DES : The Drosophila Expression System

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DES The Drosophila Expression System

• 2003.05.15
• ???? ??? ???
• ???

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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.

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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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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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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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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.

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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.

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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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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.

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pMT/V5-His A
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pMT/V5-His B
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pMT/V5-His C
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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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pMTBiP/V5-His A
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pMTBiP/V5-His B
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pMTBiP/V5-His C
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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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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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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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