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Peptide Design

There are a number of elements to consider when designing individual peptides, specifically amino acid composition, length, solubility and the application in which the peptides are to be used. See Peptide Library Design for design of large numbers of peptides in sets for high throughput synthesis.

As the length of the peptide increases, so the proportion of full-length peptide obtained from the synthesis will decrease. Optimal synthesis results are achieved for peptides up to 15 amino acids, and peptides 10-15 amino acids long are recommended for generation of peptide antisera.

The amino acid content strongly influences the purification of the peptide and the resulting solubility. The ratio of charged amino acids to uncharged and hydrophobic residues is critical in determining the ease of solubility of a peptide, and therefore its usefulness in the downstream application.

A high content of hydrophobic residues will reduce the solubility of a peptide in aqueous solution. A design with at least one charged residue every five amino acids is recommended, otherwise it is recommended to replace hydrophobic amino acids with charged or polar residues where possible.  If determination of peptide concentration is critical, the addition of a Tyrosine residue at the N- and C- terminus of the peptide is recommended if the peptide does not already contain Tyrosine or Tryptophan. 

Hydrophobic (non-polar) Ala, Ile, Leu, Met, Phe, Pro, Trp, Val
Polar (uncharged) Asn, Cys, Gly, Gln, Ser, Thr, Tyr
Charged acidic Asp, Glu, free C-terminus
Charged basic Arg, His, Lys, free N-terminus

Additionally, there are certain amino acids that can cause other problems in synthesis, solubilization and storage of peptides.

Cysteine, Methionine and Tryptophan

These residues are prone to oxidation and their presence in the sequence can cause problems with cleavage and subsequent purification of peptides. To eliminate this issue, use Ser to replace Cys and use Norleucine (Nle) to replace Met. If multiple Cys residues are present, disulphide links may form in the presence of oxygen. To minimize this, use a buffer containing reducing agent such as DTT for peptides containing free Cys, or replace Cys with Ser.

N-terminal Glutamine

Gln will cyclize to form Pyroglutamate when exposed to the acidic conditions of cleavage; to avoid this either acetylate the N-terminus, synthesize with Pyroglutamate instead of Gln to stabilize the peptide, or remove or substitute the Gln.

N-terminal Asparagine

The protecting group for Asn can be difficult to remove when at the N-terminus; to avoid problems remove Asn or substitute.

Aspartic Acid

Peptides containing Asp can undergo hydrolysis. The peptide chain may be cleaved under acidic conditions when particular amino acid pairs are present. Avoid Asp-Gly, Asp-Pro and Asp-Ser pairs if possible.

Multiple Serine or Proline

Avoid adjacent Ser residues as synthesis frequently results in a product that is low in purity and that also may contain many deletions. Proline may undergo cis/trans isomerization in solution and subsequently show low purity.

Secondary Structure

The presence of beta-sheet can lead to deletion sequences in the final peptide. Multiple or series of Gln, Ile, Leu, Phe, Thr, Tyr or Val can lead to beta-sheet formation. If possible, break up these stretches of amino acids by making replacements, such as Asn for Gln or Ser for Thr, or add Pro or Gly every third residue.

 

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