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DES : The Drosophila Expression System

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combine the advantages of both baculovirus and mammalian expression. ... S deaminase genes : bsd from Aspergillus terreus or bsr from Bacillus cereus. ... – PowerPoint PPT presentation

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Title: DES : The Drosophila Expression System


1
DES The Drosophila Expression System
  • 2003.05.15
  • ???? ??? ???
  • ???

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.

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5
Selection Vector (pCoHygro)
  • used to create stable transfectants in Drosophila
  • contains the E.coli hygromycin-B-phosphotransferas
    e gene under control of the Drosophila copia
    promoter for resistance to hygromycin-B in S2
    cells
  • When added to cultured Drosophila cells
    hygromycin B acts as an aminocyclitol to inhibit
    protein systhesis by disrupting translocation and
    promoting mistranslation

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7
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

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9
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.

10
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.

11
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.

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13
Characterization and use of the Drosophila
metallothionein promoter in 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.

14
pMT/V5-His A
15
pMT/V5-His B
16
pMT/V5-His C
17
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.

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19
pMTBiP/V5-His A
20
pMTBiP/V5-His B
21
pMTBiP/V5-His C
22
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 gt 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.

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25
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.

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28
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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