Cell Biology and Toxicology

, Volume 26, Issue 1, pp 57–68 | Cite as

Genetic modification of a baculovirus vector for increased expression in insect cells

  • Richard B. HitchmanEmail author
  • Robert D. Possee
  • Andrew T. Crombie
  • Adam Chambers
  • Kim Ho
  • Evangelia Siaterli
  • Olga Lissina
  • Heather Sternard
  • Robert Novy
  • Kathryn Loomis
  • Louise E. Bird
  • Raymond J. Owens
  • Linda A. King


Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.


Baculovirus Gene deletion Insect cells Recombinant protein expression Chitinase Cathepsin 



Autographa californica multi-nucleopolyhedrovirus


alkaline phosphatase


5-bromo-4-chloro-3-indoyl phosphatase


baculovirus expression vector system


chitinase A




days post infection


enhanced green fluorescent protein


hours post infection


multiplicity of infection


nitro blue tetrazolium


homologous region


occlusion derived virus


polymerase chain reaction


plaque-forming units


phosphate-buffered saline




restriction enzyme


relative fluorescent units


SDS-polyacrylamide gel electrophoresis


Spodoptera frugiperda 9



The Oxford Protein Production Facility is supported by the MRC and BBSRC.


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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Richard B. Hitchman
    • 1
    Email author
  • Robert D. Possee
    • 2
  • Andrew T. Crombie
    • 1
    • 3
  • Adam Chambers
    • 1
    • 6
  • Kim Ho
    • 1
    • 6
  • Evangelia Siaterli
    • 1
  • Olga Lissina
    • 1
  • Heather Sternard
    • 4
  • Robert Novy
    • 4
  • Kathryn Loomis
    • 4
  • Louise E. Bird
    • 5
  • Raymond J. Owens
    • 5
  • Linda A. King
    • 6
  1. 1.Oxford Expression Technologies LtdOxford Brookes UniversityOxfordUK
  2. 2.Centre for Ecology & HydrologyOxfordUK
  3. 3.Department of Biological SciencesUniversity of WarwickCoventryUK
  4. 4.EMD Biosciences, Inc.MadisonUSA
  5. 5.Oxford Protein Production FacilityWellcome Trust Centre for Human GeneticsOxfordUK
  6. 6.School of Life SciencesOxford Brookes UniversityOxfordUK

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