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 Molecular Biology
Unit Number:  Unit 11.16
DOI:  10.1002/0471142727.mb1116s59
Online Posting Date:  August, 2002
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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 procedure for selecting the antigenic sequence is relatively straightforward. This unit identifies ways to determine the best sequence and to chemically couple the synthetic peptide to a carrier protein to boost the immune response. Another method entails direct synthesis of a MAP covalent multimer of the simple peptide sequence. Peptides can also be coupled to carrier proteins through either their amino or carboxyl groups. Alternate protocols in this unit cover these scenarios. In addition, methods for assaying free sulfhydryl content and for reducing disulfide bonds in synthetic peptides are provided. Finally, once the coupling procedure has been performed, it is possible to determine the approximate degree of coupling by amino acid analysis, and a procedure for this is also provided.

     
 
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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
  • Figures
  • Tables
     
 
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Materials

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

  Materials
  • Keyhole limpet hemocyanin (KLH; Pierce, Sigma, Calbiochem, or Boehringer Mannheim)
  • 0.01 M sodium phosphate buffer, pH 7.5 ( appendix 22)
  • 10 mg/ml MBS in fresh N,N‐dimethylformamide (DMF)
  • 0.05 M and 0.1 M sodium phosphate buffer, pH 7.0 ( appendix 22)
  • Synthetic peptide with a reduced cysteine residue at either the N‐ or C‐terminus
  • recipe6 M guanidine˙HCl (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 (unit 10.9)
NOTE: Do not use Tris or other buffers with primary amino groups in this procedure.CAUTION: MBS is a moisture‐sensitive irritant. Read the Material Safety Data Sheet before use.

Alternate Protocol 1: Manual Inspection to Select Appropriate Peptide Sequences

  Materials
  • recipeCysteine standard stock solution (see recipe)
  • 0.1 M sodium phosphate, pH 8.0 ( appendix 22)
  • Peptide to be assayed
  • recipeEllman's reagent solution (see recipe)
  • 13 × 100–mm glass test tubes

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

  Materials
  • Synthetic peptide
  • 0.1 M sodium phosphate, pH 8.0 ( appendix 22)
  • 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 unit 10.14)

Alternate Protocol 2: Designing a Synthetic Multiple Antigenic Peptide

  • 50 mM sodium borate buffer, pH 8.0: adjust pH with HCl
  • recipeGlutaraldehyde 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.

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

  • 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC; Pierce), used fresh or stored 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.

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

  • 4‐benzoyl benzoic acid (Sigma or Aldrich)
  • Quartz spectrophotometry cuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Chou, P.Y. and Fasman, G.D. 1974. Prediction of protein conformation. Biochemistry 13:222‐245.
   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.B., 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. 272:73‐80.
   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.
   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.html
  Web site for programs to analyze protein sequences.
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