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Expression and Purification of Recombinant Proteins Using the Baculovirus System

Cheryl Isaac Murphy1,  Helen Piwnica‐Worms2,  Stefan Grünwald3,  William G. Romanow4,  Nicole Francis5,  Hua‐Ying Fan5

1Aquila Biopharmaceuticals, Worcester, Massachusetts
2Washington University School of Medicine, St. Louis, Missouri
3Pharmingen, San Diego, California
4Corning Costar, Portsmouth, New Hampshire
5Massachusetts General Hospital, Boston, Massachusetts





Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 16.11
DOI: 
10.1002/0471142727.mb1611s65
Online Posting Date: 
February, 2004
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Abstract

This unit describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production , how to maximize and scale up recombinant protein production, and how to purify recombinant proteins.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Small-Scale Expression for Initial Analysis
  • Support Protocol 1: Determining Time Course of Maximum Protein Production
  • Support Protocol 2: Metabolic Labeling of Recombinant Proteins
  • Basic Protocol 2: Large-Scale Production of Recombinant Proteins
  • Alternate Protocol 1: Co-Infection with Multiple Baculoviruses for Purification of Recombinant Protein Complexes
  • Basic Protocol 3: Purification of Recombinant Proteins Containing a Polyhistidine (6×His) Tag
  • Alternate Protocol 2: Purification of Recombinant Proteins Containing a GST Tag
  • Alternate Protocol 3: Purification of Recombinant Proteins Containing a FLAG Tag
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Small-Scale Expression for Initial Analysis

 Materials
  • Spodoptera frugiperda (Sf9) cells (UNIT 16.10)
  • TNM-FH insect medium (see recipe in UNIT 16.10) with or without 10% fetal bovine serum (FBS)
  • High-titer recombinant baculovirus stocks (UNIT 16.10)
  • Phosphate-buffered saline (PBS; APPENDIX 2)
  • 1× SDS sample buffer (UNIT 10.2A)
  • 60-mm tissue culture plates
  • 27°C incubator (humidification optional)
  • 15-ml polypropylene centrifuge tubes
  • Beckman GPR centrifuge with GH-3.7 rotor (or equivalent), 4°C
  • Boiling water bath or 100°C heating block
  • Sonicator
  • Additional reagents for quantitating protein using the Bradford method (UNIT 10.1A) and preparing insect cell cultures and viral stocks (UNIT 16.10)

Support Protocol 1: Determining Time Course of Maximum Protein Production

 Additional Materials (also see Basic Protocol 1)
  • Wild-type baculovirus (available from Dr. Max D. Summers; see UNIT 16.10 for instructions on generating recombinant baculoviruses using wild-type baculoviral DNA)

Support Protocol 2: Metabolic Labeling of Recombinant Proteins

 Additional Materials (also see Basic Protocol 1)
  • Wild-type baculovirus (available from Dr. Max D. Summers; see UNIT 16.10 for instructions on generating recombinant baculoviruses using wild-type baculoviral DNA)
  • Methionine-free or methionine-free/cysteine-free Grace's insect cell culture medium (Invitrogen)
  • EXPRE35S35S, containing [35S]methionine and [35S]cysteine (>1000 Ci/mmol; Du Pont NEN)
  • Additional reagents and equipment for one-dimensional SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 2: Large-Scale Production of Recombinant Proteins

 Materials
  • Spodoptera frugiperda (Sf9) cells (UNIT 16.10)
  • Serum-free insect cell culture medium (e.g., BaculoGold medium from Pharmingen, Sf-900 II from Invitrogen, or ExCell 401 from JRH Biosciences)
  • High-titer recombinant baculovirus (UNIT 16.10)
  • 1- to 10-liter spinner (Techne or Bellco) or shaker (Bellco) flasks
  • Two-port cap assemblies for spinner flasks (Techne or Bellco; optional)
  • Silicone tubing (Cole-Palmer) with 3/16-in. (0.48-cm) inner diameter (i.d.), 5/16-in. (0.8-cm) outer diameter (o.d.), and 1/16-in. (0.16-cm) wall
  • 0.2-µm filter units (Millipore)
  • 4-in. (10.16-cm) cable ties (Cole-Palmer)
  • Tension tool (Cole-Palmer)
  • Stir plate for multiple spinner flasks (Techne or Bellco; optional)
  • 27°C incubator
  • Air-supply pump (Bellco)
  • Additional reagents and equipment for preparing insect cell cultures and viral stocks (UNIT 16.10)

Alternate Protocol 1: Co-Infection with Multiple Baculoviruses for Purification of Recombinant Protein Complexes

 Additional Materials (see Basic Protocol 2)
  • Recombinant baculoviruses for complex subunits with one subunit carrying an affinity tag for purification
  • Shaker or spinner flask cultures (see Basic Protocol 2)

Basic Protocol 3: Purification of Recombinant Proteins Containing a Polyhistidine (6×His) Tag

 Materials
  • Insect cell lysis buffer (see recipe) containing 1× protease inhibitors (see recipe for 50×)
  • Ni-NTA agarose (Qiagen)
  • 6×His wash buffer (see recipe)
  • 6×His elution buffer (see recipe) containing imidazole as either a step or linear gradient
  • Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent), 4°C
  • Sorvall centrifuge and SS-34 rotor (or equivalent) or 0.2-µm filter
  • Suitable chromatography column
  • Additional reagents and equipment for preparing insect cell cultures and viral stocks (UNIT 16.10) and quantitation of protein by absorbance spectrometry (APPENDIX 3D)

Alternate Protocol 2: Purification of Recombinant Proteins Containing a GST Tag

 Additional Materials (also see Basic Protocol 3)
  • Glutathione agarose beads (Pharmingen or Sigma)
  • PBS wash buffer (Pharmingen)
  • GST elution buffer (see recipe)
  • 50 mM Tris·Cl, pH 8.0 (APPENDIX 2)
  • Thrombin, bovine (e.g., Sigma or Boehringer Mannheim)
  • Additional reagents and equipment for dialysis (APPENDIX 3C)

Alternate Protocol 3: Purification of Recombinant Proteins Containing a FLAG Tag

 Additional Materials (see also Basic Protocol 3)
  • Anti-FLAG M2 affinity beads (Sigma)
  • BC buffers (UNIT 16.13)
  • FLAG peptide (Sigma)
  • Additional reagents and equipment for lysing cells (UNIT 16.7) and SDS-PAGE (UNIT 10.2A)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 16.11.1
    Bellco spinner flask with two-port cap assembly.

    View Image

Literature Cited

Literature Cited
    Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., and Wingfield, P.T. (eds.) 1997. Current Protocols in Protein Science. John Wiley & Sons, New York.
    Guan, K. and Dixon, J.E. 1991. Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192:262-267.
    Haun, R.S. and Moss, J. 1993. Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli. Gene 112:37-43.
    O'Reilly, D.R., Miller, L.K., and Luckow, V.A. 1992. Baculovirus Expression Vectors. W.H. Freeman, New York.
    Phelan, M.L., Sif, S., Narlikar, G.J., and Kingston, R.E. 1999. Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits. Mol. Cell 3:247-53.
    Summers, M.D. and Smith, G.E. 1987. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin No. 1555. College Station, Texas.
    Wu, H., White, G.C., Workman, E.F., Jenzano, J.W., and Lundblad, R.L. 1992. Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin. Thrombosis Res. 67:419-427.
    Zhang, Y., Ng, H.-H., Erdjument-Bromage, H., Tempst, P., Bird, A., and Reinberg, D. 1999. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev. 13:1924-1935.
 Key Reference
    O'Reilly et al., 1992. See above.

A laboratory manual that will aid researchers in the expression and purification of recombinant proteins using the baculovirus system.

     
 
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