Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system

Abstract Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will req...

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Main Authors:	Xinying Shi, Ti Wu, Christian M. Cole, Neal K. Devaraj, Simpson Joseph
Format:	Article
Language:	English
Published:	Nature Publishing Group 2018-02-01
Series:	Scientific Reports
Online Access:	https://doi.org/10.1038/s41598-018-21739-6

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spelling	doaj-7864bd636f484af58fd311bfaf3f0ba42020-12-08T05:52:13ZengNature Publishing GroupScientific Reports2045-23222018-02-018111110.1038/s41598-018-21739-6Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression systemXinying Shi0Ti Wu1Christian M. Cole2Neal K. Devaraj3Simpson Joseph4Department of Chemistry and Biochemistry, University of CaliforniaDepartment of Chemistry and Biochemistry, University of CaliforniaDepartment of Chemistry and Biochemistry, University of CaliforniaDepartment of Chemistry and Biochemistry, University of CaliforniaDepartment of Chemistry and Biochemistry, University of CaliforniaAbstract Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.https://doi.org/10.1038/s41598-018-21739-6
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Xinying Shi Ti Wu Christian M. Cole Neal K. Devaraj Simpson Joseph
spellingShingle	Xinying Shi Ti Wu Christian M. Cole Neal K. Devaraj Simpson Joseph Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system Scientific Reports
author_facet	Xinying Shi Ti Wu Christian M. Cole Neal K. Devaraj Simpson Joseph
author_sort	Xinying Shi
title	Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system
title_short	Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system
title_full	Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system
title_fullStr	Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system
title_full_unstemmed	Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system
title_sort	optimization of clpxp activity and protein synthesis in an e. coli extract-based cell-free expression system
publisher	Nature Publishing Group
series	Scientific Reports
issn	2045-2322
publishDate	2018-02-01
description	Abstract Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.
url	https://doi.org/10.1038/s41598-018-21739-6
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Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system

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