Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system
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A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink).
Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc) and of the enhanced green fluorescent protein (eGFP). Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured.
The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in new research areas such as in vitro synthetic biology and systems biology where engineering informational processes requires a quantitative control of mRNA inactivation and protein degradation.
KeywordsReporter Protein Inactivation Rate Maturation Time Informational Process Plasmid Concentration
enhanced green fluorescent protein
- (RNase A)
see ): deGFP.
Cell-free expression has become a serious alternative to cell-based expression. In response to an increasing number of applications that require fast production of a large amount of proteins , new preparation methods and new reaction components are frequently proposed to improve protein productivity of cell-free systems and to reduce the cost of reaction [2, 3]. These systems use bacteriophage transcriptions, such as T7, and extracts with low degradation of both mRNAs and proteins to produce on the order of 1 mg/ml of proteins in batch mode after a few hours of incubation. As they become more powerful, cell-free systems are used in new applications. In vitro synthetic biology is one of the new research areas where transcription-translation extracts can be employed to engineer processes based on biological information. Cell-free elementary gene circuits [4, 5], pattern formation  and synthetic vesicles [7, 8] have been engineered with cell-free systems. However, the properties of conventional cell-free expression systems are not well adapted for this type of study that requires more than a fast and a powerful expression of proteins. In particular, the control of mRNA inactivation and protein degradation rates are essential components for the construction of interesting informational processes in vitro. The production of cycles in time or patterns in space requires a precise balance between a source and a sink [5, 9]. It is a general and an essential property of dynamical systems. Whereas most of the efforts to optimize cell-free systems have consisted in increasing protein productivity, no procedure has been proposed to change the inactivation rate of mRNAs and the degradation rate of proteins in cell-free expression systems. It is one of the main bottlenecks for the development and the study of quantitative informational processes in vitro.
In this work, an approach is presented to accelerate the global mRNA turnover of the synthesized mRNAs in a cell-free reaction and to control the degradation of the synthesized proteins. The E. coli mRNA interferase MazF was used to adjust the inactivation rate of synthesized mRNAs and the endogenous E. coli AAA + proteases were used to control the degradation of synthesized proteins in a cell-free expression system driven by the endogenous E. coli RNAP and sigma factor 70 . The experiments were carried out with eGFP and deGFP, a highly translatable version of eGFP with the same fluorescence properties . The Firefly luciferase was used as a second reporter protein for control experiments.
MazF-MazE is a toxin-antitoxin pair found in E. coli. The antitoxin MazE inhibits the activity of the toxin MazF. MazF is a small ribonuclease that inactivates mRNAs by cleaving at the ribonucleotide ACA single strand sequence . The toxin is expressed in E. coli under special conditions, such as amino acid starvation, to block protein synthesis by inactivating all the mRNAs. Ribosomes and tRNAs are not inactivated by MazF. For these reasons, we found that MazF was a convenient tool to accelerate the global mRNA turnover in a cell-free reaction without inactivating other types of RNA. The mean lifetime of deGFP mRNA, modeled by an exponential decay, could be easily tuned from 13 minutes (the mean lifetime without addition of toxin), to 0 minutes (complete inactivation). Practically, the mRNA mean lifetime was adjusted by adding a small fraction of an extract containing MazF to a cell-free reaction.
A complete control of gene expression dynamics in vitro also requires adjusting the degradation rate of the synthesized proteins while not affecting transcription and translation machineries. This aspect is of particular importance to prevent synthesized proteins from accumulating in batch mode reaction. The endogenous AAA + proteases, such as the ClpXP and ClpAP complexes, present in the E. coli extract provide an adequate solution. Synthesized proteins have to be tagged either in N-terminal or C-terminal with a short amino acid sequence to be specifically degraded by the ClpXP or ClpAP complexes . In this study, seven tags were tested on deGFP. With the 11-residue SsrA tag , the deGFP degradation rate in cell-free reaction was constant up to a concentration of one micromolar. We show that cell-free production of deGFP could be predicted when MazF and the SsrA tag were used concurrently.
The cell-free system used in this study has been developed by Shin and Noireaux . Briefly, the crude extract was prepared from E. coli BL21 Rosetta2 cells according to Kigawa et al  and Liu et al  with slight modifications. S30 buffer A (50 mM Tris, 60 mM potassium glutamate and 14 mM magnesium glutamate, pH 7.7, 2 mM DTT) was used for washing and resuspension. The crude extract was dialyzed against S30 buffer B (5 mM Tris, 60 mM potassium glutamate and 14 mM magnesium glutamate, pH 8.2, 1 mM DTT). The cells were broken with a bead beater (mini bead-beater-1, Biospecs Products Inc, Bartlesville, OK). The crude extract was stored at -80 C after dialysis. The endogenous E. coli RNA polymerase was used for expression. Preparation of the MazF crude extract was identical to the preparation of the crude extract for cell-free reaction except for the expression of MazF prior to preparation. The MazF gene was obtained by PCR using E. coli as a template and cloned under the arabinose promoter in the plasmid pBAD/His A (Invitrogen). At OD600 = 1.2, cells bearing the MazF plasmid were induced with 0.25% arabinose (final concentration) for one hour before preparing the extract. A typical concentration of 27-30 mg/ml and 22 mg/ml of proteins in the crude extract, with and without MazF respectively, was measured by Bradford assay. Both crude extracts are stable at least 1 year when stored at -80°C.
The standard cell-free reactions were composed of 33% crude extract (between 9 and 9.5 mg/ml of proteins), the other 66% containing the reaction buffer and plasmid with the following final concentrations: 50 mM Hepes pH 8, 1.5 mM ATP and GTP each, 0.9 mM CTP and UTP each, 1 mM spermidine, 0.75 mM cAMP, 0.33 mM NAD, 0.26 mM coenzymeA, 30 mM 3-phosphoglyceric acid, 0.068 mM folinic acid, 0.2 mg/ml tRNA, 1 mM IPTG, 1.5 mM amino acids. The concentrations of PEG 8000, magnesium glutamate and potassium glutamate were adjusted depending on the reporter used . The cell-free reactions with the MazF crude extract were composed of 43% MazF crude extract (between 9 and 9.5 mg/ml of proteins), the other 57% containing the reaction buffer and plasmid with the same final concentrations as the standard cell-free reactions. The plasmid concentration was adjusted depending on the experiment (a final concentration comprised between 0.1 nM and 5 nM was used in this study). The reactions were incubated at 22°C for Luc and 29°C for eGFP. The reagents used for cell-free reactions were purchased from Sigma, USB Corporation (GTP, CTP, UTP) and Roche (tRNA, amino acids). Other reagents used in this study: Ribonuclease A (Sigma), Tagetin (Epicentre Biotechnologies), MazF (Takara Bio Inc).
Protein expression and purification
The plasmid pET21a(+) (Novagen) was used for recombinant protein expression. The proteins His-MazE (6Histag in N-terminal), His-eGFP-SsrA (6Histag in N-terminal and SsrA tag in C-terminal) and His-eGFP-SsrA-DD (6Histag in N-terminal and SsrA-DD tag in C-terminal) were over-expressed in E. coli BL21 (DE3) and purified by affinity chromatography on agarose nickel beads according to the manufacturer protocol (Invitrogen). The proteins were desalted against a storage buffer (50 mM Tris HCl pH 7.5, 5% glycerol) and stored at -80°C. The concentration of the purified proteins was measured by Bradford assay. Pure recombinant eGFP (Clontech) was used to determine the concentration of His-eGFP-SsrA and His-eGFP-SsrA-DD.
All the plasmids used in this study originate from the plasmid pBEST-Luc (Promega). The list and sequences of the different regulatory parts are reported in the additional file 1. Luc refers to Firefly luciferase [GenBank: CAA59281.1], eGFP to the enhanced green fluorescent protein [GenBank: CAD97424.1], deGFP to eGFP-Del6-229 (enhanced green fluorescent protein truncated and modified in N- and C-terminal, ), UTR1 to the untranslated region containing the T7 g10 leader sequence for highly efficient translation initiation  [GenBank: M35614.1], T500 to the transcription terminator , OR2-OR1-Pr to the lambda repressor Cro promoter [GenBank: J02459.1], SsrA, SsrA-D, SsrA-DD, Crl, YbaQ, YdaM and OmpA to the tags specific to the ClpXP and ClpAP complexes . The plasmids were prepared using the standard molecular cloning procedures. Picogreen (Invitrogen) was used to measure plasmid concentration.
The fluorescence measurements (kinetics and end-point) were either performed with a Wallac Victor III plate reader (PerkinElmer, 384-well plate) or with an Olympus IX-71 inverted microscope equipped with a photo multiplier tube (Hamamatsu, H7421-40). Pure recombinant eGFP (Clontech) was used for calibration and quantification. Luc expression was measured with a custom-built luminometer . Pure Luc and Luc assay kit (Promega) were used for calibration and measurements.
Results and discussion
Luc and eGFP maturation time
where deGFPf is the fluorescent deGFP, deGFPd is the dark deGFP (the primary amino acid chain is synthesized but the reporter protein is not fluorescent), [deGFP0] = [deGFPf] + [deGFPd] = constant and 1/κ is the maturation time that includes folding of the protein and formation of the fluorophore by oxidation . Equation (1.2) is the solution to equation (1.1). Fitting of the data gave a reproducible maturation time of 1/κ = 8-8.5 minutes for deGFP in our cell-free extract. This maturation time was taken into account in all of the subsequent calculations. The same maturation time was obtained when the experiment was carried out after 1 hour of incubation at 29°C. This result was comparable to the maturation time of eGFP measured in vivo .
Endogenous messenger RNA inactivation
where deGFPf is the fluorescent deGFP, deGFPd is the dark deGFP, m is the concentration of deGFP mRNA, m0 is the concentration of active mRNAs when transcription is stopped, 1/κ is the maturation time that includes folding of the protein and formation of the fluorophore by oxidation, β is the mRNA inactivation rate and α is the protein production rate. This model describes mRNA inactivation as an exponential decay . The reference time point t = 0 is set to transcription arrest (addition of Tagetin). The initial concentration of deGFPf is set to zero. The initial concentration of deGFPd is set to 0.25 μM (this concentration is given by Figure 1B). Equation (1.6) is the solution to the equations (1.3), (1.4) and (1.5). The data were fitted to equation (1.6). No estimations of α and m0 have been determined in this work. The determination of β was independent of the product αm0. No interesting information could be extracted from the numerical constant αm0 since it depends on the time at which the assay is performed (due to m0). With a maturation time of 8 minutes for deGFP, we found an endogenous deGFP mRNA inactivation rate β of 0.077 min-1, which corresponds to an average lifetime of 13 minutes. This result is comparable to some estimation of mRNA lifetime in a commercial cell-free system .
Messenger RNA inactivation with MazF
Concurrent messenger RNA inactivation and protein degradation
In this work, the inactivation rate of mRNA and the degradation rate of proteins have been studied in a transcription-translation cell-free reaction. Methods to increase the inactivation rate of synthesized mRNAs and to induce the degradation rate of synthesized proteins have been described. These methods are quantitative, cost-effective and simple to use. Inactivation and degradation were characterized with a cell-free expression system driven by the endogenous E. coli RNA polymerase, which presents some advantages for the construction of synthetic circuitry in vitro. Structure of E. coli promoters provides much more modularity to engineer informational processes, such as gene circuits, compared to bacteriophage promoters used in conventional cell-free systems. Construction of any interesting synthetic circuitry, such as cycles in time or patterns in space, requires a fine balance between the source and the sink [5, 9]. This work is a first step to provide the necessary inactivation and degradation tools to engineer complex informational processes in vitro involving transcription and translation reactions. For instance, it would be interesting to test the production of oscillations in vitro with this system. On a broader perspective, the introduction of quantitative sinks contributes to the development of cell-free toolboxes to synthesize, to run and to study informational processes outside living organisms.
The authors thank Nadezda Monina for technical assistance in the extract preparation and Catherine Raach for reading and correcting the manuscript. This work was supported by UMN startup funds, BSF Binational Science Foundation grant 2006398, National Science Foundation grant PHY-0750133.
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