Custom Peptide Design
There are several important factors to consider when designing peptide sequence for successful antibody production. The sequence, amino acid composition and peptide length will influence whether correct chain assembly, purification and subsequent solubilization are feasible. In general, peptide sequences that are rich in hydrophobic amino acids could be difficult to dissolve in aqueous solutions, and may be unsuitable for use in biological systems.
Most peptides of biological interest are derived from native proteins. The trans-membrane region of a protein is not usually exposed and should be avoided. Similarly, any region that undergoes post-translational modification (e.g. glycosylation), should also be avoided, since antibodies raised against this sequence may not recognize the modified native protein. Frequently, the derived sequences are altered, generally in the ‘non-essential’, or say less-important, amino acid residues, although the important/essential amino acid residues are not always easy to determine. Those changes may includes
- Amino acid substitution – improve the peptide solubility and stability
- Chemical modification – improve solubility and stability, structure-function studies
- Peptide conjugation - antisera production
- Ligand attachments - chromophores, affinity ligands
- N- and C-Termini capping – avoid introducing extra charge, increase stability
- N-or C-term adding single Cys – for linking or conjugation via maleimide approach
Amino Acid Compositions
The chemical, physical, and structural properties of a peptide depend on its unique amino acid sequence and composition. Peptide solubility is an important factor to consider when designing a peptide. It is strongly influenced by the amino acid composition. Peptides containing a large percentage of hydrophobic residues, such as Leu, Val, Ile, Met, Phe, and Trp, will often have limited solubility in aqueous solution and may be completely insoluble. In contrast, the charged residues increase the peptide solubility.
Amino acid relative hydrophobicity at pH 7
Very hydrophobic: W > F > L > I > M > V
Hydrophobic: Y > C > A
Neutral (uncharged): T, S, G, N, Q, P, H
Hydrophilic (charged residues): R, K, E, D
To help ensure the solubility
- Try to reduce the number of very hydrophobic residues (W, F, L, I, M, V), ideally <25%
- Try to avoid having more than five continuous hydrophobic residues in a raw
- Try to incorporate at least one charged residue for every five residues in the sequence
- Try to avoid multiple Glutamines, since they may cause insolubility by forming hydrogen bonds between the peptides
- Consider adding a set of polar residues to the N- or C-terminus
Problematic Amino Acid
- Cys, Met, or Trp: susceptible to oxidation and/or side reactions, minimize the exposure high pH or air.
- Gln and Asn: prone to base catalyzed deamination, try to minimize or avoid having Gly next to Gln or Asn (e.g., Gln-Gly, Asn-Gly, Gly-Gln-Gly, Gly-Asn-Gly), try to minimize exposure of the peptide at high pH
- N-term Gln: can undergo cyclization to form N-terminal pyroglutamic acid, considering modify with N-terminal acetylation, extend the N-terminal by one more amino acid or use N-terminal Pyroglutamic acid instead of N-terminal Gln if possible
- Asp-Pro: can undergo acid catalyzed cleavage and prone to form aspartimide side product, try to void having Asp-Pro in the sequence when possible.
It’s difficulty to efficiently predict the aggregation of a
peptide sequence. Such sequences may form β-sheet, coiled-coils
and the special backbone-backbone hydrogen bondings. Besides
bring difficulty during synthesis, the aggregation can exclude
water and may cause the peptide solubility problem for final
application. Long stretches of beta-sheet formation prone
residues (Q, T, Y, V, I, F, W, L) may cause this problem. 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.