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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
Article

Abstract

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.

Keywords

Baculovirus Gene deletion Insect cells Recombinant protein expression Chitinase Cathepsin 

Abbreviations

AcMNPV

Autographa californica multi-nucleopolyhedrovirus

AP

alkaline phosphatase

BCIP

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

BEVS

baculovirus expression vector system

chiA

chitinase A

cath

cathepsin

dpi

days post infection

EGFP

enhanced green fluorescent protein

hpi

hours post infection

moi

multiplicity of infection

NBT

nitro blue tetrazolium

hr

homologous region

ODV

occlusion derived virus

PCR

polymerase chain reaction

pfu

plaque-forming units

PBS

phosphate-buffered saline

polh

polyhedrin

REN

restriction enzyme

RFU

relative fluorescent units

SDS-PAGE

SDS-polyacrylamide gel electrophoresis

Sf9

Spodoptera frugiperda 9

Notes

Acknowledgments

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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