Peptide Synthesis Considerations

　 The overall amino acid (a.a.) composition of a peptide is an important design variable that is frequently overlooked. A peptide's solubility is strongly influenced by composition.

　Length of Sequence
　The purity of a crude peptide typically decreases as the length increases. The yield of peptide for sequences less than 15 residues is usually satisfactory, and such peptides can typically be made without difficulty. In addition, peptides of 10-15 residues in length are satisfactory for raising antisera to linear epitopes of intact proteins.

　Hydrophobic Stretches
　Peptides with a high content of hydrophobic residues, such as leucine, valine, isoleucine, methionine, phenylalanine and tryptophan may exhibit limited solubility in aqueous solutions or be completely insoluble. Peptides rich in hydrophobic stretches may be difficult to purify and complicate experimental application. Given these considerations, it is recommended that peptide design maintain a hydrophobic a.a. content <50% and ensure that there is one charged residue for every five amino acids.

　To reduce potential complications and ensure timely synthesis we have the following limitations on synthesis: No two valine residues in a row or a Val-phe-Ile stretch.

　Considerations for acidic and basic amino acids
　At physiological pH aspartate, glutamate, lysine, and arginine all have charged side chains. A conservative replacement, such as replacing alanine with glycine, or adding a set of polar residues to the N- or C-terminus, may improve solubility.

　Difficult Amino Acids
　Peptides containing multiple cysteine, methionine or tryptophan residues are also difficult to obtain in high purity-partly because these residues are susceptible to oxidation and/or hydrogen bonding. Peptide orders placed with these three amino acids at a high frequency are likely to be problematic in synthesis. This may result in a slight delay as it is remanufactured.

　Alternatives for methionine and cysteine

　Secondary Structure
　Beta-sheet formation is another consideration in peptide design. During synthesis, beta-sheet formation causes incomplete solvation of the growing peptide and results in a high degree of deletion sequences in the final product. This problem can be avoided by selecting sequences that do not contain multiple or adjacent residues comprised of valine, isoleucine, tyrosine, phenylalanine, tryptophan, leucine, glutamine, and threonine. If sequences cannot be chosen to avoid stretches of these residues, it often helps to break the pattern by making conservative replacements, eg. inserting a glycine or proline at every third residue, replacing glutamine with asparagine, or replacing threonine with serine.

　Purity
　The purity needed for peptide depends on the length synthesized and the downstream application. For example, sequences that are fairly long (22-32) have several truncated versions of the desired peptide in the final product and hence increased purification is recommended, especially if binding assays or crystallography are a potential application. The default purification of a custom peptide is approximately 50% or "crude", however we offer HPLC purification options at 70%, 80%, 90% and 95%.

　Storage of Peptides
　Most peptides are stable at -20°C indefinitely, especially if they are lyophilized and stored in a dessicator. Allow lyophilized peptides to come to room temperature before exposing them to air to minimize hygroscopic effects. If lyophilization is not possible, store in small, working size aliquots.