AAPPTec chemists have over 20 years experience designing and preparing high quality custom peptides. They have prepared the following guidelines for designing and synthesizing highly effective custom peptides. For more information on how AAPPTec can assist you in filling your custom peptide needs, click here.
How the peptide will be utilized is one of the major factors in designing the peptide. If the goal is to synthetically replicate a naturally occurring peptide, then the peptide sequence and length are fixed. More often, though, the peptide to be prepared is a fragment of a larger peptide or protein and will be used in binding studies, epitope mapping, structure-function studies, fragment assembly or antibody production. The peptide should be long enough to include the residues responsible for activity but very long sequences should be avoided for synthesis and purification problems increase with increased peptide length and yield and purity decrease. Peptides used for epitope mapping or antibody production should generally be 10 - 15 residues.
N- and C-Terminal Modification
Peptides that are segments of a larger peptide or protein may need modification at the N- and C- terminals to accurately model the binding of the parent molecule. A fragment derived from the N-terminal of a large peptide or protein could require an amide function at the C-terminal, a C-terminal fragment might require acylation of the N-terminal. A peptide modeling a segment from the interior portion of a protein or larger peptide may require modification of both N- and C-terminals.
The solubility of the peptide product is an important consideration. If the peptide is insoluble or is poorly dissolved, it will have little or no utility. Generally, short peptides of five amino acids or less are soluble in aqueous media unless all of the residues are highly hydrophobic. Longer peptides containing 50% to 75% hydrophobic residues may be insoluble or only partially soluble in aqueous media. These can be dissolved in a small amount of a stronger solvent such as DMF, isopropanol, ethanol, acetic acid or DMSO and then slowly added to an aqueous buffer solution as long as the original solvent is compatible with application. If the peptide sequence contains more than 75% hydrophobic residues, generally it will not dissolve in aqueous media and will require very strong solvents such as formic acid or TFA for initial solubilization. In this case, expanding the size of the peptide fragment to include more hydrophilic residues should be considered.
Peptides that contain a very high proportion of hydrogen bonding residues (S, T, D, E, R, K, H, Y, N, and Q) can form an extensive network of intermolecular hydrogen bonds, resulting in gel formation in high aqueous concentrations. These peptides should be dissolved in a stronger solvent first, then slowly added to an aqueous buffer to avoid gelation.
Amino Acid Substitutions
Peptides containing Met, Trp or Cys residues are subject to oxidation which can alter the properties of the peptide. Replacing these residues with other amino acids might be considered. Methionine, for example, can often be replaced with norleucine without significant loss of activity. Tryptophan has been replaced by D-2'-naphthylalanine in some instances. Cysteine readily oxidizes, forming a disulfide bond with another cysteine residue. This can provide essential structure in the peptide, forming a dimeric or cyclic compound for instance, or lead to inactive peptide. If cysteine does not play an essential role in the structure of the peptide, it can be replaced with 2-aminobutyric acid or norleucine.
Peptides used in binding or kinetic studies usually incorporate a chromophoric or fluorescent label. Tryptophan is naturally fluorescent and may be used as an intrinsic fluorophore if the peptide contains a Trp residue. The advantage is that no modification of the peptide is needed, thus the conformation and binding ability of the peptide is not altered. Tryptophan fluorescence can be quenched by protonated aspartic acid and glutamic residues, which may limit its utility.
Other fluorescent or chromophoric labels can be incorporated at the N- or C-terminals of a peptide or on an amino acid sidechain. Incorporating such labels can cause problems, however. These labels add steric bulk and can affect the binding of the peptide to its substrate. When this is a problem, adding a linker such a 6-aminohexanoic acid between the peptide and the label can help.
When peptides are used in enzyme kinetic studies, it is useful to incorporate a reporter label. The reporter label undergoes a change in absorbance or fluorescence upon reaction of the peptide. Peptide para-nitroanalides (pNAs) are commonly used in studies of proteases. pNA undergoes a change in absorbance when it is cleaved from the peptide. The absorbance of the free pNA can be used to measure the extent of the reaction and measuring the change in absorbance over time provides kinetic data. Frequency Resonance Energy Transfer (FRET) is another popular reporter labeling system. FRET requires two labels attached to the peptide, one a fluorescence emitter and another, the absorber, with an absorption maximum near the emission maximum of the fluorescence emitter. When the emitter and the absorber are attached to the same peptide, energy is transferred from the emitter to the absorber and no fluorescence is observed. When the peptide is cleaved between the emitter and the absorber, the fluorescence of the emitter is restored. FRET is a very useful and highly sensitive technique, but the incorporation of the emitter and absorber labels should be carefully planned so that the peptide retains the desired reactivity.
For studying reactions and processes within living cells, caged peptides might be utilized. Caged peptides typically have a photolabile group attached at a key position of the peptide. The photolabile group blocks the binding of the peptide to its receptor and allows the caged peptide to permeate the cell. Rapid photolysis releases the peptide and provides a zero time point for kinetic studies. Lysine, cysteine, serine, and tyrosine derivatives have been developed. A recent attempt to utilize 4,5-dimethoxy-2-nitrobenzyl esters on the side chains of aspartic acid and glutamic acid was unsuccessful. These derivatives proved to be highly prone to cyclization during peptide synthesis and cleavage.1
Peptides for Immunology/Antibody Production
Peptides for antibody production should be carefully designed to prevent wasted time and resources. Ideally, peptides used to produce antibodies should produce a strong immunological response and should produce antibodies that have a high affinity for the target protein with low cross-reactivity. To produce effective antibodies, the peptide should represent a segment from the exposed surface of the protein. Similarly, if the protein is membrane-bound, the peptide segment should be from a section exposed on the cell surface rather than a potion normally within the lipid bilayer of the membrane. Segments containing Cys, Leu or Val, if they occur on the surface of the protein, are more likely to be antigenic sites. Kolaskar and Tongaonkar have developed a semi-empirical method to predict epitopes on proteins with about 75% accuracy.2 If the three dimensional conformation of the protein is not well known, as is the case with many proteins, then a segment from the N-terminal or C-terminal of the protein will often be suitable, for at least one protein terminal is often exposed.
If the peptide has hormonal activity, additional planning may be required. Endocrine peptides are generated from longer polypeptide precursors. Often, one polypeptide precursor will lead to several different sets of biologically active peptides depending on where and how the polypeptide is processed posttranslationally. These peptides often have important posttranslational modifications, such as C-terminal amidation. Often a Tyr residue is incorporated in the peptide to provide a labeling site for developing a radioimmunoassay.
The ideal length of peptide for preparing high quality antibodies is disputed. Generally, a peptide of 8-12 residues is considered adequate. Shorter peptides can be effective, but the chances for cross-reactivity are increased. Longer peptides might produce more than one epitope, reducing antibody specificity. Peptides of this size, though, often are not large enough to produce an immune response and need to be conjugated with a carrier protein. Adding a Cys residue to the N- or C-terminal of the peptide will facilitate conjugation of the peptide with the carrier. Cross-linking of the peptide to the carrier protein can also be achieved through a lysine residue or by linking Asp or Glu residues to Lys sidechains of the carrier. This chemistry can lead to unintended cross-linking and is not widely utilized.
AAPPTec provides custom peptide synthesis services to research and pharmaceutical laboritories worldwide. AAPPTec provides high quality custom peptides at competitive prices and within a reasonable time. To learn more about AAPPTec's custom peptide services, click here.
1 Bourgault, S.; Létourneau, M.; Fournier, A. Peptides, 2007, 28, 1074-1082. Return
2Kolaskar, A.S.; Tongaonkar, P.C. FEBS Lett., 1990, 276, 172-174. Return