Production of Antisera Using Fusion Proteins

Sara E. Dodson1, Craig J. Heilman1, Richard A. Kahn1, Allan I. Levey1

1 Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.7
DOI:  10.1002/0471142301.ns0507s40
Online Posting Date:  July, 2007
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Abstract

This unit details the use of bacterially produced fusion proteins for the production of antisera, allowing for the large‐scale generation of affinity‐purified antibodies to specific, targeted epitopes. The use of pET vectors containing a polyhistidine (His) or glutathione‐S‐transferase (GST) tag to construct bacterial expression plasmids are provided as prototypical examples of fusion protein methodology. The basic protocols provided in this unit describe: (1) transformation of E. coli for high‐yield production of soluble fusion protein, (2) purification of soluble fusion proteins for use in immunization using chelated nickel or glutathione affinity chromatography (for His‐ and GST‐tagged fusion proteins, respectively), (3) immunization of rabbits with purified fusion protein and collection of antisera, and (4) characterization of antisera for antibody specificity using immunoblotting techniques. Support protocols describe the purification of His‐tagged insoluble fusion proteins for animal immunization and the construction and use of affinity columns for purifying antibodies using soluble fusion proteins. Curr. Protoc. Neurosci. 40:5.7.1‐5.7.26. © by John Wiley & Sons, Inc.

Keywords: His6 tag; glutathione‐S‐transferase (GST); bacterial expression; soluble fusion protein; insoluble fusion protein; immunization; polyclonal antibody; affinity purification; antibody characterization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fusion Protein Expression
  • Purification of Soluble Fusion Protein
  • Basic Protocol 2: Purification of His6‐Tagged Fusion Proteins
  • Alternate Protocol 1: Purification of GST‐Fusion Proteins
  • Basic Protocol 3: Immunization Using Fusion Proteins
  • Basic Protocol 4: Characterization of Antisera
  • Support Protocol 1: Purification of Insoluble Fusion Proteins Using Immobilized Metal Affinity Chromatography (IMAC)
  • Support Protocol 2: Affinity Purification of Antisera
  • Support Protocol 3: Preparation of Affinity Columns for Purification of Anti‐Fusion Protein Antisera
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fusion Protein Expression

  Materials
  • E. coli strain BL21(DE3) (Novagen)
  • Recombinant pET‐X fusion protein expression plasmid (Novagen)
  • LB plates (see recipe) containing 100 µg/ml ampicillin (LB/amp plates)
  • LB medium (see recipe) containing 100 µg/ml ampicillin (LB/amp)
  • 1 M isopropyl thio‐β‐D‐galactosidase (IPTG; store in aliquots indefinitely at −20°C)
  • 1× SDS‐PAGE sample buffer
  • Buffer A (see recipe), ice‐cold
  • 20 mg/ml lysozyme in buffer A (see recipe for buffer A; prepare fresh), optional
  • Protease inhibitor stock solutions (store up to 1 year at −20°C), optional:
    • 1 mg/ml aprotinin in 0.01 M HEPES, pH 8.0
    • 1 mg/ml leupeptin in water
    • 1 mg/ml pepstatin A in ethanol
  • 10% (v/v) Triton X‐100, optional
  • TE buffer, pH 8.0 ( appendix 2A), ice‐cold
  • 37°C incubator
  • 14‐ml snap‐cap tubes
  • Shaker at 37°C
  • 1.5‐ml microcentrifuge tubes
  • 250‐ml flasks
  • 1‐liter baffled flask(s)
  • Refrigerated ultracentrifuge with Ti45 rotor and centrifuge buckets
  • 50‐ml conical polypropylene centrifuge tubes
  • French press
  • Additional reagents and equipment for transformation of E. coli ( appendix 1L and CPMB UNIT ), growth of E. coli on liquid (CPMB UNIT ) and solid (CPMB UNIT ) media, SDS‐PAGE under nondenaturing conditions (CPMB UNIT ), Coomassie staining of gels (CPMB UNIT ), and dialysis (CPMB APPENDIX )

Basic Protocol 2: Purification of His6‐Tagged Fusion Proteins

  Materials
  • HiTrap chelating column (GE Healthcare)
  • 0.1 M NiSO 4
  • Cleared bacterial lysate or DEAE‐Sephacel eluate
  • Buffer H (see recipe)
  • 10, 50, 100, and 300 mM imidazole in buffer H (see recipe for buffer H)
  • Syringes
  • Additional reagents and equipment for SDS‐PAGE (CPMB UNIT ) and Coomassie staining of gels (CPMB UNIT ); also see appendix 1A

Alternate Protocol 1: Purification of GST‐Fusion Proteins

  Materials
  • Fusion protein (see protocol 1)
  • Buffer C (see recipe), ice cold
  • Protease inhibitor stock solutions (store up to 1 year at −20°C):
    • 1 mg/ml aprotinin in 0.01 M HEPES, pH 8.0
    • 1 mg/ml leupeptin in water
    • 1 mg/ml pepstatin A in ethanol
  • Glutathione‐agarose bead slurry (GE Healthcare; see recipe)
  • 20% (w/v) sodium azide (1000× stock)
  • 50 mM reduced glutathione in buffer C, adjust pH to ∼7.5 with NaOH (prepare fresh)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 15‐ and 50‐ml tubes
  • Shaker
  • Refrigerated centrifuge
  • Quartz spectrophotometer cuvette
  • UV spectrophotometer
  • Spectra/Por dialysis tubing (MWCO 6000 to 8000; Spectrum; also see CPMB APPENDIX )
  • Centrifugal filter device, optional
  • Additional reagents and equipment for SDS‐PAGE (CPMB UNIT ), Coomassie staining of gels (CPMB UNIT ), protein assay (CPMB UNIT ) and dialysis (CPMB APPENDIX ); also see appendix 1A

Basic Protocol 3: Immunization Using Fusion Proteins

  Materials
  • New Zealand white rabbits (∼2.5 kg) (young adult females are suggested)
  • Purified fusion protein (see protocol 2)
  • Complete and incomplete Freund's adjuvant (CFA and IFA; e.g., Pierce)
  • Cannula or syringe
  • Additional reagents and equipment for emulsification of antigens with Freund's adjuvant and serum preparation (unit 5.5), immunoblotting (unit 5.19), and immunocytochemistry (unit 1.2)

Basic Protocol 4: Characterization of Antisera

  Materials
  • Purified fusion protein
  • Crude pre‐immune serum
  • Primary antibody solution and buffer
  • Antisera and/or affinity‐purified antibody (e.g., eluates from affinity columns)
  • Razor blade or scalpel
  • Additional equipment and reagents for immunoblot analysis (unit 5.19) and immunocytochemistry (unit 1.2)

Support Protocol 1: Purification of Insoluble Fusion Proteins Using Immobilized Metal Affinity Chromatography (IMAC)

  Materials
  • Pellet from cell lysate containing fusion protein (see protocol 1)
  • PBS containing 0.5% Triton X‐100
  • Buffer H (see recipe) containing 5, 10, 50, 200, or 500 mM imidazole and 8 M urea
  • 5‐ml HiTrap chelating HP column (GE Healthcare)
  • Buffer H (see recipe) containing 8 M urea
  • Refrigerated ultracentrifuge and centrifuge buckets
  • 50‐ml conical polypropylene centrifuge tubes
  • Dounce homogenizer
  • Syringe or FPLC pump

Support Protocol 2: Affinity Purification of Antisera

  Materials
  • Heparin
  • Crude, clarified antiserum (see protocol 4)
  • 10‐ml BL21/GST affinity column (see protocol 8)
  • 10 mM, 0.1 M, and 1 M Tris·Cl, pH 8.0 ( appendix 2A), ice‐cold
  • 0.1 M glycine, pH 3.0, ice‐cold
  • 2‐ml fusion protein affinity column (see protocol 8)
  • Glycerol
  • 20% (w/v) sodium azide (1000× stock)
  • 15‐ and 50‐ml tubes
  • Quartz spectrophotometer cuvette
  • UV spectrophotometer
  • pH paper
  • 1.5‐ml microcentrifuge tubes

Support Protocol 3: Preparation of Affinity Columns for Purification of Anti‐Fusion Protein Antisera

  Materials
  • Purified fusion protein (see protocol 3)
  • Affinity resin (Affi‐Gel 10 and 15; Bio‐Rad)
  • BL21(DE3)/GST lysate (see protocol 3)
  • 0.1 M glycine, pH 3.0
  • 0.1 M Tris·Cl, pH 8.0 ( appendix 2A)
  • 0.1 M triethylamine, pH 11.0
  • Sodium azide
  • 1‐ml pipets
  • Razor blades
  • 15‐ and 50‐ml tubes
  • Tabletop centrifuge with bucket rotors, 4°C
  • Shaker
  • 2‐ml (for fusion protein) and 10‐ml (for BL21(DE3)/GST) double‐frit chromatography columns (Pierce)
  • Pasteur pipets
  • UV spectrometer
  • Additional equipment and reagents for SDS‐PAGE (CPMB UNIT ) and Coomassie staining (CPMB UNIT )
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Figures

Videos

Literature Cited

   Angeletti, R.H. 1999. Design of useful peptide antigens. J. Biomol. Tech. 10:2‐10.
   Barrett, A.J. 2003. Proteases. Curr. Protoc. Protein Sci. 21:21.1.1‐21.1.12.
   Ciliax, B.J., Heilman, C., Edmunds, S., Hersch, S.M., and Levey, A.I. 1995. Anti‐fusion protein antibodies specific for receptor subtypes. J. Neurosci. Methods 25:431‐454.
   Frangioni, J.V. and Neel, B.G. 1993. Solubilization and purification of enzymatically active glutathione S‐transferase (pGEX) fusion proteins. Anal. Biochem. 210:179‐187.
   Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and Studier, F.W. 1987. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125‐135.
   Smith, D.B. and Johnson, K.S. 1988. Single‐step purification of polypeptides in Escherichia coli as fusions with glutathione‐S‐transferase. Gene 67:31‐40.
   Smith, G.E., Ju, G., Eriscon, B.L., Moschera, J., Lahm, H.W., Chizzonite, R., and Summers, M.D. 1985. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc. Natl. Acad. Sci. U.S.A. 82:8404‐8408.
   Studier, F.W. 2005. Protein production by auto‐induction in high density shaking cultures. Protein Expr. Purif. 41:207‐234.
   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Meth. Enzymol. 185:60‐89.
   Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J.S., and Arakawa, T. 2004. Role of arginine in protein refolding, solubilization, and purification. Biotechnol. Prog. 20:1301‐1308.
   Volkel, D., Blankenfeldt, W., and Schomburg, D. 1998. Large‐scale production, purification and refolding of the full‐length cellular prion protein from Syrian golden hamster in Escherichia coli using the glutathione S‐transferase‐fusion system. Eur. J. Biochem. 251:462‐471.
Key References
   Harlow, E. and Lane, D. 1999. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  Describes many aspects of antibody production.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 2001. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  A good source for general molecular biological techniques.
Internet References
  http://bio.dfci.harvard.edu/Tools/antigenic.pl
  Available peptide algorithms.
  http://peptideselect.invitrogen.com/peptide/
  Available Novagen pET vectors.
  http://www.emdbiosciences.com/html/NVG/pETTableMenued.html
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