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.
Peptide solubility
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.
Cross reactivity
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.
For more information on our services, please visit;
http://www.innovagen.com/custom-peptide-synthesis
http://www.innovagen.com/custom-antibody-development