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Bac-to-Bac Baculovirus Expression System. Lab. of Molecular Genetics Ryu Hyun-Mi. Bac-to-Bac Baculovirus Expression System. An efficient site-specific transposition system to generate baculovirus for high-level expression of recombinant proteins

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bac to bac baculovirus expression system

Bac-to-BacBaculovirus Expression System

Lab. of Molecular Genetics

Ryu Hyun-Mi

bac to bac baculovirus expression system2
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
bac to bac system
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, lacZα, kanamycin resistance marker

2. helper plasmid : transposase, tetracycline resistance marker

advantages
Advantages
  • Time-saving expression bacmid

-high titer virus is produced from the initial transfection.

  • Easy colony screening

-lacZα gene(blue/white screening white colony : recombinant)

  • Rapid purifacation of recombinant proteins

-pFastBacHT

pfastbacht
Strong polyhedrin promoter

N-terminal 6 X His tag( TEV protease cleavage site)

3 reading frame( pFastBacHTa, b, and c)

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