Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins

Gregory A. Grant1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.2
DOI:  10.1002/0471142735.im0902s55
Online Posting Date:  August, 2003
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Abstract

Antibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The steps to produce an effective antibody include: (1) designing the peptide sequence based on the sequence of the protein; (2) synthesizing the peptide; (3) preparing the immunogen either by coupling the synthetic peptide to a carrier protein or through the use of a multiple antigenic peptide (MAP); (4) immunizing the host animal; (5) assaying antibody titer in the host animal's serum; and (6) obtaining the antiserum and/or isolating the antibody. This unit covers steps 1 and 3. Once the coupling procedure has been performed, it is possible to determine the approximate degree of coupling by amino acid analysis. Also presented are methods for assaying free sulfhydryl content and for reducing disulfide bonds in synthetic peptides.

     
 
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Table of Contents

  • Basic Protocol 1: Computer‐Assisted Selection of Appropriate Antigenic Peptide Sequences
  • Alternate Protocol 1: Manual Inspection to Select Appropriate Peptide Sequences
  • Basic Protocol 2: Designing a Synthetic Peptide for Coupling to a Carrier Protein
  • Alternate Protocol 2: Designing a Synthetic Multiple Antigenic Peptide
  • Basic Protocol 3: Coupling Synthetic Peptides to a Carrier Protein Using a Heterobifunctional Reagent
  • Support Protocol 1: Assay of Free Sulfhydryls with Ellman's Reagent
  • Support Protocol 2: Reducing Cysteine Groups in Peptides
  • Alternate Protocol 3: Coupling Synthetic Peptides to a Carrier Protein Using a Homobifunctional Reagent
  • Alternate Protocol 4: Coupling Synthetic Peptides to a Carrier Protein Using a Carbodiimide
  • Alternate Protocol 5: Coupling Synthetic Peptides to a Carrier Protein Photochemically
  • Support Protocol 3: Calculation of the Molar Ratio of Peptide to Carrier Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Computer‐Assisted Selection of Appropriate Antigenic Peptide Sequences

  Materials
  • Protein sequence in appropriate format

Alternate Protocol 1: Manual Inspection to Select Appropriate Peptide Sequences

  Materials
  • Peptide synthesis facility
  • Additional reagents and equipment for selecting a synthetic peptide sequence (see protocol 1 or protocol 2); conjugating to a carrier protein by cross‐linking with a heterobifunctional reagent (see protocol 5; for peptides with no internal cysteine) or other cross‐linking method (see protocol 8Alternate Protocols 3, protocol 94, or protocol 105), or synthesizing a multiple antigenic peptide (see protocol 4); and acetylation and amidation (Geoghegan, )

Basic Protocol 2: Designing a Synthetic Peptide for Coupling to a Carrier Protein

  Materials
  • Keyhole limpet hemocyanin (KLH; Pierce, Sigma, Calbiochem, or Boehringer Mannheim)
  • 0.01 M sodium phosphate buffer, pH 7.5 ( appendix 2A)
  • 10 mg/ml MBS in fresh N,N‐dimethylformamide (DMF)
  • 0.05 M and 0.01 M sodium phosphate buffer, pH 7.0 ( appendix 2A)
  • Synthetic peptide with a reduced cysteine residue (see protocol 7) at either the N‐ or C‐terminus
  • 6 M guanidine·HCl (optional; see recipe)
  • Small glass vial with flat bottom
  • ∼0.9 × 15–cm gel filtration column with Sephadex G‐25 or G‐50 (Pharmacia Biotech) or Bio‐Gel P2 or P4 (Bio‐Rad) resin; or prepacked PD‐10 column (Pharmacia Biotech)
  • Additional reagents and equipment for gel filtration chromatography (Hagel, ) and dialysis ( appendix 3H)
CAUTION: MBS is a moisture‐sensitive irritant. Read the Material Safety Data Sheet before use.NOTE: Do not use Tris or other buffers with primary amino groups in this procedure.

Alternate Protocol 2: Designing a Synthetic Multiple Antigenic Peptide

  Materials
  • Cysteine standard stock solution (see recipe)
  • Peptide to be assayed
  • 0.1 M sodium phosphate, pH 8.0 ( appendix 2A)
  • Ellman's reagent solution (see recipe)
  • 13 × 100–mm glass test tubes

Basic Protocol 3: Coupling Synthetic Peptides to a Carrier Protein Using a Heterobifunctional Reagent

  Materials
  • Synthetic peptide
  • 0.1 M sodium phosphate, pH 8.0 ( appendix 2A)
  • 1 M aqueous dithiothreitol (DTT)
  • 1 N HCl
  • 100‐ or 250‐µl polypropylene tubes
  • Nitrogen gas source
  • Additional reagents and equipment for reversed‐phase HPLC of peptides (see Henzel and Stults, )

Support Protocol 1: Assay of Free Sulfhydryls with Ellman's Reagent

  • 50 mM sodium borate buffer, pH 8.0 (pH adjusted with HCl)
  • Glutaraldehyde solution (see recipe)
  • 1 M glycine in 50 mM sodium borate buffer, pH 8.0
NOTE: Do not use Tris or other buffers with primary amino groups in this procedure.

Support Protocol 2: Reducing Cysteine Groups in Peptides

  • 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC; Pierce), use fresh or store desiccated and frozen
  • 0.1 N HCl
NOTE: Buffers containing amino or carboxyl groups should not be used in this procedure. According to some reports, buffers containing phosphate groups should also be avoided. Water is the safest choice as a solvent.

Alternate Protocol 3: Coupling Synthetic Peptides to a Carrier Protein Using a Homobifunctional Reagent

  • Quartz spectrophotometry cuvettes
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Figures

Videos

Literature Cited

   Chou, P.Y. and Fasman, G.D. 1974. Prediction of protein conformation. Biochemistry 13:222‐245.
   Crabb, J.W., West, K.A., Dodson, W.S., and Hulmes, J.D. 1997. Amino acid analysis. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.), pp. 11.9.1‐11.9.41 John Wiley & Sons, New York.
   Creighton, T.E. 1993. Proteins: Structure and Molecular Properties, 2nd ed. W.H. Freeman, New York.
   Gailit, J. 1993. Restoring free sulfhydryl groups in synthetic peptides. Anal. Biochem. 214:334‐335.
   Getz, E.M., Xiao, M., Chakrabarty, T., Cooke, R., and Selvin, P.R. 1999. A comparison between the sulfhydryl reductants tris(2‐carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273:73‐80.
   Geoghegan, K.F. 1996. Modification of amino groups. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.) pp. 15.2.1‐15.2.18. John Wiley & Sons, New York.
   Gorka, J., McCourt, D.W., and Schwartz, B.D. 1989. Automated synthesis of a C‐terminal photoprobe using combined Fmoc and t‐Boc synthesis strategies on a single automated peptide synthesizer. Peptide Res. 2:376‐380.
   Hagel, L. 1998. Gel‐filtration chromatography. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.), pp. 8.3.1‐8.3.30. John Wiley & Sons, New York.
   Henzel, W.J. and Stults, J.T. 2001. Reversed‐phase isolation of peptides. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.), pp. 11.6.1‐11.6.16. John Wiley & Sons, New York.
   Krystek, S.R. Jr., Metzler, W.J., and Novotny, J. 1995a. Hydrophobicity profiles for protein sequence analysis. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.), pp. 2.2.1‐2.2.13. John Wiley & Sons, New York.
   Krystek, S.R. Jr., Metzler, W.J., and Novotny, J. 1995b. Protein secondary structure prediction. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.), pp. 2.3.1‐2.3.20. John Wiley & Sons, New York.
   Kyte, J. and Doolittle, R.F. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105‐132.
   Mints, L., Hogue Angeletti, R., and Nieves, E. 1997. Analysis of MAPS peptides. ABRF News 8:22‐26.
   Posnett, D.N., McGrath, H., and Tam, J.P. 1988. A novel method for producing anti‐peptide antibodies. Production of site‐specific antibodies to the T‐cell antigen beta‐chain. J. Biol. Chem. 263:1719‐1725.
   Tam, J.P. 1988. Synthetic peptide vaccine design: Synthesis and properties of a high density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85:5409‐5413.
   Van Regenmortel, M.H.V., Briand, J.P., Muller, S., and Plaué, S. 1988. Synthetic polypeptides as antigens. In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19 (R.H. Burdon and P.H. van Knippenberg, eds.). Elsevier/North‐Holland, Amsterdam.
Key References
   Van Regenmortel et al., 1988. See above.
  Comprehensive treatment of theory and method.
   Tam, J.P. 1988. High density multiple antigen peptide system for preparation of anti‐peptide antibodies. Methods Enzymol. 168:7‐15.
  Original methods article for MAPs.
Internet Resource
  http://expasy.org.tools
  Web site for programs to analyze protein sequences.
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