Niess,A., Siemann-Herzberg,M. and Takors,R. (2019) Protein Production in Escherichia Coli Is Guided by the Trade-off between Intracellular Substrate Availability and Energy Cost. Microbial Cell Factory, 18.
Failmezger,J., Scholz,S., Blombach,B. and Siemann-Herzberg,M. (2018) Cell-Free Protein Synthesis From Fast-Growing Vibrio Natriegens. Frontiers in Microbiology, 9.
Failmezger,J., Ludwig,J., Niess,A. and Siemann-Herzberg,M. Quantifying Ribosome Dynamics in Escherichia Coli Using Fluorescence. FEMS MICROBIOLOGY LETTERS, 364.
AbstractRibosomes are a crucial component of the physiological state of a cell.
Therefore, we aimed to monitor ribosome dynamics using a fast and easy
fluorescence readout. Using fluorescent-labeled ribosomal proteins, the
dynamics of ribosomes during batch cultivation and during nutritional
shift conditions was investigated. The fluorescence readout was compared
to the cellular rRNA content determined by capillary gel electrophoresis
with laser-induced fluorescence detection during exponentially
accelerating and decelerating growth. We found a linear correlation
between the observed fluorescence and the extracted rRNA content
throughout cultivation, demonstrating the applicability of this method.
Moreover, the results show that ribosome dynamics, as a result of
slowing growth, are accompanied by the passive effect of dilution of
preexisting ribosomes, de novo ribosome synthesis and ribosome
degradation. In light of the challenging task of deciphering ribosome
regulatory mechanisms, our approach of using fluorescence to follow
ribosome dynamics will allow more comprehensive studies of biological
Niess,A., Failmezger,J., Kuschel,M., Siemann-Herzberg,M. and Takors,R. Experimentally Validated Model Enables Debottlenecking of in Vitro Protein Synthesis and Identifies a Control Shift under in Vivo Conditions. ACS SYNTHETIC BIOLOGY, 6, 1913–1921.
AbstractCell-free (in vitro) protein synthesis (CFPS) systems provide a
versatile tool that can be used to investigate different aspects of the
transcription-translation machinery by reducing cells to the basic
functions of protein formation. Recent improvements in reaction
stability and lysate preparation offer the potential to expand the scope
of in vitro biosynthesis from a research tool to a multifunctional and
versatile platform for protein production and synthetic biology. To
date, even the best-performing CFPS systems are drastically slower than
in vivo references. Major limitations are imposed by ribosomal
activities that progress in an order of magnitude slower on the mRNA
template. Owing to the complex nature of the ribosomal machinery,
conventional ``trial and error'' experiments only provide little
insight into how the desired performance could be improved. By applying
a DNA-sequence-oriented mechanistic model, we analyzed the major
differences between cell-free in vitro and in vivo protein synthesis. We
successfully identified major limiting elements of in vitro translation,
namely the supply of ternary complexes consisting of EFTu and tRNA.
Additionally, we showed that diluted in vitro systems suffer from
reduced ribosome numbers. On the basis of our model, we propose a new
experimental design predicting 90\% increased translation rates, which
were well achieved in experiments. Furthermore, we identified a shifting
control in the translation rate, which is characterized by availability
of the ternary complex under in vitro conditions and the initiation of
translation in a living cell. Accordingly, the model can successfully be
applied to sensitivity analyses and experimental design.
Failmezger,J., Rauter,M., Nitschel,R., Kraml,M. and Siemann-Herzberg,M. Cell-Free Protein Synthesis from Non-Growing, Stressed Escherichia Coli. SCIENTIFIC REPORTS, 7.
AbstractCell-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.
Failmezger,J., Nitschel,R., Sanchez-Kopper,A., Kraml,M. and Siemann-Herzberg,M. Site-Specific Cleavage of Ribosomal RNA in Escherichia Coli-Based Cell-Free Protein Synthesis Systems. PLoS One, 11.
AbstractCell-free protein synthesis, which mimics the biological protein
production system, allows rapid expression of proteins without the need
to maintain a viable cell. Nevertheless, cell free protein expression
relies on active in vivo translation machinery including ribosomes and
translation factors. Here, we examined the integrity of the protein
synthesis machinery, namely the functionality of ribosomes, during (i)
the cell-free extract preparation and (ii) the performance of in vitro
protein synthesis by analyzing crucial components involved in
translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and
ribosomal protein Si, we show that processing of a cell-free extract
results in no substantial alteration of the translation machinery.
Moreover, we reveal that the 16S rRNA is specifically cleaved at helix
44 during in vitro translation reactions, resulting in the removal of
the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate
in the cell-free system. We demonstrate that the specific cleavage of
the 16S rRNA is triggered by the decreased concentrations of Mg2+. In
addition, we provide evidence that helix 44 of the 30S ribosomal subunit
serves as a point-of-entry for ribosome degradation in Escherichia coli.
Our results suggest that Mg2+ homeostasis is fundamental to preserving
functional ribosomes in cell-free protein synthesis systems, which is of
major importance for cell-free protein synthesis at preparative scale,
in order to create highly efficient technical in vitro systems.
Oelschlaeger,P., Lange,S., Schmitt,J., Siemann,M., Reuss,M. and Schmid,R. Identification of Factors Impeding the Production of a Single-Chain Antibody Fragment in Escherichia Coli by Comparing in Vivo and in Vitro Expression. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 61, 123–132.
AbstractThe atrazine-specific single-chain variable antibody fragments (scFv)
K411B was produced by expression in either the cytoplasm or the
periplasm of Escherichia coli BL21 (DE3). For periplasmic production,
the pelB leader was N-terminally fused to scFv, whereas the unfused
variant resulted in cytoplasmic expression. The extent of protein
accumulation differed significantly. Expression of scFv with leader was
2.3 times higher than that of the protein without leader. This was
further investigated by generating the respective translation profiles
using coupled in vitro transcription/translation assays, the results of
which were in agreement. This comparative approach was also applied to
functionality: Periplasmic expression and in vitro expression resulted
in only 10\% correctly folded scFv, indicating that the oxidizing
environment of the periplasm did not increase proper folding. Thus, the
data obtained in vitro confirmed the findings observed in vivo and
suggested that the discrepancy in expression levels was due to different
translation efficiencies. However, the in vivo production of scFv with
enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP)
was only successful in the cytoplasm, although in vitro the expression
with and without the leader rendered the same production profile as for
scFv. This indicated that neither the translation efficiency nor the
solubility but other factors impeded periplasmic expression of the
Arnold,S., Siemann,M., Scharnweber,K., Werner,M., Baumann,S. and Reuss,M. Kinetic Modeling and Simulation of in Vitro Transcription by Phage T7 RNA Polymerase. BIOTECHNOLOGY AND BIOENGINEERING, 72, 548–561.
AbstractThis study provides a mathematical model of T7 RNA polymerase (T7 RNAP)
kinetics under in vitro conditions targeted at application of this model
to simulation of dynamic transcription performance. A functional
dependence of transcript synthesis rate is derived based on: (a)
essential reactant concentrations, including T7 RNAP and its promoter,
substrate nucleotides, and the inhibitory byproduct inorganic
pyrophosphate; (b) a distinction among vector characteristics such as
recognition sequences regulating transcription initiation and
termination, respectively; and (c) specific properties of the nucleotide
sequence including both transcript length and nucleotide composition.
Inactivation kinetics showed a half-life of T7 RNAP activity of 50 min
under the conditions applied in vitro using the isolated enzyme. Model
parameters and their precision are estimated using dynamic simulation
and nonlinear regression analysis. The particular novelty of this model
is its capability to incorporate linear genomic sequence information for
simulation of nonlinear in vitro transcription kinetics. (C) 2001 John
Wiley & Sons, Inc.
Schindler,P., Baumann,S., Reuss,M. and Siemann,M. In Vitro Coupled Transcription Translation: Effects of Modification in Lysate Preparation on Protein Composition and Biosynthesis Activity. ELECTROPHORESIS, 21, 2606–2609.
AbstractCell-free extracts (lysates) from Escherichia coli were used for protein
synthesis in vitro. Essential steps of the lysate preparation were
modified and analyzed with respect to their impact on in vitro protein
synthesis capacity, using the green fluorescent protein (GFP) as a
target protein. Variably manufactured lysates of low, medium and higher
protein synthesis activity, were examined by high resolution
two-dimensional get electrophoresis to determine whether the
modifications result in substantial alterations in protein composition
of the final lysate. The total number of proteins calculated from the
get maps did not vary for lysates with different activity and thus
cannot serve as an evaluation parameter. Ribosomal proteins RP-S1,
RP-L9, and RP-L10 were found in stoichiometric amounts for each of these
lysates and in equal concentrations in comparison among the different
lysates. Conversely, depending on the activity profiles, up to 7
different isoforms of the elongation factor EF-Ts were detected in the
Schindler,P., Macherhammer,F., Arnold,S., Reuss,M. and Siemann,M. Investigation of Translation Dynamics under Cell-Free Protein Biosynthesis Conditions Using High-Resolution Two-Dimensional Gel Electrophoresis. ELECTROPHORESIS, 20, 806–812.
AbstractA cell-free extract from Escherichia coli, generated through a routine
procedure according to Chen and Zubay (Methods Enzymol. 1983, 101,
674-690), was used for an in vitro protein synthesis. High-resolution
two-dimensional gel electrophoresis (2-DE) was exploited to investigate
the protein composition of the cell-extract and its dynamic development
during a 24 h-period of cell-free protein synthesis performed in a
membrane reactor device. Green fluorescent protein (GFP) was chosen as a
target protein to be produced in a cell-free reactor because of its
functional activity, which can easily be monitored by measurement of
fluorescence, and because of its high sensitivity. GFP synthesis was
observed by a standard fluorescence assay and was correlated to a
quantitative assessment of the silver-stained GFP spot appearing on 2-DE
gel maps. A constant protein synthesis rate was obtained for at least 8
h of process operation. While declining continuously, protein synthesis
stopped entirely after 24 h. Both, the total protein content and total
number of detectable spots were found to decrease over the reaction
time, due to proteolytic digestion and protein precipitation. Certain
proteins taking part in the translation process, such as the elongation
factors (EF-Tu, EF-Ts) and the ribosomal protein RP-L9, were identified
by Edman N-terminal sequencing and have thus been considered for
reaction evaluation. The dynamics obtained during the entire process
suggest that these translational factors were likewise affected by
Katanaev,V., Spirin,A., Reuss,M. and Siemann,M. Formation of Bacteriophage MS2 Infectious Units in a Cell-Free Translation System. FEBS LETTERS, 397, 143–148.
AbstractWe show that a simple cell-free translation system from Escherichia
coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells,
The plaques appearing on the E. coli host strain are morphologically
indistinguishable from those derived from normal phage MS2 infection,
This effect is strictly translation-dependent, since an incomplete
translation system or the system inhibited by antibiotics leads to no
infection, The cell-free based infection is maximal under conditions
favouring the highest synthesis of maturation protein (one of the four
phage-encoded proteins), The infection is abolished when RNase A or
trypsin treatment is included before addition of cells, Similarly, due
to RNA and maturation protein degradation, the continued incubation of
the translation mixture under protein synthesis conditions significantly
decreases infectivity, These findings suggest the formation of `minimal
infectious units', simple complexes of MS2 RNA and maturation protein,
Here we describe the first example of bacteriophage infectious unit
formation directly performed in a cell-free translation system, A
possible application of this phenomenon might be the construction of
newly designed RNA vector delivery systems and, moreover, could be an
approach for molecular evolution studies.