Protein production in Escherichia coli is guided by the trade-off between intracellular substrate availability and energy cost

Abstract Background In vivo protein formation is a crucial part of cellular life. The process needs to adapt to growth conditions and is exploited for the production of technical and pharmaceutical proteins in microbes such as Escherichia coli. Accordingly, the elucidation of basic regulatory mechanisms controlling the in vivo translation machinery is of primary interest, not only to improve heterologous protein production but also to elucidate fundamental regulation regimens of cellular growth. Results The current modeling analysis elucidates the impact of diffusion for the stochastic supply of crucial substrates such as the elongation factor EFTu, and tRNA species, all regarded as key elements for ensuring optimum transcriptional elongation. Together with the consideration of cellular ribosome numbers, their impact on the proper functioning of the translation machinery was investigated under different in vivo and in vitro conditions and utilizing the formation of non-native GFP and native EFTu as target proteins. The results show that translational elongation was diffusion limited. However, this effect was much more pronounced for the translation of non-native proteins than for the formation of codon-optimized native proteins. Conclusions Cellular ATP requirements constrain the options of improving protein production. In the case of non-native protein sequences, an optimized tRNA supply may be the most economical solution, as cells necessarily have to invest in ATP-costly ribosome synthesis to boost translation and increase growth rates

Cell-Free Protein Synthesis From Fast-Growing Vibrio natriegens

Vibrio natriegens constitutes one of the fastest-growing nonpathogenic bacteria and a potential novel workhorse for many biotechnological applications. Here, we report the development of a Vibrio-based cell-free protein synthesis system (CFPS). Specifically, up to 0.4 g L-1 eGFP could be successfully synthesized in small-scale batch reactions using cell-free extract obtained from fast-growing V. natriegens cultures. Versatile CFPS system characterization attained by combining the analyses of key metabolites for translation and ribosomes revealed limitations regarding rRNA stability and critical substrate consumption (e.g., amino acids). Alternatively, rRNA showed increased stability by inducing Mg2+homeostasis in the reaction. Although the enormous translation capacity of the CFPS system based on the available ribosome concentration could not yet be fully exploited, its potential was successfully demonstrated by activating an endogenous transcription unit with V. natriegensRNA polymerase (RNAP) for protein expression. This allowed the use of in vitro screening for promoter strength, a critical factor for efficient gene expression in vitro and in vivo. Three different promoters were tested and output signals corresponded well with the expected affinity for V. natriegens RNAP. This established CFPS toolbox may provide a foundation to establish V. natriegens as a valuable platform in biotechnology as well as synthetic biology

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

In order to produce the atrazine-specific scFv K411B, it was expressed in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the scFv was N-terminally fused to the pelB leader, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly: The expression level of the scFv with leader was 2.3 times higher than that of the protein without leader. To further investigate this, the respective translation profiles were generated by coupled in vitro transcription/translation assays and gave according results. Periplasmic expression resulted in only 10% correctly folded scFv. The same percentage was obtained when the scFv was expressed in vitro, 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 the scFv with 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. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein

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

<div><p>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 <i>in vivo</i> 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 <i>in vitro</i> 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 <i>in vitro</i> 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 <i>Escherichia coli</i>. 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 <i>in vitro</i> systems.</p></div

Primer extension analysis of intact and cleaved 16S rRNA.

<p>Intact and cleaved 16S rRNA were purified from a 2% agarose gel. Primers specific to regions 1470 to 1491 bp (P1), 895 to 915 bp (P2), 431 to 450 bp (P3) were annealed and cDNA synthesis was performed. RNA was digested and cDNA was visualized on a 1% agarose gel.</p