Peptide sequence is crucial to creating an antibody that will specifically recognize native proteins with high affinity. We usually use peptides that are 12 to 20 amino acids in length.
The chemical, physical, and structural properties of a peptide depend on its unique amino acid sequence and composition. For this reason, peptide sequences can be challenging to synthesize, purify, and/or solubilize. In general, peptide sequences that are rich in hydrophobic amino acids can be difficult to dissolve in aqueous solutions, and may be unsuitable for use in biological systems. The following tips will be useful in modifying your peptide sequence to assist in solubility, synthesis, and/or purification:
- Minimize difficult amino acids such as multiple Cys, Met, Arg, or Trp in the sequence.
- Minimize hydrophobic amino acids or break up a string of hydrophobic amino acids with a polar amino acid.
- Avoid N-terminal glutamic acid so that it cannot cyclize to form pyroglutamine.
- A single antigenic determinant (i.e. the smallest immunogenic peptide) could be between 5 and 8 amino acids. However, longer peptides have a greater conformational similarity to the native protein and are therefore more likely to induce antibodies that recognize the natural protein. Since at least one epitope is recommended, ideal peptides are 12-20 amino acids in length.
- The peptide sequence must be selected from an accessible region of the protein. The trans-membrane region of a protein is not usually exposed and should thus be avoided. Similarly, any region that undergoes post-translational modification (e.g. glycosylation), should also be avoided, since antibodies raised against this sequence may not recognize the modified native protein.
- Synthetic peptides should be able to adopt a conformation that mimics its structure within the protein.
- Multiple glutamines should be avoided, since they may cause insolubility by forming hydrogen bonds between the peptides.
- A single cysteine in the selected sequence is useful for conjugation to a carrier protein. The cysteine should be at the N or C terminus for optimal conjugation. Inter- or intra- peptide disulphide bonds may be formed due to the presence of additional cysteine residues in the sequence, thus leading to insolubility and structural alteration of the peptide.
- Proline and tyrosine residues enhance the immunogenicity of a peptide as they induce conformational change of peptide that mimics the native structure. One or more proline, arginine or histidine residues in the middle of the sequence will greatly reduce peptide aggregation during synthesis.
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