Cell-free protein synthesis is a versatile protein production system.
Performance of the protein synthesis depends on highly active
cytoplasmic extracts. Extracts from E. coli are believed to work best;
they are routinely obtained from exponential growing cells, aiming to
capture the most active translation system. Here, we report an active
cell-free protein synthesis system derived from cells harvested at
non-growth, stressed conditions. We found a downshift of ribosomes and
proteins. However, a characterization revealed that the stoichiometry of
ribosomes and key translation factors was conserved, pointing to a fully
intact translation system. This was emphasized by synthesis rates, which
were comparable to those of systems obtained from fast-growing cells.
Our approach is less laborious than traditional extract preparation
methods and multiplies the yield of extract per cultivation. This
simplified growth protocol has the potential to attract new entrants to
cell-free protein synthesis and to broaden the pool of applications. In
this respect, a translation system originating from heat stressed,
non-growing E. coli enabled an extension of endogenous transcription
units. This was demonstrated by the sigma factor depending activation of
parallel transcription. Our cell-free expression platform adds to the
existing versatility of cell-free translation systems and presents a
tool for cell-free biology.
The work was funded by the Bundesministerium fur Bildung und Forschung
(grant number: 031A157D, BMBF, Berlin, Germany). The authors thank
Alexander NieB for helpful discussions regarding the manuscript.
%0 Journal Article
%1 ISI:000416398300004
%A Failmezger, Jurek
%A Rauter, Michael
%A Nitschel, Robert
%A Kraml, Michael
%A Siemann-Herzberg, Martin
%C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND
%D 2017
%I NATURE PUBLISHING GROUP
%J SCIENTIFIC REPORTS
%K myown proteinsynthesis
%R 10.1038/s41598-017-16767-7
%T Cell-free protein synthesis from non-growing, stressed Escherichia coli
%U https://doi.org/10.1038/s41598-017-16767-7
%V 7
%X Cell-free protein synthesis is a versatile protein production system.
Performance of the protein synthesis depends on highly active
cytoplasmic extracts. Extracts from E. coli are believed to work best;
they are routinely obtained from exponential growing cells, aiming to
capture the most active translation system. Here, we report an active
cell-free protein synthesis system derived from cells harvested at
non-growth, stressed conditions. We found a downshift of ribosomes and
proteins. However, a characterization revealed that the stoichiometry of
ribosomes and key translation factors was conserved, pointing to a fully
intact translation system. This was emphasized by synthesis rates, which
were comparable to those of systems obtained from fast-growing cells.
Our approach is less laborious than traditional extract preparation
methods and multiplies the yield of extract per cultivation. This
simplified growth protocol has the potential to attract new entrants to
cell-free protein synthesis and to broaden the pool of applications. In
this respect, a translation system originating from heat stressed,
non-growing E. coli enabled an extension of endogenous transcription
units. This was demonstrated by the sigma factor depending activation of
parallel transcription. Our cell-free expression platform adds to the
existing versatility of cell-free translation systems and presents a
tool for cell-free biology.
@article{ISI:000416398300004,
abstract = {{Cell-free protein synthesis is a versatile protein production system.
Performance of the protein synthesis depends on highly active
cytoplasmic extracts. Extracts from E. coli are believed to work best;
they are routinely obtained from exponential growing cells, aiming to
capture the most active translation system. Here, we report an active
cell-free protein synthesis system derived from cells harvested at
non-growth, stressed conditions. We found a downshift of ribosomes and
proteins. However, a characterization revealed that the stoichiometry of
ribosomes and key translation factors was conserved, pointing to a fully
intact translation system. This was emphasized by synthesis rates, which
were comparable to those of systems obtained from fast-growing cells.
Our approach is less laborious than traditional extract preparation
methods and multiplies the yield of extract per cultivation. This
simplified growth protocol has the potential to attract new entrants to
cell-free protein synthesis and to broaden the pool of applications. In
this respect, a translation system originating from heat stressed,
non-growing E. coli enabled an extension of endogenous transcription
units. This was demonstrated by the sigma factor depending activation of
parallel transcription. Our cell-free expression platform adds to the
existing versatility of cell-free translation systems and presents a
tool for cell-free biology.}},
added-at = {2018-01-25T13:38:08.000+0100},
address = {{MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}},
affiliation = {{Siemann-Herzberg, M (Reprint Author), Univ Stuttgart, Inst Biochem Engn, Stuttgart, Germany.
Failmezger, Jurek; Rauter, Michael; Nitschel, Robert; Kraml, Michael; Siemann-Herzberg, Martin, Univ Stuttgart, Inst Biochem Engn, Stuttgart, Germany.}},
article-number = {{16524}},
author = {Failmezger, Jurek and Rauter, Michael and Nitschel, Robert and Kraml, Michael and Siemann-Herzberg, Martin},
author-email = {{siemann@ibvt.uni-stuttgart.de}},
biburl = {https://puma.ub.uni-stuttgart.de/bibtex/256049d06cf91a57572d2af99069b00b4/siemannherzberg},
da = {{2018-01-25}},
doc-delivery-number = {{FN9YH}},
doi = {{10.1038/s41598-017-16767-7}},
funding-acknowledgement = {{Bundesministerium fur Bildung und Forschung {[}031A157D]}},
funding-text = {{The work was funded by the Bundesministerium fur Bildung und Forschung
(grant number: 031A157D, BMBF, Berlin, Germany). The authors thank
Alexander NieB for helpful discussions regarding the manuscript.}},
interhash = {f2fef7d3761cfe467db0f2759461d4d6},
intrahash = {56049d06cf91a57572d2af99069b00b4},
issn = {{2045-2322}},
journal = {{SCIENTIFIC REPORTS}},
journal-iso = {{Sci Rep}},
keywords = {myown proteinsynthesis},
keywords-plus = {{RIBOSOME MODULATION FACTOR; TRANSCRIPTION-TRANSLATION SYSTEM; FREE
EXPRESSION SYSTEM; STATIONARY-PHASE; EXTRACT PREPARATION;
RNA-POLYMERASE; FED-BATCH; GENE; EFFICIENT; BACTERIA}},
language = {{English}},
month = {{NOV 28}},
number-of-cited-references = {{59}},
oa = {{gold}},
publisher = {{NATURE PUBLISHING GROUP}},
research-areas = {{Science \& Technology - Other Topics}},
times-cited = {{0}},
timestamp = {2018-06-14T11:14:34.000+0200},
title = {{Cell-free protein synthesis from non-growing, stressed Escherichia coli}},
type = {{Article}},
unique-id = {{ISI:000416398300004}},
url = {https://doi.org/10.1038/s41598-017-16767-7},
usage-count-last-180-days = {{1}},
usage-count-since-2013 = {{1}},
volume = {{7}},
web-of-science-categories = {{Multidisciplinary Sciences}},
year = {{2017}}
}