Bac-to-Bac Baculovirus Expression System - PowerPoint PPT Presentation

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Bac-to-Bac Baculovirus Expression System

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  1. Bac-to-BacBaculovirus Expression System Lab. of Molecular Genetics Ryu Hyun-Mi

  2. 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

  3. 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

  4. Site-specific transposition

  5. Diagram of the Bac-to-Bac system

  6. 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

  7. Strong AcMNPV polyhedrin promoter Large MCS pFastBac1

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

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

  10. END

  11. 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