Protein Expression in Drosophila Schneider Cells

Benting, Jürgen; Lecat, Sandra; Zacchetti, Daniele; Simons, Kai

doi:10.1006/abio.1999.4409

Cited by 40 publications

(28 citation statements)

References 37 publications

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“…The S2 cells are of embryonic origin and can incorporate large amounts of plasmid DNA (27). They can be cultured in serum-free medium, avoiding contamination with serum proteins.…”

mentioning

confidence: 99%

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

Schmetzer

Moldenhauer

Riesenberg³

et al. 2005

The Journal of Immunology

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“…The S2 cells are of embryonic origin and can incorporate large amounts of plasmid DNA (27). They can be cultured in serum-free medium, avoiding contamination with serum proteins.…”

mentioning

confidence: 99%

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

Schmetzer

Moldenhauer

Riesenberg³

et al. 2005

The Journal of Immunology

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“…Dcert gene was cloned in to pRMHA-C in-frame with the C-terminal FLAG tag and expressed in S2 cells by using standard techniques (44). CPE and ceramides were estimated from isolated membranes from dcert 1 and w1118 flies by using a combination of HPLC and mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

Ceramide transfer protein function is essential for normal oxidative stress response and lifespan

Rao

Yuan

Allegood

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

136

129

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Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein.We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display >70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal.aging ͉ Drosophila ͉ membrane fluidity ͉ sphingolipids ͉ sphingomyelin

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“…We decided to overexpress each protein separately in a heterologous system (Schneider cells) in order to purify them to determine their three-dimensional structure. Schneider 2 (S2) cells are suitable hosts for the expression of large amounts of recombinant eukaryotic protein, since they are inducible, nonlytic, stable, and able to reach a cell density as high as 3 ϫ 10 7 cells/ml (10-fold higher than Sf9 cells) (27)(28)(29). hPtc and hSmo cDNA were subcloned in S2 cell expression vectors in which the sequence MAP was previously inserted in place of the existent tags V5 and His.…”

Section: Establishment Of Schneider Cell Lines Stably Expressing Humamentioning

confidence: 99%

Human Receptors Patched and Smoothened Partially Transduce Hedgehog Signal When Expressed in Drosophila Cells

Rivoyre

Ruel

Varjosalo

et al. 2006

Journal of Biological Chemistry

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Protein Expression in Drosophila Schneider Cells

Cited by 40 publications

References 37 publications

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

Ceramide transfer protein function is essential for normal oxidative stress response and lifespan

Human Receptors Patched and Smoothened Partially Transduce Hedgehog Signal When Expressed in Drosophila Cells

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