Peptides are complex molecules and each sequence is unique with regarding to its sequence, amino acid composition and length of a peptide. While some peptides are difficult to synthesize, otheres are relatively straight forward, but difficult to purify. 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 BSI to help you in designing your peptide, click here to request for sequence analysis or visit our Gizmo for online interative analysis. Remember! You design. We can Help!

Design of Peptide Sequence

1. Shorten the sequence
Generally speaking, as peptide length increases, the yield and purity of the crude product becomes lower. Most peptides of 15 residues or fewer can be synthesized without major difficulty, but with peptides longer than 20 residues, yields generally decrease.

2. Decrease the number of hydrophobic residues
Peptides with a predominance of hydrophobic residues, especially in the region of 7-12 amino acids from the C-terminus, often have assembly problems. This is thought to cause ß-sheet formation between peptide chains during synthesis, which causes incomplete coupling. In these cases, replacing one or more residues with a more polar residue, or by adding a Gly or Pro residue to help break up the regular peptide structures may help.

3. Minimize difficult residues
Peptides with multiple cysteine, methionine, arginine and tryptophan are often difficult to synthesize. Serine can be used as a non-oxidizing replacement for cysteine, and norleucine can be used as a methionine replacement. Lysine can be used in place of arginine while tyrosine or phenylalanine or other hydrophobic residues such as leucine are sometimes adequate replacements for tryptophan.

OPTIONS TO IMPROVE SOLUBILITY
1. Change N or C terminus
For acidic peptides, those having an overall negative charge at pH7, we recommend a peptide format Acetyl-peptide-COOH (acetyl group at amino terminus and free acid at carboxy terminus) to maximize the negative charge. For basic peptides (i.e. peptide has an overall positive charge pH7), we recommend a peptide format of H-peptide-amide (free N terminal amino group, and amide C-terminus) to maximize the positive charge.

2. Shorten or lengthen sequence
Some sequences contain a large number of hydrophobic residues such as Trp, Phe, Val, Ile, Leu, Met, Tyr and Ala. Generally we see solubility problems in peptides where >50% of the residues are these hydrophobic amino acids. In order to increase the polarity of the peptide, it may be useful to lengthen the sequence, provided the added amino acids increase peptide polarity. Alternatively, the sequence may be shortened to eliminate hydrophobic residues and, hence, increase peptide polarity. The more polar the peptide, the more likely it is to be soluble in aqueous buffers.

3. Add solubilizing residues
For some peptides, it is possible to arbitrarily add a set of polar residues to improve solubility. We recommend for acidic
peptides to add Glu-Glu to the N or C terminus and for basic peptides to add Lys-Lys to the N or C terminus. If a charged group cannot be tolerated, we recommend the addition of

Ser-Gly-Ser to the N or C-terminus. Obviously there are cases where the N and C termini cannot be altered, and this approach would not be applicable.

4. Alter sequence by substituting one or more residues
Peptide solubility may be improved by changing some residues within the sequence. Often, a single replacement (e.g., replacing alanine with glycine) can dramatically improve solubility but remains relatively conservative.

5. Alter the sequence by selecting a different frame or overlapping peptides
If a number of sequential or overlapping peptides of set length forming a sequence are to be made, a change in the starting point of each peptide may make a difference. This creates a better balance between hydrophobic and hydrophilic residues in individual peptides, or by separating difficult residues into