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Issue 2, 2016
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Metabolomic analysis of riboswitch containing E. coli recombinant expression system

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Abstract

In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional–translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.

Graphical abstract: Metabolomic analysis of riboswitch containing E. coli recombinant expression system

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Publication details

The article was received on 16 Sep 2015, accepted on 19 Nov 2015 and first published on 23 Nov 2015


Article type: Paper
DOI: 10.1039/C5MB00624D
Citation: Mol. BioSyst., 2016,12, 350-361
  • Open access: Creative Commons BY license
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    Metabolomic analysis of riboswitch containing E. coli recombinant expression system

    H. Muhamadali, Y. Xu, R. Morra, D. K. Trivedi, N. J. W. Rattray, N. Dixon and R. Goodacre, Mol. BioSyst., 2016, 12, 350
    DOI: 10.1039/C5MB00624D

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