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Protein degradation in a TX-TL cell-free expression system using ClpXP protease

Sun, Zachary Z. and Kim, Jongmin and Singhal, Vipul and Murray, Richard M. (2015) Protein degradation in a TX-TL cell-free expression system using ClpXP protease. . (Unpublished)

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An in vitro S30-based Escherichia coli expression system (“Transcription-Translation”, or “TX-TL”) has been developed as an alternative prototyping environment to the cell for synthetic circuits [1-5]. Basic circuit elements, such as switches and cascades, have been shown to function in TX-TL, as well as bacteriophage assembly [2, 6]. Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and transformation steps [7]. However, most published results have been obtained in a “batch mode” reaction, where factors that play an important role for in vivo circuit dynamics – namely protein degradation and protein dilution – are severely hindered or are not present. This limits the complexity of circuits built in TX-TL without steady-state or continuous-flow solutions [8-10]. However, alternate methods that enable dilution either require extra equipment and expertise or demand lower reaction throughput. We explored the possibility of supplementing TX-TL with ClpXP, an AAA+ protease pair that selectively degrades tagged proteins [11], to provide finely-tuned degradation. The mechanism of ClpXP degradation has been extensively studied both in vitro and in vivo [12-15]. 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 [16], and ClpXP is known to require significant amounts of ATP to unfold different protein targets [17, 18]. We find that ClpXP’s 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.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
Sun, Zachary Z.0000-0002-9425-2924
Kim, Jongmin0000-0002-2713-1006
Singhal, Vipul0000-0003-1670-1824
Murray, Richard M.0000-0002-5785-7481
Additional Information:The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Preprint first posted online May 22, 2015. Date: 14 July 2014. 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. We thank Jan Kostecki for protein purification and size exclusion chromatography assistance, Rohit Sharma for initial testing of AAA+ degradation mechanisms, Robert Sauer and Karl Schmitz for advice in purifying ClpX, and Kyle Martin for laboratory assistance. This material is based upon work supported in part by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C-0065 (DARPA/CMO). Z.Z.S. is also supported by a UCLA/Caltech Medical Scientist Training Program fellowship, and a National Defense Science and Engineering Graduate fellowship. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-12-C-0065
UCLA/Caltech Medical Scientist Training ProgramUNSPECIFIED
National Defense Science and Engineering Graduate (NDSEG) FellowshipUNSPECIFIED
Record Number:CaltechAUTHORS:20150608-094354039
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Official Citation:Protein degradation in a TX-TL cell-free expression system using ClpXP protease Zachary Sun, Jongmin Kim, Vipul Singhal, Richard M Murray doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:58075
Deposited On:08 Jun 2015 18:15
Last Modified:10 Nov 2021 21:58

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