PeptideCAD™ is our proprietary software that we use as an aid in designing immunogenic peptides as antigens for antibody production. The process of selecting peptide sequences suitable for immunisation is a rather complex one with many aspects to consider. Some of these are listed in the below.
Antibody target domain
If there is prior knowledge of the target protein, such as information on regions and domains of particular interest to target - or to avoid - then this will be the basis for any design. This type of information has to be provided by you.
Whatever sequence you finally decide on, the resulting peptide has to be soluble. So far, there is no reliable algorithm for predicting the solubility of a peptide. Looking at the number of charged amino acids, and their positions within the peptide will however be a fairly good indication on whether or not solubilisation may be an issue. When a peptide is deemed to have solubility issues, that can’t be over come by simply shifting the sequence a few amino acids up or down, or by truncating it slightly, then one can opt to add a spacer and a few hydrophilic residues in order to improve on this.
Sequence surface exposure
A sequence that is exposed on the protein surface is typically more versatile, and finding such a sequence is desirable. When the 3D structure of the protein is known, verifying that a certain sequence is located on the surface becomes a trivial task. In most cases however one has to resort to predictions.
Modification of peptide termini
Peptides carry a +1 charge at the N-terminus and a -1 charge at the C-terminus. Unless one is specifically targeting a neo-epitope or the absolute end of the native protein, then blocking N- or C-terminus will make the peptide mimic the protein better.
Antigenicity / Immunogenicity
The peptide-conjugate nearly always illicits an immune response, even if the peptide sequence itself scores low in an antigenicity algorithm. However, used with some discretion, a prediction of antigenicity may very well be included as the “swing vote” in the final selection of a suitable sequence.
Repeats and other sequences of low complexity, GTP binding sites, RGD recognition motifs, etc. should be avoided. Otherwise, potential cross reactivity with arbitrary proteins is not an issue. However, when you have a limited number of proteins for which potential cross reactivity – desired or not - is crucial, then careful selection becomes important. Since anti-peptide antibodies target short linear epitopes, and similarities could be found anywhere within a given protein, simply aligning a set of protein sequences will not suffice. Each protein has to be scanned for similarities with a number of small sequences.
Peptide conjugation feasibility
Peptides typically need to be conjugated to a larger carrier molecule in order to illicit an immune response. Typically this is achieved by use a functional group of the peptide (amino, carboxyl or thiol) and a hetero bi-functional crosslinker. Since it is desirable to have a single functional group located at the terminus of the peptide, it is common to add a Cysteine to the peptide sequence.
Peptide synthesis feasibility
In general, any peptide can be made. However, if obvious difficulties can be avoided then the risk for potential delays is minimized. For example, although the peptide as a whole is deemed soluble, it may contain a highly hydrophobic stretch that potentially causes synthesis or purification issues.
Although PeptideCAD™ is a highly useful tool that simplifies the peptide design process considerably, the selecting of peptide sequences suitable for immunisations is always done by our experienced staff. That said; Typically the best results are achieved when we can work together and combine your knowledge of the target protein with the PeptideCAD™ software and our experience from thousands of anti-peptide antibody projects.
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