Limiting factors of the translation machinery

Freischmidt, Axel; Meysing, Maren; Liss, Michael A.; Wagner, Ralf; Kalbitzer, Hans Robert; Horn, Gudrun

doi:10.1016/j.jbiotec.2010.07.017

Cited by 7 publications

(7 citation statements)

References 28 publications

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“…Given that the large number of proteins involved in translation would make a quantitative assessment impractical, we chose to focus on proteins that have previously been shown to be critical for in vitro translation systems, specifically EF-Tu, EF-Ts, EF-G and RPS1 [25,26]. We identified two signature peptides representing each elongation factor and RPS1 by MS (Table 1), which were monitored by adding stable isotopic labeled proteins for their relative quantification.…”

Section: Resultsmentioning

confidence: 99%

Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

et al. 2016

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Cell-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 S1, 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.

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Section: Resultsmentioning

confidence: 99%

Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

et al. 2016

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“…We speculate that the addition of a large quantity of aa-tRNA CUA is perturbing the subtle equilibrium of all biomolecules involved in the translation mechanism, such as ribosomes, aaRSs and tRNAs, and several initiation, elongation and release factors [ 74 ], reducing its efficiency. Even though the exact concentrations and ratios of these components are not known, and vary between lysates, efforts have been made to investigate the composition of S30 lysate and potential factors that are limiting translation in CF systems [ 75 , 76 ]. While current S30 lysates seem to present optimal ratios between the all components necessary for an ordinary translation reaction [ 75 ], the addition of aa-tRNA CUA seems to disturb this balance.…”

Section: Resultsmentioning

confidence: 99%

“…Even though the exact concentrations and ratios of these components are not known, and vary between lysates, efforts have been made to investigate the composition of S30 lysate and potential factors that are limiting translation in CF systems [ 75 , 76 ]. While current S30 lysates seem to present optimal ratios between the all components necessary for an ordinary translation reaction [ 75 ], the addition of aa-tRNA CUA seems to disturb this balance. Based on the number of tRNA molecules present in each cell at specific growth rates and doubling times [ 77 ], we estimated the total tRNA concentration of our lysate to be ~10 µM.…”

Section: Resultsmentioning

confidence: 99%

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

et al. 2020

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The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics.

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“…Cell-free expression mixtures based on S30 cell lysates contain proteins at a concentration of $15 mg/ml, 49,50 which is estimated to be equivalent to $0.1À1 mM. We, therefore, investigated a positively charged and a negatively charged protein, BSA and lysozyme, respectively, at a concentration of 3 mM and compared this with the much lower concentrations of 3 lM and 3 nM, which is within the typical range for interdroplet bilayer experiments with ion channels such as a-hemolysin.…”

Section: B Pure Proteins and Polymersmentioning

confidence: 99%

Cell-free protein expression systems in microdroplets: Stabilization of interdroplet bilayers

2013

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Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study, we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) polyethylene glycol 8000 (PEG 8000) and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can, therefore, be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channels.

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Limiting factors of the translation machinery

Cited by 7 publications

References 28 publications

Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

Cell-free protein expression systems in microdroplets: Stabilization of interdroplet bilayers

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