Protein production using the baculovirus‐insect cell expression system

Contreras-Gómez, Antonio; Sánchez-Mirón, A.; Camacho, F. Garcı́a; Grima, E. Molina; Chisti, Yusuf

doi:10.1002/btpr.1842

Cited by 126 publications

(108 citation statements)

References 213 publications

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“…These viruses exhibit biphasic morphology in the form of occluded and budded viruses pre-ordained for primary larval infection and horizontal spread within the host, respectively. These have been employed extensively as protein expression vectors (Contreras-Gó mez et al, 2014;Hitchman et al, 2011;Kost et al, 2005;Murphy et al, 2004) and as biopesticide agents against major lepidopteran crop pests (Heimpel et al, 1973;Summers, 2006;Sun & Peng, 2007). Spodoptera litura nucleopolyhedrovirus (SpltNPV) has been exploited as a commercially viable alternative to obliterate the polyphagous insect pest Spodoptera litura, which infects more than 120 plant species spanning several regions of Asia and Oceania (Nathan & Kalaivani, 2005;Okada, 1977;Zhang, 1994).…”

Section: Introductionmentioning

confidence: 99%

Temporal expression profiling of novel Spodoptera litura nucleopolyhedrovirus-encoded microRNAs upon infection of Sf21 cells

Kharbanda

Jalali

Ojha

et al. 2015

Journal of General Virology

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Section: Introductionmentioning

confidence: 99%

Temporal expression profiling of novel Spodoptera litura nucleopolyhedrovirus-encoded microRNAs upon infection of Sf21 cells

Kharbanda

Jalali

Ojha

et al. 2015

Journal of General Virology

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“…1A (GST-N⌬173 Tec, GST-PH-TH, GST-SH3-SH2, and the SH1 kinase domain) were expressed in SF9 insect cells using the baculovirus expression system (53). GST-N⌬173 Tec, GST-PH-TH, and GST-SH3-SH2 were purified by glutathione affinity chromatography (GSTrap TM FF, GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

Venuta

Wegehingel

Sehr

et al. 2016

Journal of Biological Chemistry

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Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.Although the majority of secretory proteins carry signal peptides for endoplasmic reticulum/Golgi-dependent secretion, a set of important growth factors and cytokines involved in tumor-induced angiogenesis and inflammatory responses makes use of alternative routes. These processes have collectively been termed "unconventional protein secretion" (1-5). FGF2 and IL1␤ are prominent examples for secretory proteins that make use of such pathways (4 -12). The molecular mechanism by which IL1␤ is secreted from cells is debated and may differ in some aspects between various kinds of immune cells (4,5,10,(13)(14)(15)(16). By contrast, the mechanism of the unconventional secretory pathway of FGF2 is emerging with great molecular detail (12). It is based upon direct translocation of folded species of FGF2 across plasma membranes (9,(17)(18)(19)(20). Hallmarks of this process are (i) FGF2 recruitment at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P 2 2 (9, 21, 22), (ii) FGF2 oligomerization and membrane pore formation (11,12,23,24), and (iii) extracellular trapping of FGF2 mediated by membrane-proximal heparan sulfate proteoglycans (1,2,25,26). Recently, these hallmarks of FGF2 secretion have also been implicated in unconventional secretion of HIV-Tat (HIV trans-activator of transcription) (12, 27-30). Based on a genome-wide RNAi screening approach, two additional factors physically associated with the plasma membrane have been identified to play a role in FGF2 secretion, Tec kinase (9, 11, 12, 24, 31) and ATP1A1 (12, 32-34), the ␣ subunit of the Na/K-ATPase (35). Although t...

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“…Several reactor systems with low shear stress and high rates of oxygen and carbon dioxide transfer have been designed or adapted for the BEVS, including the rotating wall reactor [25] and the wave reactor [26]. However, most processes still use a stirred-tank reactor (STR) due to the simpler process control and scalability [27,28].…”

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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Protein production using the baculovirus‐insect cell expression system

Cited by 126 publications

References 213 publications

Temporal expression profiling of novel Spodoptera litura nucleopolyhedrovirus-encoded microRNAs upon infection of Sf21 cells

Temporal expression profiling of novel Spodoptera litura nucleopolyhedrovirus-encoded microRNAs upon infection of Sf21 cells

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

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

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