MultiBac: Multigene Baculovirus‐Based Eukaryotic Protein Complex Production

Christoph Bieniossek1, Timothy J. Richmond1, Imre Berger1

1 Institute 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 protocol 1)
  • Linearized MultiBac vectors (see 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 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 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 protocol 4)
  • 2× TY medium (see recipe)
  • Antibiotics (Table 5.20.2; see recipe)
  • Spodoptea frugiperda Sf21 cells ( protocol 8)
  • 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 5.0.2   MaterialsSteps for Bacmid Preparation and MultiBac Derivatives Involved

    Step Vectors Antibiotic Host strain
    Inserting genes into vectors
    pFL Ampicillin TOP10
    BD In‐Fusion pKL Kanamycin TOP10
    pUCDM Chloramphenicol BW23473
    pSPL Spectinomycin BW23473
    Multigene cassette generation
    Derivatives of:
    Double fusion, pFL and pUCDM Ampicillin, chloramphenicol TOP10
    Cre‐mediated pKL and pUCDM Kanamycin, chloramphenicol TOP10
    pFL and pSPL Ampicillin, spectinomycin TOP10
    pKL and pSPL Kanamycin, spectinomycin TOP10
    Derivatives of:
    Triple fusion, pFL, pUCDM and pSPL Ampicillin, chloramphenicol, TOP10
    Cre‐mediated pKL, pUCDM and pSPL Spectinomycin TOP10
    Kanamycin, chloramphenicol
    Derivatives of:
    Multiplication pFL Ampicillin TOP10
    pKL Kanamycin TOP10
    pUCDM Chloramphenicol BW23473
    pSPL Spectinomycin BW23473
    Composite MultiBac bacmid generation
    loxP MultiBac bacmid and derivatives of pUCDM Ampicillin, kanamycin, tetracycline, chloramphenicol DH10MultiBacCre
    integration MultiBac bacmid and derivatives of pUCDM Ampicillin, kanamycin, tetracycline, spectinomycin DH10MultiBacCre
    Tn7 MultiBac bacmid d and derivatives e of pFL Ampicillin, gentamycin, tetracycline DH10MultiBac
    transposition MultiBac bacmid d and derivatives e of pKL Kanamycin, gentamycin, tetracycline DH10MultiBac

     dBacmids with or without donor derivatives integrated in loxP site.
     eDouble and triple acceptor‐donor fusions included.

Basic Protocol 6: Virus Amplification and Protein Expression

  Materials
  • V 0 (or V 1) virus of interest (see protocol 5)
  • Spodoptea frugiperda Sf21 cells ( protocol 8)
  • 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

  • Fluorescence spectrophotometer and cuvettes
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Figures

Videos

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.
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   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.
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   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.
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   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.
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Key References
  Berger et al., 2004. See above.
  These papers illustrate the MultiBac technology, including expression of several heterologous protein complexes.
  Fitzgerald et al., 2006, 2007. See above.
   Roy, P. 2004. Baculovirus solves a complex problem. Nat. Biotechnol. 22:1527‐1528.
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