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Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette.

Zwick F, Lale R, Valla S - Microb. Cell Fact. (2012)

Bottom Line: 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.Our 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands Vei 6/8, N-7491, Trondheim, Norway.

ABSTRACT

Background: The 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.

Results: We 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.

Conclusion: Our 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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Phosphoglucomutase enzyme activities and celB transcript amounts for extra-chromosomally (a) and chromosomally (b) expressed combination constructs. Enzyme activities in white, transcript amounts in black, all values are relative to the wild-type (arbitrarily set to 1). The values are the average of at least two biological replicas. E. coli DH5α was used as host.
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Figure 4: Phosphoglucomutase enzyme activities and celB transcript amounts for extra-chromosomally (a) and chromosomally (b) expressed combination constructs. Enzyme activities in white, transcript amounts in black, all values are relative to the wild-type (arbitrarily set to 1). The values are the average of at least two biological replicas. E. coli DH5α was used as host.

Mentions: As for any expression system individual proteins are expressed at quite varying levels from Pm, and β-lactamase is not among the highly expressed proteins. In the original identification of the X, P and U variants the bla gene was used as a reporter and it is therefore of interest to study if the variant combinations would also stimulate the expression of genes other than bla. We selected the bacterial celB gene (encoding phosphoglucomutase) and the human gm-csf gene (encoding cytokine granulocyte-macrophage colony-stimulating factor) as representative examples for such an analysis. Both of these genes were previously (in contrast to bla) shown to be efficiently expressed from wild-type xylS/Pm. CelB was earlier found to be the clearly dominating protein on a crude gel when expressed from wild-type xylS/Pm[21] and also GM-CSF could be visualized on a protein gel when expressed from a plasmid with elevated copy number [14]. The bla gene in the constructs described above was therefore substituted with either the celB or the gm-csf gene. Phosphoglucomutase enzyme activities were then measured, while GM-CSF protein levels were visualized by Western Blotting (Figures 4a and 5a). In case of gm-csf another strain (RV308) was used as host to enable comparison of expression levels to previously published results [14]. Comparisons of ampicillin tolerances and analyses of gm-csf expression at the transcript and protein levels in DH5α andRV308 indicated that absolute values are slightly higher in RV308, while all relative values, compared to wild-type, were similar in both strains (data not shown).


Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette.

Zwick F, Lale R, Valla S - Microb. Cell Fact. (2012)

Phosphoglucomutase enzyme activities and celB transcript amounts for extra-chromosomally (a) and chromosomally (b) expressed combination constructs. Enzyme activities in white, transcript amounts in black, all values are relative to the wild-type (arbitrarily set to 1). The values are the average of at least two biological replicas. E. coli DH5α was used as host.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526546&req=5

Figure 4: Phosphoglucomutase enzyme activities and celB transcript amounts for extra-chromosomally (a) and chromosomally (b) expressed combination constructs. Enzyme activities in white, transcript amounts in black, all values are relative to the wild-type (arbitrarily set to 1). The values are the average of at least two biological replicas. E. coli DH5α was used as host.
Mentions: As for any expression system individual proteins are expressed at quite varying levels from Pm, and β-lactamase is not among the highly expressed proteins. In the original identification of the X, P and U variants the bla gene was used as a reporter and it is therefore of interest to study if the variant combinations would also stimulate the expression of genes other than bla. We selected the bacterial celB gene (encoding phosphoglucomutase) and the human gm-csf gene (encoding cytokine granulocyte-macrophage colony-stimulating factor) as representative examples for such an analysis. Both of these genes were previously (in contrast to bla) shown to be efficiently expressed from wild-type xylS/Pm. CelB was earlier found to be the clearly dominating protein on a crude gel when expressed from wild-type xylS/Pm[21] and also GM-CSF could be visualized on a protein gel when expressed from a plasmid with elevated copy number [14]. The bla gene in the constructs described above was therefore substituted with either the celB or the gm-csf gene. Phosphoglucomutase enzyme activities were then measured, while GM-CSF protein levels were visualized by Western Blotting (Figures 4a and 5a). In case of gm-csf another strain (RV308) was used as host to enable comparison of expression levels to previously published results [14]. Comparisons of ampicillin tolerances and analyses of gm-csf expression at the transcript and protein levels in DH5α andRV308 indicated that absolute values are slightly higher in RV308, while all relative values, compared to wild-type, were similar in both strains (data not shown).

Bottom Line: 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.Our 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands Vei 6/8, N-7491, Trondheim, Norway.

ABSTRACT

Background: The 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.

Results: We 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.

Conclusion: Our 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.

Show MeSH
Related in: MedlinePlus