Protein Production Using the Baculovirus Expression System

Sarah L. Irons1, Adam C. Chambers2, Olga Lissina2, Linda A. King1, Robert D. Possee2

1 Department of Biological & Medical Sciences, Oxford Brookes University, Oxford, 2 Oxford Expression Technologies Ltd., Oxford
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.5
DOI:  10.1002/cpps.45
Online Posting Date:  February, 2018
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Abstract

Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed ‘BacMam’, with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus—all steps needed before commencing gene expression and protein production. We also cover the use of small‐scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale‐up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.

Keywords: BEVS; baculovirus; protein production; insect cells; BacMam

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Recombinant Baculovirus by Co‐Transfection of Insect Cells with Transfer Vector and Virus DNA (Homologous Recombination System)
  • Basic Protocol 2: Amplification of Recombinant Viruses
  • Support Protocol 1: Production of High Titer P2 or P3 Virus Inoculum
  • Basic Protocol 3: Titration of Virus Stocks by Plaque Assay
  • Basic Protocol 4: Initial Test of Protein Production in Recombinant Baculovirus‐Infected Insect Cells
  • Alternate Protocol 1: Analysis of Recombinant Protein Production in Virus‐Infected Suspension Cell Cultures
  • Basic Protocol 5: Scaling Up Protein Production in Virus‐Infected Insect Cells
  • Alternate Protocol 2: Recombinant Protein Production in Wave Bioreactor
  • Basic Protocol 6: Transduction of Mammalian Cells with BacMam Vectors
  • Basic Protocol 7: Protein Purification Considerations
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Recombinant Baculovirus by Co‐Transfection of Insect Cells with Transfer Vector and Virus DNA (Homologous Recombination System)

  Materials
  • Sf9 insect cells in serum‐free growth medium, 0.45 × 106 cells/ml, 2 ml per dish
  • Serum‐free insect cell culture medium (e.g., ESF 921, Expression Systems LLC, US, or OET Ltd, UK)
  • Purified baculovirus DNA (e.g., flashBAC™ DNA, OET Ltd), 100 ng per co‐transfection
  • Sterile baculovirus transfer vector DNA containing the gene of interest (GOI), 500 ng per co‐transfectionThe DNA must be sterile and of a quality suitable for transfection into cells.
  • lacZ control transfer vector DNA, 500 ng per co‐transfection.This vector is provided in flashBAC™ DNA kits and comprises the lacZ coding region under control the of the polh promoter. It serves as useful control, as transfected cells turn blue in the presence of X‐gal.
  • TC100 insect cell culture medium (Gibco®, Thermo Fisher Scientific)
  • BaculoFECTIN II transfection reagent (OET Ltd, UK), or similarIf a different reagent is used, refer to the manufacturer's instructions to modify to the protocol below.
  • 200 units/ml of penicillin and 200 μg/ml of streptomycin
  • 2% (w/v) X‐gal in dimethylformamide
  • 35‐mm tissue culture dishes
  • Incubator at 28°C (non‐CO 2)
  • Sterile pipets, pipet tips, and bijoux or other polystyrene containers mix transfection componentsDo not use microcentrifuge tubes made of polypropylene.
  • Plastic sandwich box
  • 1% Virkon (Amtec), or similar disinfectant
  • Inverted phase‐contrast microscope
  • Tissue culture hood

Basic Protocol 2: Amplification of Recombinant Viruses

  Materials
  • 50‐ to 100‐ml culture of healthy log‐phase Sf9 insect cells in appropriate serum‐free growth medium ( protocol 1)
  • P0 seed stock of recombinant virus ( protocol 1)
  • 1% Virkon, or similar disinfectant
  • Fetal bovine serum (FBS), for storage of P1 stock
  • Shaking culture flask of appropriate size to generate high surface area to volume ratio (e.g., 250‐ml disposable Erlenmeyer flask)
  • Incubator shaker (110 to 130 rpm) at ∼28˚C, or shaker platform in an incubator or warm room at 28°C
  • Inverted phase‐contrast microscope

Support Protocol 1: Production of High Titer P2 or P3 Virus Inoculum

  Additional Materials (also see protocol 2)
  • Sf9 cells in serum‐free medium
  • P1 or P2 virus stock of known titer (Basic Protocols protocol 22 and protocol 43)
  • Shaking flasks (e.g., 1‐ or 2‐liter disposable Erlenmeyer flasks)

Basic Protocol 3: Titration of Virus Stocks by Plaque Assay

  Materials
  • Sf21 cells in serum‐supplemented medium, in log‐phase (if unavailable, use Sf9 cells)
  • Serum‐supplemented medium (e.g., TC100 with 10% FCS)Note that batches of serum vary considerably, so each should be tested to ensure it supports Sf21 cell growth.We do not normally heat‐inactivate.
  • Virus stock to be titrated ( protocol 2 or protocol 3)
  • 1% Virkon
  • 2% (w/v) low‐gelling temperature (LGT) agarose (for cell culture; e.g., Lonza SeaPlaque) in sterile dH 2O, sterilized by autoclaving
  • Phosphate‐buffered saline (PBS, pH 6.2; e.g., Sigma), sterilized by autoclaving
  • 0.5% (w/v) Neutral Red stock solution (see recipe)
  • 35‐mm tissue culture dishes or 12‐well plates

Basic Protocol 4: Initial Test of Protein Production in Recombinant Baculovirus‐Infected Insect Cells

  Additional Materials (also see protocol 1)
  • Sf9 or Tni cells in log‐phase
  • Recombinant baculovirus stock ( protocol 2 or protocol 3)
  • StrataClean resin (Agilent Technologies)
  • Rocking platform
  • Boiling water bath
  • Tube rotator
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, ) and immunoblot analyses (unit 10.10; Ni, Xu, & Gallagher, )

Alternate Protocol 1: Analysis of Recombinant Protein Production in Virus‐Infected Suspension Cell Cultures

  Additional Materials (also see protocol 5)
  • Sf21 cells (1.5 × 106 cell/ml), Sf9 cells (2 × 106 cell/ml), or Tni cells (1 × 106 cell/ml) in appropriate medium (see protocol 1)
  • Recombinant baculovirus stock ( protocol 2 or protocol 3)
  • Shaker flasks (e.g., 125‐ml Erlenmeyer flasks)
  • Hemacytometer or automated cell counter and slides

Basic Protocol 5: Scaling Up Protein Production in Virus‐Infected Insect Cells

  Additional Materials (also see protocol 2 and protocol 3)
  • Sf9 cells (2 × 106 cell/ml) or Tni cells (1 × 106 cell/ml) in growth medium (see protocol 1)
  • Recombinant virus stock, high titer (108 pfu/ml or greater)
  • Protease inhibitor cocktail set III (Calbiochem), or similar
  • 3‐liter Fernbach flask with filter capThese flasks can be re‐used after washing and sterilization, but we recommend using a new cap each time.

Alternate Protocol 2: Recombinant Protein Production in Wave Bioreactor

  Additional Materials (also see protocol 7)
  • ReadyToProcess WaveTM25 bioreactor system (GE Healthcare), including a computer with the UNICORN™ system control software installed
  • 10‐liter Optical Cellbag (GE Healthcare)
  • Compressed gas cylinders (air, oxygen, and CO 2) and appropriate connective tubing (an air compressor can be used in place of a compressed air cylinder)
  • Peristaltic pump
  • ReadyMate connector
  • pH meter

Basic Protocol 6: Transduction of Mammalian Cells with BacMam Vectors

  Additional Materials (also see protocol 5)
  • Mammalian cell line of interest (e.g., HK‐2 or CHO cells)
  • Suitable cell culture medium, supplemented with serum as necessary
  • BacMam virus stock: recombinant baculovirus stock of known titer, with the gene of interest under control of a mammalian promoter (e.g., CMV)
  • 6‐well or 12‐well tissue culture plates
  • Humidified 37°C incubator with supply of 5% CO 2
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Figures

Videos

Literature Cited

  Bonning, B. C., Roelvink, P. W., Vlak, J. M., Possee, R. D., & 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. The Journal of General Virology, 73, 1551–1556. doi: 10.1099/0022‐1317‐75‐7‐1551.
  Boyce, F. M., & Bucher, N. L. (1996). Baculovirus‐mediated gene transfer into mammalian cells. Proceedings of the National Academy of Sciences of the United States of America, 93, 2348–2352. doi: 10.1073/pnas.93.6.2348.
  Gallagher, S. R. (2012). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Gröner, A., Granados, R. R., & Burand, J. P. (1984). Interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines. Intervirology, 21, 203–209. doi: 10.1159/000149522.
  Harrison, R. L., & Summers, M. D. (1995). Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production. The Journal of General Virology, 76, 1451–1459. doi: 10.1099/0022‐1317‐76‐6‐1451.
  Hink, W. F., Thomsen, D. R., Davidson, D. J., Meyer, A. L., & Castellino, F. J. (1991). Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines. Biotechnology Progress, 7, 9–14. doi: 10.1021/bp00007a002.
  Hitchman, R. B., Possee, R. D., Siaterli, E., Richards, K. S., Clayton, A. J., Bird, L. E., … King, L. A. (2010). Improved expression of secreted and membrane‐targeted proteins in insect cells. Biotechnology and Applied Biochemistry, 56(3), 85–93. doi: 10.1042/BA20090130.
  Hitchman, R. B., Possee, R. D., & King, L. A. (2012). High‐throughput baculovirus expression in insect cells. Methods in Molecular Biology (Clifton, N.J.), 824, 609–627. doi: 10.1007/978‐1‐61779‐433‐9_33.
  Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P., & Strauss, M. (1995). Efficient gene transfer into human hepatocytes by baculovirus vectors. Proceedings of the National Academy of Sciences of the United States of America, 92, 10099–10103. doi: 10.1073/pnas.92.22.10099.
  Ignoffo, C. M., & Rafajko, R. R. (1972). In vitro attempts to infect primate cells with the nucleopolyhedrosis virus of Heliothis. Journal of Invertebrate Pathology, 20, 321–325. doi: 10.1016/0022‐2011(72)90163‐2.
  Kadwell, S. H., & Overton, L. K. (2016). Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors. Methods in Molecular Biology (Clifton, N.J.), 1350, 263–284. doi: 10.1007/978‐1‐4939‐3043‐2_12.
  Kool, M., Voncken, J. W., van Lier, F. L. J., Tramper, J., & Vlak, J. M. (1991). Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. Virology, 183, 739–746. doi: 10.1016/0042‐6822(91)91003‐Y.
  McIntosh, A. H., & Shamy, R. (1980). Biological studies of a baculovirus in a mammalian cell line. Intervirology, 13, 331–341. doi: 10.1159/000149143.
  Miltenburger, H. G., & Reimann, R. (1980). Viral pesticides: Biohazard evaluation on the cytogenetic level. Developments in Biological Standardization, 46, 217–222.
  Murphy, C. I., & Piwnica‐Worms, H. (2001). Overview of the Baculovirus Expression System. Current Protocols in Protein Science, 00, 5.4.1–5.4.4. doi: 10.1002/0471140864.ps0504s00.
  Ni, D., Xu, P., & Gallagher, S. (2017). Immunoblotting and immunodetection. Current Protocols in Protein Science, 88, 10.10.1‐10.10.37. doi: 10.1002/cpps.32.
  Pennock, G. D., Shoemaker, C., & Miller, L. K. (1984). Strong and regulated expression of Escherichia coli beta‐galactosidase in insect cells with a baculovirus vector. Molecular and Cellular Biology, 4, 399–406. doi: 10.1128/MCB.4.3.399.
  Possee, R. D., & King, L. A. (2016). Baculovirus Transfer Vectors. Methods in Molecular Biology (Clifton, N.J.), 1350, 51–71. doi: 10.1007/978‐1‐4939‐3043‐2_3.
  Reimann, R., & Miltenburger, H. G. (1982). Cytogenetic studies in mammalian cells after treatment with insect pathogenic viruses [Baculoviridae]. I. In vivo studies with rodents. Entomophaga, 27, 267–276. doi: 10.1007/BF02374810.
  Roelvink, P. W., Van Meer, M. M. M., De Kort, C. A. D., Possee, R. D., Hammock, B. D., & Vlak, J. M. (1992). Dissimilar expression of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes. The Journal of General Virology, 73, 1481–1489. doi: 10.1099/0022‐1317‐73‐6‐1481.
  Schlaeger, E. J. (1996). Medium design for insect cell culture. Cytotech, 20, 57–70. doi: 10.1007/BF00350389.
  Shoji, I., Aizaki, H., Tani, H., Ishii, K., Chiba, T., Saito, I., … Matsuura, Y. (1997). Efficient gene transfer into various mammalian cells, including non‐hepatic cells, by baculovirus vectors. The Journal of General Virology, 78, 2657–2664. doi: 10.1099/0022‐1317‐78‐10‐2657.
  Singh, V. (1999). Disposable bioreactor for cell culture using wave‐induced agitation. Cytotech, 30, 149–158. doi: 10.1023/A:1008025016272.
  Smith, G. E., Summers, M. D., & Fraser, M. J. (1983). Production of human beta interferon in insect cells infected with a baculovirus expression vector. Molecular and Cellular Biology, 3, 2156–2165. doi: 10.1128/MCB.3.12.2156.
  Tjia, S. T., zu Altenschildesche, G. M., & Doerfler, W. (1983). Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells. Virology, 125, 107–117. doi: 10.1016/0042‐6822(83)90067‐3.
Key References
  King, L. A. & Possee, R. D. (1992). The baculovirus expression system. A laboratory guide. Chapman & Hall, London.
  A comprehensive manual that describes how to set up and use the baculovirus expression system.
Internet Resources
  https://oetltd.com/
  Information on FlashBAC system, supporting publications, reagent sales.
  https://oetltd.wordpress.com/
  A blog run by OET providing hints, tips and guidance (and some historical perspectives) on working with insect cells and protein expression.
  https://www.gelifesciences.com/
  Information on WAVE bioreactors.
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