Protein Expression in the Baculovirus System

Alain Bernard1, Mark Payton1, Kathryn R. Radford1

1 Glaxo Institute for Molecular Biology, Geneva
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.5
DOI:  10.1002/0471140864.ps0505s00
Online Posting Date:  May, 2001
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Abstract

Insect cell‐recombinant baculovirus co‐cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post‐translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large‐scale production of recombinant proteins in the baculovirus expression system. A method for large‐scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end‐point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small‐scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS‐PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed‐batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.

     
 
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Table of Contents

  • Basic Protocol 1: Large‐Scale Production of Viral Stock
  • Basic Protocol 2: Determination of Expression Kinetics
  • Basic Protocol 3: Production in Bioreactors
  • Alternate Protocol 1: Production in Perfusion Cultures
  • Support Protocol 1: Titrating Viral Stocks with the Plaque Assay
  • Support Protocol 2: Titrating Viral Stocks with the End‐Point Assay
  • Support Protocol 3: Harvesting
  • Support Protocol 4: Preparing Samples for Protein Analysis
  • Support Protocol 5: Monitoring Nutrient Levels in Culture Media
  • Support Protocol 6: Determination of Cell Growth and Viability
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Large‐Scale Production of Viral Stock

  Materials
    Spodoptera frugiperda (Sf9) cells
  • Insect cell medium: TC100 (GIBCO/BRL) or IPL41 (GIBCO/BRL), with 5% to 10% (w/v) fetal calf serum (FCS); or Sf‐900 II (GIBCO/BRL), serum free
  • Pure recombinant virus (at 107 pfu/ml)
  • 1‐liter to 10‐liter capacity shaker flasks or spinner culture flasks
  • 27°C shaker incubators, humidity controlled
  • Low‐speed centrifuge (Sorvall RC3B or equivalent)
  • 1‐liter glass conical‐bottom centrifuge bottles (Corning or Bellco), sterile
  • Additional reagents and equipment for monitoring cell growth by cell counting and determining cell viability with trypan blue (see protocol 10), monitoring nutrient levels in culture media (see protocol 9), and titration of virus (see protocol 5Support Protocols 1 and protocol 62)

Basic Protocol 2: Determination of Expression Kinetics

  Materials
    Cell line: Spodoptera frugiperda (Sf9, Sf21; ATCC, Pharminogen, Invitrogen) orTrichoplusia ni (Tn5 line; High Five cell line, Invitrogen)
  • Insect cell medium: TC100 (GIBCO/BRL) or IPL41 (GIBCO/BRL), serum free or with 5% to 10% (w/v) fetal calf serum (FCS); or Ex‐Cell 401 or Ex‐Cell 405 (JRH Biosciences) or Sf‐900 II (GIBCO/BRL), serum free
  • Recombinant viral stock (see protocol 1)
  • 1.5‐ to 3‐liter shaker flasks or spinner culture flasks
  • 27°C incubator, humidity controlled
  • Additional reagents and equipment for monitoring cell growth by cell counting and determining cell viability with trypan blue (see protocol 10), monitoring nutrient levels in culture media (see protocol 9), and preparing samples for protein analysis (see protocol 8)

Basic Protocol 3: Production in Bioreactors

  Materials
  • Insect cell medium: TC100 (GIBCO/BRL) or IPL41 (GIBCO/BRL), serum free or with 5% to 10% (w/v) fetal calf serum (FCS); or Ex‐Cell 401 (JRH Biosciences) or Sf‐900 II (GIBCO/BRL), serum free
  • Inoculum (15% to 20% bioreactor culture volume): spinner or shaker flask culture of uninfected insect cells and in full exponential growth phase
  • Recombinant viral stock (see protocol 1)
  • Bioreactor fitted with temperature, pH, agitation, air flow, and pO 2 control (see Table 97.80.4711 and Fig. )
  • Millipak filter, sterile (Millipore)
  • Viral stock and inoculum transfer vessel fitted with transfer line (see Fig. )
  • Peristaltic pump (optional)
  • Additional reagents and equipment for checking sterility and monitoring cell growth and determining cell viability with trypan blue (see protocol 10)

Alternate Protocol 1: Production in Perfusion Cultures

  • Cell retention device: spin filter, internal membrane filter, external membrane filter, or external centrifuge
  • Fermentor level controller (contact probe)
  • Medium feed and filtrate polypropylene holding tanks (10 to 15 times reactor volume; Nalgene)
  • Load cell or electronic balance for feed tank (Mettler or equivalent)
  • Peristaltic pumps (Watston‐Marlow or equivalent)
  • Additional reagents and equipment for determining cell viability with trypan blue (see protocol 10), and preparing samples for protein analysis (see protocol 8)

Support Protocol 1: Titrating Viral Stocks with the Plaque Assay

  Materials
  • Sf9 cells (30 ml, 8 × 105 cells/ml)
  • TC100 medium (GIBCO/BRL), with and without 5% (w/v) fetal calf serum (FCS)
  • Test sample: viral stock or culture supernatant
  • 3% (w/v) low gelling/melting temperature agarose, autoclaved
  • 2× TC100 medium/10% (w/v) FCS
  • 0.3% (w/v) neutral red, diluted 1:20 in 1× phosphate‐buffered saline (PBS, appendix 2E; filter sterilize and store in the dark)
  • 6‐well cluster dishes, sterile
  • Pipet tips, sterile
  • Tubes, sterile
  • Humid box

Support Protocol 2: Titrating Viral Stocks with the End‐Point Assay

  Materials
  • Sf9 cells (1 × 106 cells/ml), grown in Sf‐900 II medium (GIBCO/BRL)
  • Test sample: viral stock or culture supernatant
  • Chromogenic substrate for detecting recombinant protein (optional, if available; e.g., Xgal for β‐galactose)
  • 96‐well microtiter plate, sterile
  • Multitip pipettor
  • Pipet tips, sterile
  • Humid box

Support Protocol 3: Harvesting

  Materials
  • Protease inhibitor mix (see recipe)
  • 1‐liter conical‐bottom centrifuge bottles (Bellco), sterile
  • Low‐speed centrifuge (Sorvall RC3B or equivalent), continuous feed centrifuge (Heareus Contifuge or equivalent), or tangential flow microfiltration system (Microgon KrosFloII or equivalent; 0.04 m2/liter culture)

Support Protocol 4: Preparing Samples for Protein Analysis

  Materials
  • Test sample
  • SDS sample buffer (unit 10.1) or detergent lysis buffer (see recipe)
  • Low‐speed centrifuge

Support Protocol 5: Monitoring Nutrient Levels in Culture Media

  Materials
  • Fresh cell culture sample
  • 0.2% (w/v) trypan blue solution (Sigma)
  • Ethanol
  • Hemacytometer
  • Inverted microscope (×10 objective, phase contrast)
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Figures

Videos

Literature Cited

   Belyaev, A.S. and Roy, P. 1993. Development of baculo virus triple and quadruple expression vectors: Co‐expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus‐like particles in insect cells. Nucl. Acids Res. 21:1219‐1223.
   Bonning, B.C., Roelvink, P.W., Vlak, J.M., Possee, R.D., and Hammock, B.D. 1994. Superior expression of juvenile‐hormone‐esterase and beta‐galactosidase from the basic protein promoter of Autographa californica nuclear‐polyhedrosis virus compared to the p10 protein and polyhedrin promoters J. Gen. Virol. 73:1551‐1556.
   Chazenbalk, G.D. and Rapoport, B. 1995. Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins. J. Biol. Chem. 270:1539‐1543.
   Deramoudt, F.X., Monnet, S., Rabaud, J.N., Quiot, J.M., Cerutti, M., Devauchelle, G., and Kaczorek, M. 1994. Production of a recombinant protein in a high density insect cell cytoflow reactor. In Animal Cell Technology: Products of Today, Prospects for Tomorrow. (R.E. Spier, J.B. Griffiths, and W. Berthold, ed.) pp 222‐226. Butterworth.
   Doyle, A., Griffiths, J.B., and Newell, D.G. 1994. Biochemistry of cells in culture. In Cell & Tissue Culture: Laboratory Procedures. John Wiley & Sons, Chichester, UK.
   Hink, W.F., Thomsen, D.R., Davidson, D.J., Meyer, A.L., and Castellino, F.J. 1991. Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines. Biotechnol. Prog. 7:9‐14.
   Hu, W.S. and Wang, D.I.C. 1986. Mammalian cell culture technology: A review from an engineering perspective. In Mammalian Cell Technology (W.G. Thilly, ed.) pp. 167‐199. Butterworths, London.
   Jäger, V., Grabenhorst, E., Kobold, A., Deutschmann, S.M., and Conradt, H.S. 1992. High density perfusion culture of insect cells for the production of recombinant glycoproteins. In Baculovirus and Recombinant Protein Production Processes (J.M. Vlak, E.J. Schlager, and A.R. Bernard, eds.) pp. 274‐284. Roche Editions, Basel.
   Jarvis, D.L. and Aurelio, G., Jr. 1994. Long‐term stability of baculoviruses stored under various conditions. BioTechniques 16:508‐513.
   Kool, M., Voncken, J.W., van Lier, F.L.J., Tramper, J., and Vlak, J.M. 1991. Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. Virology 183:739‐746.
   Luckow, V.A. and Summers, M.D. 1988. Trends in the development of baculovirus expression vectors. Bio/Technology 6:47‐55.
   Maiorella, B., Dorin, G., Carion, A., and Harano, D. 1991. Crossflow microfiltration of animal cells. Biotechnol. Bioeng. 37:121‐126.
   McCutchen, B.F., Choudary, P.V., Crenshaw, R., Maddox, D., Hammock, B.D., and Maeda, S. 1991. Development of a recombinant baculo virus expressing an insect‐selective neurotoxin: Potential for pest control. Bio/Technology 9:848‐852.
   McGlynn, E., Becker, M., Mett, H., Reutener, S., Cozens, R., and Lydon, N.B. 1992. Large‐scale purification and characterisation of a recombinant epidermal growth‐factor receptor protein‐tyrosine kinase. Modulation of activity by multiple factors. Eur. J. Biochem. 207:265‐275.
   Miller, L.K. 1988. Baculoviruses as gene expression vectors. Annu. Rev. Microbiol. 42:177‐199.
   Murhammer, D.W. and Goochee, C.F. 1988. Scaleup of insect cell cultures: Protective effects of Pluronic F68. Bio/Technology 6:1411‐1418.
   Murphy, C.I. and Piwnica‐Worms, H. 1994a. Preparation of insect cell cultures and baculovirus stocks. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.10.1‐16.10.8. John Wiley & Sons, New York.
   Murphy, C.I. and Piwnica‐Worms, H. 1994b. Generation of recombinant baculoviruses and analysis of recombinant protein expression. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.11.1‐16.11.19. John Wiley & Sons, New York.
   Ozturk, S.S., Meyerhoff, M.E., and Palsson, B.O. 1989. Measurement of ammonia and glutamine in cell culture media by gas sensing electrodes. Biotechnol. Tech. 3:217‐222.
   Prehaud, C. Harris, R.D., Fulop, V., Koh, C.L., Wong, J., Flamand, A., and Bishop, D.H. 1990. Expression, characterization, and purification of a phosphorylated rabies nucleoprotein synthesized in insect cells by baculovirus vectors. Virology 178:486‐497.
   Rankl, N.B., Rice, J.W., Gurganus, T.M., Barbee, J.L., and Burns, D.J. 1994. The production of an active protein kinase C‐delta in insect cells is greatly enhanced by the use of the basic protein promoter. Protein Expression Purif. 5:346‐356.
   Rice, J.W., Rankl, N.B., Gurganus, T.M., Marr, C.M., Barna, J.B., Waters, M.M., and Bruns, D.J. 1993. A comparison of large‐scale Sf9 insect cell growth and protein production: Stirred vessel vs. airlift. BioTechniques 15:1052‐1059.
   Roy, P. 1990. Use of baculovirus expression vectors: Development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus. FEMS Microbiol. Immunol. 2:223‐234.
   Sridhar, P. and Hasnain, S.E. 1993. Differential secretion and glycosylation of recombinant human chorionic gonadotropin (beta‐HCG) synthesized using different promoters in the baculo virus expression vector system. Gene 131:261‐264.
   Sridhar, P., Panda, A.K., Pal, R., Talwar, G.P., and Hasnain, S.E. 1993. Temporal nature of the promoter and not relative strength determines the expression of an extensively processed protein in a baculovirus system. FEBS Lett. 315:282‐286.
   Stewart, L.M.D., Hirst, M., Lopez‐Ferber, M., Merryweather, A.T., Cayley, P.J., and Possee, R.D. 1991. Construction of an improved baculo virus insecticide containing an insect‐specific toxin gene. Nature 352:85‐88.
   Summers, M.D. and Smith, G.E. 1987. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experimental Station Bulletin No. 1555. College Station, Texas.
   Tokashiki, M. and Takamatsu, H. 1993. Perfusion culture apparatus for suspended mammalian cells. Cytotechnology 13:149‐159.
   van‐Lier, F.L.J., van‐Duijnhoven, G.C.F., de‐Vaan, M.M.J.A.C.M., Vlak, J.M., and Tramper, J. 1994. Continuous beta‐galactosidase production in insect cells with a p10 gene based baculo virus vector in a two‐stage bioreactor system. Biotechnol. Prog. 10:60‐64.
   Wang, M.Y., Kwong, S., and Bentley, W.E. 1993. Effects of oxygen/glucose/glutamine feeding on insect cell baculo virus protein expression: A study on epoxide‐hydrolase production. Biotechnol. Prog. 9:355‐361.
   Wickham, T.J., Davis, T., Granados, R.R., Hammer, D.A., Shuler, M.L., and Wood, H.A. 1991. Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection. Biotechnol. Lett. 13:483‐488.
Key References
   Summers and Smith, 1987. See above.
  These manuals give excellent background information on the insect cell and baculovirus biology, expression vectors and strategies for optimization. Includes very practical aspects of many elementary but specialized procedures.
   O'Reilly, D., Miller, L.K., and Luckow, V.A. 1994. Baculovirus expression vectors: A laboratory manual. Oxford University Press, New York.
   King, L.A. and Possee, R.D. 1992. The baculovirus expression system. A laboratory guide. Chapman & Hall, London.
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