Abstract
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.
Highlights
Cell-free protein synthesis systems have emerged as a powerful tool for quick small-scale protein production
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, EF-Tu, EF-Ts, EF-G and ribosomal protein S1 (RPS1) [25,26]
The integrity and the concentration of ribosomes in the cell-free extract were determined by a previously described ribosomal RNA (rRNA) analysis approach, which is based on total RNA extraction and analysis by capillary gel electrophoresis with laser-induced fluorescence detection (CGELIF) [27]
Summary
Cell-free protein synthesis systems have emerged as a powerful tool for quick small-scale protein production. Compared with in vivo protein expression, these systems have many advantages. Cell-free gene expression systems tolerate the incorporation of noncanonical amino acids [1], expanding the potential of biological protein synthesis [2,3,4]. The open and modular features of in vitro translation systems allow easy automation, direct access to the catalytic system, and high-throughput production [5,6,7]. Driven by the low productivity of early in vitro translation reactions, substantial efforts have been made.
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