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Article Dans Une Revue BioTech Année : 2021

Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

Résumé

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.
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Origine : Publication financée par une institution
Licence : CC BY - Paternité

Dates et versions

hal-03810131 , version 1 (11-01-2024)

Identifiants

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Marina Snapyan, Sylvain Robin, Garabet Yeretssian, Michèle Lecocq, Frédéric Marc, et al.. Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery. BioTech, 2021, 10 (4), pp.24. ⟨10.3390/biotech10040024⟩. ⟨hal-03810131⟩
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