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DES ® : The Drosophila Expression System

DES ® : The Drosophila Expression System. 2003.05.15 약학대학 면역학 연구실 이승호. Overview of Stable Insect Expression Systems. combine the advantages of both baculovirus and mammalian expression. simple to use with straightforward techniques for transfection and selection.

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DES ® : The Drosophila Expression System

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  1. DES® : The Drosophila Expression System 2003.05.15 약학대학 면역학 연구실 이승호

  2. Overview of Stable Insect Expression Systems • combine the advantages of both baculovirus and mammalian expression. • simple to use with straightforward techniques for transfection and selection. • often achieve higher levels of expression than mammalian systems • particularly useful for production of secreted proteins. • the reliability and reproducibility of expression levels • once a stable cell line is produced, the expression levels are maintained over time. • produce protein from healthy, logarithmically growing cells, resulting in high quality protein.

  3. DES® : The Drosophila Expression System • A non-lytic system that combines the best features of mammalian and insect expression systems for simple, efficient production of recombinant protein. • uses the well-characterized Drosophila Schneider S2 cells. • Expression vectors are available for either constitutive or inducible expression. • uses simple expression vectors to allow stable or transient expression of recombinant proteins.

  4. Selection Vector(pCoHygro) • used to create stable transfectants in Drosophila • contains the E.coli hygromycin-B-phosphotransferase gene under control of the Drosophila copia promoter for resistance to hygromycin-B in S2 cells • When added to cultured Drosophila cells hygromycin Bacts as an aminocyclitol to inhibit protein systhesis by disrupting translocation and promoting mistranslation

  5. Selection Vector(pCoBlast) • contains the Streptomyces griseochromogenes bsd gene under control of the Drosophila copia promoter to confer resistance to blasticidin in S2 cells • Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells. Resistance is conferred by expression of either one of two blasticidin S deaminase genes : bsd from Aspergillus terreus or bsr from Bacillus cereus. These deaminases convert blastididin S to a non-toxic deaminohydroxyderivative

  6. DES® Cells and Media • Drosophila S2 cells • used for heterologous protein expression using DES®. • derived from a primary culture of late stage (20-24 hours old) Drosophila melanogaster embryos. • grows rapidly at room temperature without CO2 . • easily adapted to suspension culture.

  7. Advantages • The DES®vectors contain many features to simplify cloning, detection, and purification. • a C-terminal tag that adds the V5 epitope for detection • a polyhistidine (6xHis) sequence for quick and easy purification • pMT/V5-His is available Gateway® (pMT-DEST48) and topoisomerase-activated (pMT/V5-His TOPO®). • Once the expression construct is inside the S2 cell, hundreds of copies of the expression plasmid containing gene of interest will spontaneously integrate into the genome. After just a few weeks of selection, a polyclonal cell line is established that stably expresses high levels of protein faster than in mammalian systems.

  8. DES® Inducible Kits • provides the expression vector pMT/V5-His and a choice of Blasticidin (pCoBlast) or hygromycin (pCoHygro) selection. • uses the Drosophila metallothionein gene promoter induced in S2 cells upon addition of copper sulfate or cadmium chloride to the culture medium.

  9. Characterization and use of the Drosophila metallothionein promoterin cultured Drosophila melanogaster cells. Bunch TA, Grinblat Y, Goldstein LS. Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138. The promoter from the metallothionein gene may be a useful conditional promoter for the construction of chimeric genes to be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed chimeric gene containing the metallothionein promoter were examined. Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels. The level of induction depends on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing the metallothionein promoter shows a similar induction when transformed into the cells.

  10. pMT/V5-His A

  11. pMT/V5-His B

  12. pMT/V5-His C

  13. DES® Inducible/Secreted Kits • provides the vector pMT/Bip/V5-His for inducible, secreted expression of recombinant proteins and a choice of Blasticidin (pCoBlast) or hygromycin (pCoHygro) selection. • uses the Drosophila metallothionein gene promoter induced in S2 cells upon addition of copper sulfate or cadmium chloride to the culture medium. • The N-terminal signal sequence from the insect BiP gene is provided to direct the recombinant fusion protein through the secretory pathway of S2 cells into the culture medium.

  14. pMTBiP/V5-His A

  15. pMTBiP/V5-His B

  16. pMTBiP/V5-His C

  17. DES® TOPO® Expression Kit(Fast cloning saves time) • offers one-step cloning of PCR products directly into an inducible DES® expression vector. • uses the linearized, topoisomerase 1-activated pMT/V5-His- TOPO® vector for fast and easy cloning and subsequent high-level expression. • Topoisomerase activation of this vector allows PCR products to be ligated in just 5 minutes on bench top to produce > 85% recombinants. • The pMT/V5-His- TOPO® vector has all of the features of the pMT/V5-His- vector to simplify protein expression, detection, purification in Drosophila S2 cells.

  18. pMT-DEST48 Gateway®Destination Vector(The power of Gateway®) • designed for rapid cloning with a Gateway® entry clone using lambda phage site-specific recombination and subsequent expression in Drosophila S2 cells. • Gateway® Technology allows transfer of gene of interest between different vectors by recombination, eliminating the need for restriction endonucleases and ligase. You simply clone gene of interest into an entry vector and then move it into the destination vector of choice for expression. • As part of the DES Expression System, pMT-DEST48 uses the Drosophila metallothionein gene promoter induced in S2 cells upon addition of copper sulfate or cadmium chloride to the culture medium. • attR sites for efficient recombination with any attL-flanked Gateway® entry vector.

  19. DES® Constitutive Kits(Constitutive expression option) • DES® Constitutive Kit provides the vector pAc5.1/V5-His for high-level constitutive expression of recombinant proteins and a choice of Blasticidin (pCoBlast) or hygromycin (pCoHygro) selection. • offers the strong, constitutive promoter from the Drosophila actin 5C gene. • Small size to improve DNA yields and increase subcloning efficiency.

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