a9f
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spelling	oai:doaj.org-article:b562b93482b448648e8e49b9b80c6a9f2021-11-18T08:01:57ZCondensation of an additive-free cell extract to mimic the conditions of live cells.1932-620310.1371/journal.pone.0054155https://doaj.org/article/b562b93482b448648e8e49b9b80c6a9f2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23326590/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified the requirement for specific salts and buffers for functional cell-free translation extracts. The S30 fraction of Escherichia coli cell extracts without additives exhibited sufficient cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared cell extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of cell-free protein expression activity. Such a condensed cell extract may be useful for investigating the differences between cells and reconstituted materials and may contribute to the development of methods to synthesize cells from cell extracts in the future.Kei FujiwaraShin-ichiro M NomuraPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 1, p e54155 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kei Fujiwara Shin-ichiro M Nomura Condensation of an additive-free cell extract to mimic the conditions of live cells.
description	The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified the requirement for specific salts and buffers for functional cell-free translation extracts. The S30 fraction of Escherichia coli cell extracts without additives exhibited sufficient cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared cell extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of cell-free protein expression activity. Such a condensed cell extract may be useful for investigating the differences between cells and reconstituted materials and may contribute to the development of methods to synthesize cells from cell extracts in the future.
format	article
author	Kei Fujiwara Shin-ichiro M Nomura
author_facet	Kei Fujiwara Shin-ichiro M Nomura
author_sort	Kei Fujiwara
title	Condensation of an additive-free cell extract to mimic the conditions of live cells.
title_short	Condensation of an additive-free cell extract to mimic the conditions of live cells.
title_full	Condensation of an additive-free cell extract to mimic the conditions of live cells.
title_fullStr	Condensation of an additive-free cell extract to mimic the conditions of live cells.
title_full_unstemmed	Condensation of an additive-free cell extract to mimic the conditions of live cells.
title_sort	condensation of an additive-free cell extract to mimic the conditions of live cells.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/b562b93482b448648e8e49b9b80c6a9f
work_keys_str_mv	AT keifujiwara condensationofanadditivefreecellextracttomimictheconditionsoflivecells AT shinichiromnomura condensationofanadditivefreecellextracttomimictheconditionsoflivecells
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Condensation of an additive-free cell extract to mimic the conditions of live cells.

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