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MultiBac: Multigene Baculovirus‐Based Eukaryotic Protein Complex Production

Christoph Bieniossek1,  Timothy J. Richmond1,  Imre Berger1

1Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology ETH, Zurich, Switzerland

Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 5.20
DOI: 
10.1002/0471140864.ps0520s51
Online Posting Date: 
February, 2008
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Abstract

Multiprotein complexes are an emerging focus in current biology, resulting in a demand for advanced heterologous expression systems. This unit provides protocols for the expression of eukaryotic multiprotein complexes using multigene expression vectors. Homologous and site‐specific recombinases facilitate their assembly. Thus, modification of individual subunits for revised expression studies is achieved with comparative ease. The strategy outlined here employs the MultiBac baculoviral expression system for multiprotein complexes as an example. Baculoviral expression does not require particular safety precautions due to the replication incompetence of baculovirus in mammalian hosts. The MultiBac system provides for improved protein production due to deletion of specific viral genes (V‐cath, chiA). Most of the steps described in this unit are tailored for high‐throughput approaches. The general strategy of rapidly combining encoding DNAs by recombination into multigene expression vectors for protein complex expression can also be applied to other prokaryotic or mammalian expression systems. Curr. Protoc. Protein Sci. 51:5.20.1‐5.20.26. © 2008 by John Wiley & Sons, Inc.

Keywords: protein complex; eukaryotic expression; baculovirus; multigene vector assembly; recombination; MultiBac system

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Design and Preparation of Target DNAs for Expression of a Defined Multiprotein Complex
  • Basic Protocol 2: In‐Fusion Cloning of Target DNAs into Transfer Vectors
  • Basic Protocol 3: Generating Multigene Expression Cassettes
  • Basic Protocol 4: Cre‐loxP‐Mediated or Tn7‐Dependent Integration of Transfer Vectors into the Multibac Baculoviral Genome
  • Basic Protocol 5: Initial Insect Cell Infection with Composite Bacmid and Virus Generation
  • Basic Protocol 6: Virus Amplification and Protein Expression
  • Support Protocol 1: Production of Electrocompetent DH10MultiBacCre Cells
  • Support Protocol 2: Maintenance of Sf21 Insect Cell Cultures
  • Monitoring Protein Expression
  • Support Protocol 3: Expression Analysis by SDS‐PAGE
  • Support Protocol 4: Expression Analysis by Using Co‐Expressed Fluorescent Marker
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Design and Preparation of Target DNAs for Expression of a Defined Multiprotein Complex

 Materials
  • MultiBac transfer vectors (Table 5.20.1)
  • Template DNA (genomic, plasmid, or cDNA)
  • PCR primers
  • BD In‐Fusion recombinase (BD Biosciences)
  • Restriction enzymes and buffers depending on cloning strategy (e.g., DpnI; NEB or Fermentas)
  • Phusion high‐fidelity DNA polymerase and 5× HF or 5× GC reaction buffer (Finnzymes or NEB)
  • 2.5 mM dNTPs
  • PCR purification kit (e.g., QIAprep, QIAGEN)
  • Gel extraction kit (e.g., MinElute, QIAGEN), optional
  • PCR thermal cycler (appendix 4J)
  • 37°C water bath
  • Additional reagents and equipment for agarose gel electrophoresis (appendix 4F)

Basic Protocol 2: In‐Fusion Cloning of Target DNAs into Transfer Vectors

 Materials
  • BD In‐Fusion recombinase and 10× reaction buffer (BD Bioscience)
  • 10× BSA
  • PCR‐amplified inserts (see Basic Protocol 1)
  • Linearized MultiBac vectors (see Basic Protocol 1)
  • E. coli competent cells (e.g., TOP10 or BW23474)
  • TYE agar plates (see recipe)
  • Antibiotics (Table 5.20.1; see recipe)
  • 37°C heating block (optional 42° or 50°C)
  • 37°C incubator
  • Minispin purification columns
  • Additional reagents and equipment for agarose gel electrophoresis (appendix 4F) and/or PCR thermal cycler (appendix 4J)

Basic Protocol 3: Generating Multigene Expression Cassettes

 Materials
  • Donor and acceptor plasmids (see Basic Protocol 2)
  • Cre recombinase and 10× reaction buffer (NEB)
  • Restriction enzymes and buffers (PmeI, AvrII, BstZ17I, SpeI and/or NruI; NEB or Fermentas)
  • T4 DNA ligase and 10× T4 DNA ligase buffer (NEB)
  • Competent E. coli cells (e.g., TOP10 or BW23474)
  • 2× TY medium (see recipe)
  • TYE agar plates (see recipe)
  • Antibiotics (Table 5.20.1; see recipe)
  • Additional reagents and equipment for agarose gel electrophoresis (appendix 4F)

Basic Protocol 4: Cre‐loxP‐Mediated or Tn7‐Dependent Integration of Transfer Vectors into the Multibac Baculoviral Genome

 Materials
  • MultiBac transfer vectors with multigene expression cassettes (see Basic Protocol 3)
  • Electrocompetent DH10MultiBac cells (no Cre expressed) or electrocompetent DH10MultiBacCre cells (containing Cre)
  • E. coli competent cells (e.g., TOP10 (pir−) from Invitrogen, Open Biosystems or BW23474 (pir+) from Novagen, Baylor College)
  • 2× TY medium (see recipe)
  • TYE agar plates (see recipe)
  • Antibiotics (Table 5.20.1; see recipe)
  • 1 M isopropylthiogalactoside (IPTG)
  • 5‐Bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside (X‐gal)
  • Electroporator and cuvettes
  • 37°C incubator
  • Additional reagents and equipment for agarose gel electrophoresis (appendix 4F)

Basic Protocol 5: Initial Insect Cell Infection with Composite Bacmid and Virus Generation

 Materials
  • Colonies containing MultiBac bacmid of interest (see Basic Protocol 4)
  • 2× TY medium (see recipe)
  • Antibiotics (Table 5.20.2; see recipe)
  • Spodoptea frugiperda Sf21 cells (Support Protocol 2)
  • Sf‐900 II serum‐free medium (Sf‐900 II SFM; Invitrogen)
  • FuGENE (Roche)
  • 37°C shaker incubator
  • Sterile hood with UV illumination
  • 6‐well (35‐mm diameter) tissue culture plates
  • 27°C incubator
  • 1.5‐ml microcentrifuge tubes
  • Parafilm
  • 15‐ml sterile tubes (e.g., Falcon)
     
    Table 5.20.2 Steps for Bacmid Preparation and MultiBac Derivatives Involved
    StepVectorsAntibioticHost strain
    Inserting genes into vectors
    pFLAmpicillinTOP10
    BD In‐FusionpKLKanamycinTOP10
    pUCDMChloramphenicolBW23473
    pSPLSpectinomycinBW23473
    Multigene cassette generation
    Derivatives of:
    Double fusion,pFL and pUCDMAmpicillin, chloramphenicolTOP10
    Cre‐mediatedpKL and pUCDMKanamycin, chloramphenicolTOP10
    pFL and pSPLAmpicillin, spectinomycinTOP10
    pKL and pSPLKanamycin, spectinomycinTOP10
    Derivatives of:
    Triple fusion,pFL, pUCDM and pSPLAmpicillin, chloramphenicol,TOP10
    Cre‐mediatedpKL, pUCDM and pSPLSpectinomycinTOP10
    Kanamycin, chloramphenicol
    Derivatives of:
    MultiplicationpFLAmpicillinTOP10
    pKLKanamycinTOP10
    pUCDMChloramphenicolBW23473
    pSPLSpectinomycinBW23473
    Composite MultiBac bacmid generation
    loxPMultiBac bacmid and derivatives of pUCDMAmpicillin, kanamycin, tetracycline, chloramphenicolDH10MultiBacCre
    integrationMultiBac bacmid and derivatives of pUCDMAmpicillin, kanamycin, tetracycline, spectinomycinDH10MultiBacCre
    Tn7MultiBac bacmida and derivativesb of pFLAmpicillin, gentamycin, tetracyclineDH10MultiBac
    transpositionMultiBac bacmida and derivativesb of pKLKanamycin, gentamycin, tetracyclineDH10MultiBac
     aBacmids with or without donor derivatives integrated in loxP site.
     bDouble and triple acceptor‐donor fusions included.

Basic Protocol 6: Virus Amplification and Protein Expression

 Materials
  • V0 (or V1) virus of interest (see Basic Protocol 5)
  • Spodoptea frugiperda Sf21 cells (Support Protocol 2)
  • Sf‐900 II serum‐free medium (Sf‐900 II SFM; Invitrogen)
  • Liquid nitrogen
  • 27°C temperature‐controlled, shaker incubator
  • 500‐ml and 2‐liter Erlenmeyer flasks dedicated for insect cell use only
  • 15‐ and 50‐ml sterile tubes (e.g., Falcon)
  • Benchtop centrifuge (Hettich or equivalent)
  • Hemacytometer
  • Light microscope

Support Protocol 1: Production of Electrocompetent DH10MultiBacCre Cells

 Materials
  • pBADZHisCre plasmid (Table 5.20.1)
  • DH10MultiBac cells (Table 5.20.1)
  • 2× TY medium (see recipe)
  • Low‐salt TYE agar plates (see recipe)
  • Antibiotics (Table 5.20.1; see recipe)
  • 1 M isopropylthiogalactoside (IPTG)
  • 5‐Bromo‐4‐chloro‐indolyl‐β‐d‐galactopyranoside (X‐gal)
  • Low‐salt TYE medium (see recipe)
  • l‐Arabinose
  • 10% glycerol solution, sterile and ice cold
  • Liquid nitrogen
  • Electroporator and cuvettes
  • 37°C incubator
  • Spectrophotometer
  • 1.5‐ml microcentrifuge

Support Protocol 2: Maintenance of Sf21 Insect Cell Cultures

 Materials
  • S. frugiperda Sf21 cells (Invitrogen, Novagen)
  • Sf‐900 II serum‐free medium (Sf‐900 II SFM; Invitrogen)
  • DMSO
  • Liquid nitrogen
  • Shaker incubator (temperature‐controlled, 27°C)
  • 50‐ml, 500‐ml, and 2‐liter Erlenmeyer flasks dedicated for insect cell–use only
  • Hemacytometer
  • Light microscope
  • 4.5‐ml cryotube vials (Nunc or equivalent)
  • 37°C water bath
  • Sterile hood with UV illumination

NOTE: It is recommended to start a fresh cell culture approximately every 2 to 3 months.

Support Protocol 3: Expression Analysis by SDS‐PAGE

 Materials
  • Expression culture
  • Lysis buffer (e.g., PBS, see recipe)
  • SDS gel loading dye (see recipe)
  • Microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • Sonicator
  • 95°C heating block
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and staining (unit 10.5)

Support Protocol 4: Expression Analysis by Using Co‐Expressed Fluorescent Marker

 Additional Materials (also see Support Protocol 3)
  • Fluorescence spectrophotometer and cuvettes
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Figures

  • Figure 5.20.1
    MultiBac transfer vectors and multigene assembly. (A) MultiBac transfer vectors are called acceptor or donor. All vectors contain identical and exchangeable dual expression cassettes with polylinkers I1, I2 for integration of encoding DNA, a multiplication module M, and a loxP imperfect inverted repeat. Acceptors (pFL, pKL) contain the Tn7 transposition sequences (Tn7L, Tn7R), standard replicons and a gentamycin resistance marker. Acceptor pKL is a low to medium copy number vector due to the pBR322‐derived origin and contains a kanamycin resistance marker. pFL is a high copy number (ColE1 origin) vector and confers ampicillin resistance (Table 5.20.1). Donors (pUCDM, pSPL) contain conditional replicons derived from R6Kγ and confer chloramphenicol (pUCDM) or spectinomycin (pSPL) resistance (Table 5.20.1). (B) Multigene transfer vector assembly is carried out by inserting genes into I1 and/or I2 by In‐Fusion recombination or restriction digestion followed by ligation. The resulting dual expression cassette (identical in all vectors) is excised by digestion with PmeI and AvrII (sites must be unique) and inserted into a second transfer vector digested with BstZ17I and SpeI. Ligation yields a multigene transfer vector. The process is iterative. (C) Alternatively, acceptor and donor derivatives can be fused by in vitro Cre‐loxP reaction to yield plasmid dimers. Likewise, acceptors can be fused with two donors to yield acceptor‐donor‐donor trimers (not shown).

    View Image
  • Figure 5.20.2
    Composite bacmid generation. Composite MultiBac bacmid is generated by in vivo Cre‐loxP fusion of donor derivatives directly into the MultiBac baculoviral genome in DH10MultiBacCre cells. Acceptor derivatives or acceptor‐donor fusions are integrated by Tn7 transposition in DH10MultiBac cells harboring either the original MultiBac baculoviral genome or a composite bacmid with a donor integrated into the loxP site present on the bacmid.

    View Image
  • Figure 5.20.3
    Co‐expression of fluorescence marker. (A) Expression of fluorescent protein eYFP integrated via donor fusion into the MultiBac baculoviral genome can be used as a tool to monitor heterologous expression. eYFP is under control of a late viral promoter, polh, typically used for heterologous expression by baculovirus. (B) The fluorescence emission spectrum of the expressed protein is shown at 488 nm excitation. Typical progression of eYFP expression is shown in the inset bar graph (dpa: days after cell proliferation arrest). Co‐expression of eYFP does not noticeably reduce the yield of heterologous protein product (Berger et al., 2004). Fluorescence measurement of eYFP can be conveniently used to determine optimal time points for harvesting cells when protein production is maximal.

    View Image

Literature Cited

Literature Cited
    Belyaev, A.S. and Roy, P. 1993. Development of baculovirus 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. Nucleic Acids Res. 21:1219‐1223.
    Benoit, R.M., Wilhelm, R.N., Scherer‐Becker, D., and Ostermeier, C. 2006. An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Expr. Pur. 45:66‐71.
    Berger, I., Fitzgerald, D.J., and Richmond, T.J. 2004. Baculovirus expression system for heterologous multiprotein complexes. Nature Biotech. 22:1583‐1587.
    Bertolotti‐Ciarlet, A., Ciarlet, M., Crawford, S.E., Conner, M.F., and Estes, M.K. 2003. Immunogenicity and protective efficacy of rotavirus 2/6 virus‐like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice. Vaccine 21:3885‐3900.
    Fitzgerald, D.J., Berger, P., Schaffitzel, C., Yamada, K., Richmond, T.J., and Berger, I. 2006. Protein complex expression by using multigene baculoviral vectors. Nature Methods 3:1021‐1032.
    Fitzgerald, D.J., Schaffitzel, C., Berger, P., Wellinger, R., Bieniossek, C., Richmond, T.J., and Berger, I. 2007. Multiprotein expression strategy for structural biology of eukaryotic complexes. Structure 15:275‐279.
    Giot, L., Bader, J.S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y.L., Ooi, C.E., Godwin, B., Vitols, E., Vijayadamodar, G., Pochart, P., Machineni, H., Welsh, M., Kong, Y., Zerhusen, B., Malcolm, R., Varrone, Z., Collis, A., Minto, M., Burgess, S., McDaniel, L., Stimpson, E., Spriggs, F., Williams, J., Neurath, K., Ioime, N., Agee, M., Voss, E., Furtak, K., Renzulli, R., Aanensen, N., Carrolla, S., Bickelhaupt, E., Lazovatsky, Y., DaSilva, A., Zhong, J., Stanyon, C.A., Finley, R.L. Jr., White, K.P., Brayerman, M., Jarvie, T., Gold, S., Leach, M., Knight, J., Shimkets, R.A., McKenna, M.P., Chant, J., and Rothberg, J.M. 2003. A protein interaction map of Drosophila melanogaster. Science 302:1727‐1736.
    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.
    Jarvis, D.L. and Aurelio, G. 1994. Long‐term stability of baculoviruses stored under various conditions. BioTechniques 16:508‐513.
    King, L.A. and Possee, R.D. 1992. The Baculovirus Expression System. A Laboratory Guide. Chapman & Hall, London.
    Li, M.Z. and Elledge, S.J. 2007. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods 4:251‐256.
    Luckow, V.A. and Summers, M.D. 1988. Trends in the development of baculovirus expression vectors. Biotechnology 6:47‐55.
    Luckow, V.A., Lee, S.C., Barry, G.F., and Olins, P.O. 1993. Efficient generation of infectious recombinant baculoviruses by site‐specific transposon‐mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67:4566‐4579.
    Miller, L.K. 1988. Baculoviruses as gene expression vectors. Annu. Rev. Microbiol. 42:177‐199.
    Murphy, C.I. and Piwnica‐Worms, H. 1994a. Preparation of insect cell cultures and baculovirus stocks. Curr. Protoc. Mol. Biol. 65:16.10.1‐16.10.8.
    Murphy, C.I. and Piwnica‐Worms, H. 1994b. Generation of recombinant baculoviruses and analysis of recombinant protein expression. Curr. Protoc. Mol. Biol. 65:16.11.1‐16.11.19.
    O'Reilly, D.R., Miller, L.K., and Luckov, V.A. 1994. eds. Baculovirus Expression Vectors. A Laboratory Manual. Oxford University Press, New York.
    Parrish, J.R., Gulyas, K.D., and Finley, R.L. 2006. Yeast two‐hybrid contributions to interactome mapping. Curr. Opin. Biotechnol. 17:387‐393.
    Patterson, G., Day, R.N., and Piston, D. 2001. Fluorescent protein spectra. J. Cell Sci. 114:837‐838.
    Pijlman, G.B., de Vrij, J., van den End, F.J., Vlak, J.M., and Martens, D.E. 2004. Evaluation of baculovirus expression vectors with enhanced stability in continuous cascade insect‐cell bioreactors. Biotechnol. Bioeng. 87:743‐753.
    Rual, J.F., Venkatesan, K., Hao, T., Hirozane‐Kishikawa, T., Dricot, A., Li, N., Berriz, G.F., Gibbons, F.D., Dreze, M., Ayivi‐Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M., Milstein, S., Rosenberg, J., Goldberg, D.S., Zhang, L.V., Wong, S.L., Franklin, G., Li, S., Albala, J.S., Lim, J., Fraughton, C., Llamosas, E., Cevic, S., Bex, C., Lamesch, P., Sikorski, R.S., Vandenhaute, J., Zoghbi, H.Y., Smolyar, A., Bosak, S., Sequerra, R., Doucette‐Stamm, L., Cusick, M.E., Hill, D.E., Roth, F.P., and Vidal, M. 2005. Towards a proteome‐scale map of the human protein‐protein interaction network. Nature 437:1173‐1178.
    Simon, O., Williams, T., Caballero, P., and Lopéz‐Ferber, M. 2006. Dynamics of deletion genotypes in an experimental insect virus population. Proc. Biol. Sci. 273:783‐790.
    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.
    Tan, S., Kern, R.C., and Selleck, W. 2005. The pST44 polycistronic expression system for producing protein complexes in E. coli. Protein Expr. Pur. 40:385‐395.
    Tolia, N.H. and Joshua‐Tor, L. 2006. Strategies for protein co‐expression in Escherichia coli. Nat. Methods 3:385‐395.
    Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi‐Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J.M. 2000. A comprehensive analysis of protein‐protein interactions in Saccharomyces cerevisiae. Nature 403:623‐627.
 Key References
    Berger et al., 2004. See above.
    Fitzgerald et al., 2006, 2007. See above.
    Roy, P. 2004. Baculovirus solves a complex problem. Nat. Biotechnol. 22:1527‐1528.

These papers illustrate the MultiBac technology, including expression of several heterologous protein complexes.

     
 
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