ArticleMicrobial cell factories2017
The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.
Article in Microbial cell factories, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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7 citing papers in PubMed, 16 citations in OpenAlex.
- High-level production of γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system inEngineering microbiology · 2023Article
- Plasmids for Controlled and Tunable High-Level Expression in E. coli.Applied and environmental microbiology · 2022Article
- Overcoming the Solubility Problem in E. coli: Available Approaches for Recombinant Protein Production.Methods in molecular biology (Clifton, N.J.) · 2022Review
- Overproducing the BAM complex improves secretion of difficult-to-secrete recombinant autotransporter chimeras.Microbial cell factories · 2021Article
- Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance.Microbial cell factories · 2021Article
- Bicistronic Design-Based Continuous and High-Level Membrane Protein Production inACS synthetic biology · 2019Article
- E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3).Microbial cell factories · 2018Article
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8 authors at 3 institutions in 2 countries.
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Abstract
backgroundTo optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector.
resultsBy incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein.
conclusionsHere, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).
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