ArticleMicrobial cell factories2012
Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette.
Article in Microbial cell factories, 2012. 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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Who cites it
7 citing papers in PubMed, 15 citations in OpenAlex.
- Physical decoupling of XylS/Pm regulatory elements and conditional proteolysis enable precise control of gene expression in Pseudomonas putida.Microbial biotechnology · 2020Article
- The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.Microbial biotechnology · 2017Review
- Genome engineering for improved recombinant protein expression in Escherichia coli.Microbial cell factories · 2014Review
- Genetically modified microorganism Spingomonas paucimobilis UT26 for simultaneously degradation of methyl-parathion and γ-hexachlorocyclohexane.Ecotoxicology (London, England) · 2014Article
- Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter.BMC microbiology · 2013Article
- Recombinant polypeptide production in E. coli: towards a rational approach to improve the yields of functional proteins.Microbial cell factories · 2013Review
- A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli.Microbial cell factories · 2013Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli. Activation of transcription from the Pm promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements xylS (X), Pm (P) and the 5'-untranslated region (U) that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels.
resultsWe combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene bla (resistance to penicillin). Combination of optimized elements stimulated bla expression up to 75-fold (X, P and U combined) relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, celB (encoding phosphoglucomutase) and the human growth factor gene gm-csf. Protein production from these genes is much more efficient than from bla in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for bla. We also integrated a single copy of the expression cassette with each gene into the E. coli chromosome and found that the expression level from this single copy was higher for bla than for the wild-type plasmid system, while it was lower for celB and gm-csf.
conclusionOur results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in E. coli. For one reporter gene (bla) this allowed for more protein production from a single gene copy integrated on the chromosome, compared to the wild-type plasmid system. The approach described here should in principle be applicable for improvement of any expression cassette.
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