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Protein degradation in a TX-TL cell-free expression system using ClpXP protease |
Abstract |
We explored the possibility of supplementi … We explored the possibility of supplementing an in vitro S30-based Escherichia coli expression system (or âTX-TLâ) with ClpXP, an AAA+ protease pair that selectively degrades tagged proteins, to provide finely-tuned degradation. The mechanism of ClpXP degradation has been extensively studied both in vitro and in vivo. However, it has not been characterized for use in synthetic circuits -- metrics such as toxicity, ATP usage, degradation variation over time, and cellular loading need to be determined. In particular, TX-TL in batch mode is known to be resource limited, and ClpXP is known to require significant amounts of ATP to unfold different protein targets. We find that ClpXPâs protein degradation dynamics is dependent on protein identity, but can be determined experimentally. Degradation follows Michaels- Menten kinetics, and can be fine tuned by ClpX or ClpP concentration. Added purified ClpX is also not toxic to TX-TL reactions. Therefore, ClpXP provides a controllable way to introduce protein degradation and dynamics into synthetic circuits in TX-TL.
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NOTE: This is a technical report for future inclusion in work pending submission, review, and publication. Therefore, this work has not been peer-reviewed and is presented as-is. been peer-reviewed and is presented as-is. +
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Authors | Zachary Z. Sun, Jongmin Kim, Vipul Singhal, Richard M. Murray + |
Funding | Biomolecular Breadboards for Prototyping and Debugging Synthetic Biocircuits + |
ID | 2014g + |
Source | Technical Report, 7 July 2014 + |
Tag | sksm14-clpxp + |
Title | Protein degradation in a TX-TL cell-free expression system using ClpXP protease + |
Type | Technical Report + |
Categories | Papers |
Modification date This property is a special property in this wiki.
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15 May 2016 06:14:45 + |
URL This property is a special property in this wiki.
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http://www.cds.caltech.edu/~murray/preprints/sksm14-clpxp.pdf + |
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Protein degradation in a TX-TL cell-free expression system using ClpXP protease + | Title |
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