Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

Viitanen, Mikko I; Vasala, Antti; Neubauer, Peter; Alatossava, Tapani

doi:10.1186/1475-2859-2-2

Cited by 42 publications

(22 citation statements)

References 23 publications

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“…Data analysis by ANN showed the significant effects of both pH and temperature on hEGF production, although changes in pH were found to be more effective. ANN analysis recommended 29 °C as the optimal temperature for hEGF production in P. pastoris and the yield of hEGF was expected to be decrease at temperatures higher than 31 °C, possibly due to poor stability of the protein at higher temperatures, releasing of proteases from dead cells, and misfolding of the protein 34, 35…”

Section: Discussionmentioning

confidence: 99%

Production of recombinant human epidermal growth factor in Pichia pastoris

Eissazadeh

Moeini

Dezfouli

et al. 2017

Brazilian Journal of Microbiology

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This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 μg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.

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Section: Discussionmentioning

confidence: 99%

Production of recombinant human epidermal growth factor in Pichia pastoris

Eissazadeh

Moeini

Dezfouli

et al. 2017

Brazilian Journal of Microbiology

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“…In all the studies performed above we used purified lactose but other sources of lactose can be used. Waste pollutants derived from the dairy industry, like cheese whey, were previously used for the production of recombinant proteins with expression levels similar to those obtained with IPTG (Viitanen et al 2003; De León-Rodríguez et al 2006). …”

Section: Discussionmentioning

confidence: 99%

High level expression and facile purification of recombinant silk-elastin-like polymers in auto induction shake flask cultures

et al. 2013

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Silk-elastin-like polymers (SELPs) are protein-based polymers composed of repetitive amino acid sequence motifs found in silk fibroin (GAGAGS) and mammalian elastin (VPGVG). These polymers are of much interest, both from a fundamental and applied point of view, finding potential application in biomedicine, nanotechnology and as materials. The successful employment of such polymers in such diverse fields, however, requires the ready availability of a variety of different forms with novel enhanced properties and which can be simply prepared in large quantities on an industrial scale. In an attempt to create new polymer designs with improved properties and applicability, we have developed four novel SELPs wherein the elastomer forming sequence poly(VPGVG) is replaced with a plastic-like forming sequence, poly(VPAVG), and combined in varying proportions with the silk motif. Furthermore, we optimised a simplified production procedure for these, making use of an autoinduction medium to reduce process intervention and with the production level obtained being 6-fold higher than previously reported for other SELPs, with volumetric productivities above 150 mg/L. Finally, we took advantage of the known enhanced stability of these polymers in developing an abridged, non-chromatographic downstream processing and purification protocol. A simple acid treatment allowed for cell disruption and the obtention of relative pure SELP in one-step, with ammonium sulphate precipitation being subsequently used to enable improved purity. These simplified production and purification procedures improve process efficiency and reduce costs in the preparation of these novel polymers and enhances their potential for application.

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“…the well-known expensive inducer isopropyl β- d -1 thiogalactopyranoside (IPTG) [ 3 , 13 ]. However, induction with IPTG stresses the cells, as IPTG in higher concentrations is known to be toxic [ 13 , 18 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes

Slouka

Kopp

Hutwimmer³

et al. 2018

Microb Cell Fact

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BackgroundThe bacterium E. coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes.ResultsClassical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein.ConclusionsTime dependent monitoring of IB QA—titer, purity, IB bead size—showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate—time dependently—quality attributes in upstream processing to enable robust downstream processing.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0997-5) contains supplementary material, which is available to authorized users.

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Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

Cited by 42 publications

References 23 publications

Production of recombinant human epidermal growth factor in Pichia pastoris

Production of recombinant human epidermal growth factor in Pichia pastoris

High level expression and facile purification of recombinant silk-elastin-like polymers in auto induction shake flask cultures

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes

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