Tnao38, high five and Sf9—evaluation of host–virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression

Wilde, Monika; Klausberger, Miriam; Palmberger, Dieter; Ernst, Wolfgang; Grabherr, Reingard

doi:10.1007/s10529-013-1429-6

Cited by 55 publications

(59 citation statements)

References 30 publications

(35 reference statements)

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“…It is well documented that Sf9 cells are a preferred host for the production of baculovirus vectors, and High Five cells for the production of recombinant proteins (Krammer et al, 2010;Taticek et al, 2001;Wilde et al, 2014). This was a consequence of the high TCA cycle activity, mirroring the very efficient metabolism that characterizes the Sf9 cell line .…”

Section: Discussionmentioning

confidence: 99%

“…High Five cells produce higher levels of lactate and ammonia, however this phenotype is dependent on the culture media (Ikonomou et al, 2003;Rhiel et al, 1997). Regarding productivity, it is recognized that Sf9 cells are a better host for the production of infectious baculovirus vectors, while High Five cells are preferred for high level expression of recombinant proteins (Krammer et al, 2010;Taticek et al, 2001;Wilde et al, 2014). Moreover, the low levels of ammonia produced by Sf9 cells are related with the presence of a detoxification and recycling mechanism driven by glutamate synthase (GOGAT) activity (Doverskog et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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The role of host cell physiology in the productivity of the baculovirus‐insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines

Monteiro

Bernal

Alves

2016

Biotech & Bioengineering

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The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five cells. This analysis was conducted in comparison with Sf9 cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five cells. High Five cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 cells, in which the glucose carbon incorporation in the TCA cycle was significantly higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The role of host cell physiology in the productivity of the baculovirus‐insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines

Monteiro

Bernal

Alves

2016

Biotech & Bioengineering

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“…Today, the most popular insect cell lines for protein expression are derived from Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (High FiveTM), Bombyx mori and Drosophila melanogaster (S2). Recombinant protein production in S2 cells is achieved by stable transformation (Dyring 2011;Wilde 2014). The other cell lines are used to host the baculovirus expression vector system (BEVS), mainly Nucleopolyhedrovirus (NPV) like Autographa californica (AcMNPV) or Bombyx mori (BmNPV).…”

Section: Insect Biotechnologymentioning

confidence: 99%

“…Thereby, AcMNPV is the more versatile tool, as BmNPV has a narrower host range and is limited to B. mori cells (Palomares 2005). For the production of recombinant proteins, the two most popular systems are the S2 cells and the baculovirus expression vector system (BEVS) using AcMNPV, still the gold standard in insect-based expression systems for recombinant proteins (Sprick 2017;Wilde 2014).…”

Section: Insect Biotechnologymentioning

confidence: 99%

1st Gießen Symposium of Insect Biotechnology, October 9-10, 2017 (published online)

2017

Zeitschrift Für Naturforschung C

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The genome of entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus encodes for up to 25 biosynthesis gene clusters (BGCs), representing up to 7.5% of the total genome. While several natural products have been identified and isolated from these bacteria during the last ten years due to efforts from several labs (Bode 2009, Challinor and Bode 2016, Vizcaino 2013, the majority of these compounds are still unknown. Therefore, these natural products are not accessible for a detailed analysis of their function in the natural ecosystem or as drugs in a more applied way (antibiotics, anticancer compounds, biotechnology etc) and methods for faster access to these natural products is desirable.We have analysed the deletion of known global regulators from Gram-negative bacteria on the natural product biosynthesis in Xenorhabdus and Photorhabdus (Engel 2017) and did identify the RNA chaperon Hfq playing a major role in natural product biosynthesis. In hfq mutants of several strains analysed so far, all natural products were reduced 10 to 100-fold, most of them below the limit of detection (Tobias 2017). Hfq is known for binding small regulatory RNAs and deliver them to target mRNAs and thereby can activate or reduce protein levels. When we applied our well-established promoter exchange approach (Bode 2015) to these hfq mutants, we were able to produce only the expected compound class upon activation of the promoter. We could transform the wildtype strains (as producers of multiple compounds) via the hfq strains (as no-producers of any compound) into single producers of a desired natural product class. Since an extract generated from these strains contains just one compound class, it can be tested directly against a variety of different targets allowing a simplified functional assignment of its biological function and without actual isolation of the natural product.Moreover, since the natural product can be produced on-demand in its natural environment due to its inducible promoter, the determination of the individual compound's contribution and role in the overall ecosystem can be addressed easily.As an additional way to generate chemical diversity from microorganisms, we have developed the eXchange Unit (XU) technology that allows the simple assembly of building blocks derived from natural non-ribosomal peptide synthetases (NRPS) to generate cyclic, linear, acylated peptides and/or peptides containing D-amino acids. Due to the definition of the XU and their assembly at a specific sequence, the yields even for non-natural peptides are between 1-40 mg/L in E. coli enabling detailed bioactivity testing. The XU technology can also be applied to the original producers enabling the simple modification of NRPS encoding BGCs as well as the artificial fusion of adjacent BGCs.Both, the latest results about hfq mediated production of individual compound classes and the XU technology will be presented. Justus-Liebig-University Giessen, Bioinformatics and Systems Biology, 35392 Gießen, Germany Corresponding author...

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“…Recombinant protein production in S2 cells is achieved by stable transformation [5][6][7]. The other cell lines are used to host the baculovirus expression vector system (BEVS), which remains the gold standard in insect-based expression systems for recombinant proteins [8,9]. However, the BEVS is used not only for the production of recombinant proteins but also as a gene shuttle for stem cells [10] and for vaccine manufacturing [9].…”

Section: Introductionmentioning

confidence: 99%

The components of shear stress affecting insect cells used with the baculovirus expression vector system

Weidner

Druzinec

Mühlmann

et al. 2017

Zeitschrift Für Naturforschung C

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Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory-and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic ® . At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

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Tnao38, high five and Sf9—evaluation of host–virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression

Cited by 55 publications

References 30 publications

The role of host cell physiology in the productivity of the baculovirus‐insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines

The role of host cell physiology in the productivity of the baculovirus‐insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines

1st Gießen Symposium of Insect Biotechnology, October 9-10, 2017 (published online)

The components of shear stress affecting insect cells used with the baculovirus expression vector system

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