Designing Peptides
While peptide synthesis can be straightforward, it is important to consider several factors before synthesis commences. The sequence, amino acid composition and length of a peptide will influence whether correct assembly and purification are feasible. These factors also determine the solubility of the final product. The following summary highlights some important points that should be considered in the design of a peptide. If you would like Pepmic to help you in designing your peptide, our technical support department can assist you.

Design of Peptide Sequence
Peptides can be designed de novo, but most peptides of biological interest are derived from N-terminal, C-terminal, or internal sequences of native proteins. Unfortunately, there are valid reasons why certain native sequences sometimes need to be altered. Even for relatively short sequences, there are essential and non-essential (or less important) amino acid residues, although the relative importance of the individual amino acid residues is not always easy to determine. The "not-so-straightforward" rule of thumb is to make the changes in the "non-essential residues". These changes may include amino acid substitution (e.g., for solubility, stability, etc.), chemical modification (e.g., for stability, structure-function studies), attachment of ligands (e.g., chromophores, affinity ligands), conjugation (antisera production), etc. For internal sequences, it may be necessary to cap either or both the N- and C-termini to avoid introducing a charge where there is none in the native sequence. For bulky ligands, it is also common practice to attach a spacer between the peptide and the ligands to minimize the influence of the ligand on the folding of the peptide.

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 
The overall amino acid composition of a peptide is an important design variable that is frequently overlooked. Peptide solubility is strongly influenced by amino acid composition. Peptides with a high content of hydrophobic residues, such as Leu, Val, Ile, Met, Phe and Trp, will either have limited solubility in aqueous solution or be completely insoluble. Under these conditions, it will be difficult to use the peptide in experiments, and it may be difficult to purify the peptide if necessary. It is advisable to keep the hydrophobic amino acid content below 50% and to make sure that there is at least one charged residue for every five amino acids. At physiological pH Asp, Glu, Lys, and Arg all have charged side chains. A single conservative replacement, such as replacing Ala with Gly, or adding a set of polar residues to the N- or C-terminus, may also improve solubility.

Difficult Amino Acids 
Peptides containing multiple Cys, Met, or Trp residues are also difficult to obtain in high purity, partly because these residues are susceptible to oxidation and/or side reactions. If possible, one should choose sequences to minimize these residues. Alternatively, conservative replacements can be made for some residues. For instance, Norleucine can be used as a replacement for Met, and Ser is sometimes used as a less reactive replacement for Cys. If a number of sequential or overlapping peptides from a protein sequence are to be made, making a change in the starting point of each peptide may create a better balance between hydrophilic and hydrophobic residues. A change in the number of Cys, Met, and Trp residues contained in individual peptides may produce a similar effect. As an example, if the stop or start point for choosing peptides separates two Cys residues into two peptides, this may allow for better synthesis and purer final product.

Secondary Structure
β-sheet formation is a final consideration in peptide design. During synthesis, β-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 choosing sequences which do not contain multiple and adjacent residues comprised of Val, Ile, Tyr, Phe, Trp, Leu, Gln, and Thr. If sequences cannot be chosen to avoid stretches of these residues, it often helps to break the pattern by making conservative replacements, for example, inserting a Gly or Pro at every third residue, replacing Gln with Asn, or replacing Thr with Ser.