BactoBac Baculovirus Expression System

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Bac-to-Bac Baculovirus Expression System

• Lab. of Molecular Genetics
• Ryu Hyun-Mi

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Bac-to-Bac Baculovirus Expression System

• An efficient site-specific transposition system
  to generate baculovirus for high-level expression
  of recombinant proteins
• AcMNPV Autographa califonica multiple nuclear
  polyhedrosis virus

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Bac-to-Bac system

• Major components
• pFastBac donor plasmid vector into which the
  gene(s) of interest will be cloned
• PPH, MSC, SV40 PA, gentamycin resistance marker
• DH10Bac E.coli strain(host for pFastBac)
• 1. baculovirus shuttle vector(bacmid)
  mini-attTn7 target site, lacZa, kanamycin
  resistance marker
• 2. helper plasmid transposase, tetracycline
  resistance marker

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Site-specific transposition
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Diagram of the Bac-to-Bac system
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Advantages

• Time-saving expression bacmid
• -high titer virus is produced from the initial
  transfection.
• Easy colony screening
• -lacZa gene(blue/white screening? white colony
  recombinant)
• Rapid purifacation of recombinant proteins
• -pFastBacHT

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pFastBac1

• Strong AcMNPV polyhedrin promoter
• Large MCS

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pFastBacHT

• Strong polyhedrin promoter
• N-terminal 6 X His tag( TEV protease cleavage
  site)
• 3 reading frame( pFastBacHTa, b, and c)

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pFastBacDual

• Two strong baculovirus promoter(PH and p10)
• Two large MCS
• SV40 polyadenylation signal and HSV tk
  polyadenylation signal for transcription
  termination

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END
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Plaque Assay

• Neutral red paled pink
• Pfu/ml (of original stock) 1/dilution factor x
  number of plaques x 1/(ml of inoculum/plate))
• pfu plaque forming unit