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Role of chaperones and a cytoplasmatic protease in recombinant protein production in E. coli at suboptimal growth temperature

ABSTRACT: Recent studies have revealed that at lower cultivation temperatures (25°C) much higher percentage of correctly folded recombinant proteins can be extracted from inclusion bodies. The goal of our research was to investigate mechanisms determining characteristics of non-classical inclusion bodies production using gene expression profiling. Two strains of recombinant E. coli [BL21 (DE3)] grown at three different temperatures (25°C, 37°C and 42°C) were included in experiment.

ORGANISM(S): Escherichia coli

PROVIDER: GSE25561 | GEO | 2010/11/24

SECONDARY ACCESSION(S): PRJNA134285

REPOSITORIES: GEO

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Role of chaperones and a cytoplasmatic protease in recombinant protein production in E. coli at suboptimal growth temperature

Project description:Recent studies have revealed that at lower cultivation temperatures (25°C) much higher percentage of correctly folded recombinant proteins can be extracted from inclusion bodies. The goal of our research was to investigate mechanisms determining characteristics of non-classical inclusion bodies production using gene expression profiling. Two strains of recombinant E. coli [BL21 (DE3)] grown at three different temperatures (25°C, 37°C and 42°C) were included in experiment. Gene expression was studied in two recombinant strains (production and control strain), grown at three different temperatures (25, 37 and 42 oC) in three biological replicates. Cells were harvesed at OD=4, except for the cells grown at 25 oC, here they were harvested at OD=4 and OD=10.

2010-11-24 | E-GEOD-25561 | biostudies-arrayexpress

Metabolomic analysis of riboswitch containing E. coli recombinant expression system

Project description: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.

2017-11-16 | MTBLS393 | MetaboLights

Time series of Escherichia coli BL21(DE3) after temperature upshift from 37°C to 42°C, tiling arrays

Project description:Heat-responsive and time-resolved changes in transcriptome of E. coli BL21(DE3) Experimentally mapped transcriptome structure of Escherichia coli BL21(DE3) by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 10 nt).

2020-04-12 | GSE148034 | GEO

Microaerobic fermentation alters lactose metabolism in Escherichia coli

Project description:We report the effect of oxygenation state in lactose grown escherichia coli producing recombinant proteins. To shed more light on the mechanistic correlation between the uptake of lactose and dissolved oxygen, a comprehensive study has been undertaken with the E. coli BL21 (DE3) strain. Differences in consumption pattern of lactose, metabolites, biomass and product formation due to aerobiosis have been investigated. Transcriptomic profiling of metabolic changes due to aerobic process and microaerobic process during protein formation phase has been studied and the results provide a deeper understanding of protein production in E. coli BL21 (DE3) strains with lactose based promoter expression systems.This study also provides a scientific understanding of escherichia coli metabolism upon oxygen fluctuations.

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RNA-seq of E. coli BL21 (DE3) Evolved in High pH (pH = 9) Terrific Broth Media

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Project description:Calcium-activated potassium channels are important membrane proteins that help in maintaining the cellular physiology and cell signalling. Till date no Calcium-activated potassium channels have been identified in Leishmania donovani. It’s gene was cloned into pET vector and expressed in E. coli BL21 (DE3). The recombinant protein was purified using nickel affinity chromatography.

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Time series of Escherichia coli BL21(DE3) growth curve, tiling arrays

Project description:Experimentally mapped transcriptome structure of Escherichia coli BL21(DE3) by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 10 nt).

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Amino acid supplementation for enhancing recombinant protein production in E. coli

Project description:Amino acid starvation during recombinant protein production (RPP) induces metabolic stress to cellular host which results in reduced productivity of the recombinant bioprocess. In present study, supplementation of amino acids in a chemically defined medium showed several folds increase in recombinant product titers in E. coli BL21 DE3 strain. To understand, the effect of amino acid supplementation in alleviating cellular stress during RPP a deep insight of cellular physiology must be studied. Here, we performed transcriptomic analysis of E. coli expressing a recombinant protein in two different conditions: with (5 mM of all 20 amino acids) as test and without supplementation of amino acids as control. RNA-seq data revealed downregulation of several genes associated with stress in test culture confirming the critical role of amino acids supplementation in improving RPP.

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