Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins

Sevastsyanovich, Yanina R.; Alfasi, Sara; Overton, Tim W.; Hall, Richard M.; Jones, Jo J.; Hewitt, Christopher J.; Cole, Jeff

doi:10.1111/j.1574-6968.2009.01738.x

Cited by 43 publications

(68 citation statements)

References 24 publications

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“…Low IPTG concentrations and reduced growth temperature have been shown to increase the yield of correctly folded membrane proteins expressed from P lac /P tac controlled plasmids [28,29]. However, neither of the two changes increased the yields of glycoprotein (Additional file 2).…”

Section: Resultsmentioning

confidence: 99%

Production of glycoprotein vaccines in Escherichia coli

Kowarik²,

et al. 2010

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BackgroundConjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.ResultsTwo different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.ConclusionsThe presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

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Section: Resultsmentioning

confidence: 99%

Production of glycoprotein vaccines in Escherichia coli

Kowarik²,

et al. 2010

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“…Recombinant protein production is a complex biotechnological process, but it is relatively well document-ed compared with metabolites-oriented bioprocesses, due to the fact that reporter molecules, such as green fluorescent protein (GFP), can be fused to the protein of interest, providing a direct read-out system. Population heterogeneity was found to affect microbial recombinant protein production processes at both the levels of cell viability and productivity [60][61][62]. Microbial stress in this case is strongly dependent on the solubility of the target protein and on the extent of inclusion body formation.…”

Section: Production Of Metabolites and Recombinant Systems: Robustnesmentioning

confidence: 95%

Microbial heterogeneity affects bioprocess robustness: Dynamic single‐cell analysis contributes to understanding of microbial populations

Delvigne

Goffin

2013

Biotechnology Journal

127

102

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Heterogeneity or segregation of microbial populations has been the subject of much research, but the real impact of this phenomenon on bioprocesses remains poorly understood. The main reason for this lack of knowledge is the difficulty in monitoring microbial population heterogeneity under dynamic process conditions. The main concepts resulting in microbial population heterogeneity in the context of bioprocesses have been summarized by two distinct hypotheses. The first involves the individual history of microbial cells or the "path" followed during their residence time inside the process equipment. The second hypothesis involves a coordinated response by the microbial population as a bet-hedging strategy, in order to cope with process-related stresses. The respective contribution of each hypothesis to microbial heterogeneity in bioprocesses is still unclear. This illustrates the fact that, although microbial phenotypic heterogeneity has been thoroughly investigated at a fundamental level, the implications of this phenomenon in the context of microbial bioprocesses are still subject to debate. At this time, automated flow cytometry is the best technique for investigating microbial heterogeneity under process conditions. However, dedicated software and relevant biomarkers are needed for the proper integration of flow cytometry as a bioprocess control tool.

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“…For higher eukaryotes, chromosomal integration of the gene remains the only solution. In addition, many research groups encountered a reduction in expression of the desired protein during long cultivations when using plasmid-based systems, which is caused by plasmid loss and overgrowth of plasmid-free bacteria [142] or loss of functional T7 RNA polymerase as a result of chromosomal mutations [171]. Moreover, plasmid maintenance imposes a metabolic burden on the host cells, which results in a reduced growth rate and viability [123].…”

Section: Plasmid-based Rppmentioning

confidence: 99%

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Waegeman¹,

Soetaert²

2011

J Ind Microbiol Biotechnol

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Different hosts have been used for recombinant protein production, ranging from simple bacteria, such as Escherichia coli and Bacillus subtilis, to more advanced eukaryotes as Saccharomyces cerevisiae and Pichia pastoris, to very complex insect and animal cells. All have their advantages and drawbacks and not one seems to be the perfect host for all purposes. In this review we compare the characteristics of all hosts used in commercial applications of recombinant protein production, both in the area of biopharmaceuticals and industrial enzymes. Although the bacterium E. coli remains a very often used organism, several drawbacks limit its possibility to be the first-choice host. Furthermore, we show what E. coli strains are typically used in high cell density cultivations and compare their genetic and physiological differences. In addition, we summarize the research efforts that have been done to improve yields of heterologous protein in E. coli, to reduce acetate formation, to secrete the recombinant protein into the periplasm or extracellular milieu, and to perform post-translational modifications. We conclude that great progress has been made in the incorporation of eukaryotic features into E. coli, which might allow the bacterium to regain its first-choice status, on the condition that these research efforts continue to gain momentum.

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Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins

Cited by 43 publications

References 24 publications

Production of glycoprotein vaccines in Escherichia coli

Production of glycoprotein vaccines in Escherichia coli

Microbial heterogeneity affects bioprocess robustness: Dynamic single‐cell analysis contributes to understanding of microbial populations

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

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