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

Scientific Reports
Xinying ShiSimpson Joseph

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.

References

Apr 26, 1974·Biochimica Et Biophysica Acta·G L Jendrasiak, J H Hasty
Feb 5, 2000·Nature·M B Elowitz, S Leibler
Sep 29, 2000·Science·I LevchenkoT A Baker
Sep 10, 2003·Proceedings of the National Academy of Sciences of the United States of America·Sophie PautotD A Weitz
Jun 28, 2005·Journal of Microbiological Methods·Hiromichi ItohToshikazu Shiba
Oct 15, 2005·Physical Biology·Vincent NoireauxAlbert Libchaber
Jun 25, 2009·The Journal of Biological Chemistry·Joseph H DavisRobert T Sauer
Jun 26, 2010·Journal of Biological Engineering·Jonghyeon Shin, Vincent Noireaux
Jul 3, 2010·Journal of Biological Engineering·Jonghyeon Shin, Vincent Noireaux
Apr 8, 2011·Annual Review of Biochemistry·Robert T Sauer, Tania A Baker
Jul 9, 2011·Biochimica Et Biophysica Acta·Tania A Baker, Robert T Sauer
Nov 5, 2013·Methods in Enzymology·Vaishnavi Rajagopal, Jon R Lorsch
Apr 11, 2014·Nature·Arthur PrindleJeff Hasty
Jul 8, 2015·Journal of Molecular Biology·Mauricio H PontesEduardo A Groisman
Jan 29, 2016·ACS Synthetic Biology·Jonathan GaramellaVincent Noireaux
Feb 11, 2016·Proceedings of the National Academy of Sciences of the United States of America·Kim A Sharp
Feb 27, 2016·Chembiochem : a European Journal of Chemical Biology·Sampreeti ChowdhuriNeal K Devaraj
Oct 30, 2016·Trends in Biochemical Sciences·Germán Rivas, Allen P Minton

❮ Previous
Next ❯

Citations

Apr 23, 2019·Protein Science : a Publication of the Protein Society·Yiqing YangHeedeok Hong
Jul 23, 2020·Proceedings of the National Academy of Sciences of the United States of America·Ahanjit BhattacharyaNeal K Devaraj
Feb 18, 2020·Industrial & Engineering Chemistry Research·April M MiguezMark P Styczynski

❮ Previous
Next ❯

Methods Mentioned

BETA
fluorescence imaging
Fluorescence
Assay

Software Mentioned

ImageJ
Micromanager
ClpXP
Graphpad Prism

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.