218 11 Small Functional Peptides and Their Application in Superfunctionalizing Proteins
more resistant to proteolytic degradation than terminal fusions [12]; (iii) the
peptide might need to be structurally stabilized to exhibit its function as it is
especially the case in vaccine development using epitope tags or small molecule
mimicking peptides [13–16]; or (iv) the specific function delivered by the pep-
tide needs per se to be introduced into the middle of the protein as it is the case
for the engineering of cleavable proteins by insertion of a protease cleavage site
[5, 9, 17, 18].
For these reasons, the focus of the subsequent discussion will be on proteins
where the peptides were integrated into the sequence rather than positioned at
the N‐ or C‐terminal end.11.2 Permissive Sites and Their Identification in a Protein
in a Protein
Sites within proteins at which large insertions are tolerated without loss of
structural integrity and activity represent an extreme in the spectrum of
sequence flexibility and have been called permissive sites [19]. Although it was
originally assumed that permissive sites generally correspond to surface regions
at which the added sequences do not disrupt overall folding [20], it is in fact
hard to predict rationally where insertions will be tolerated even in the pres-
ence of detailed structural knowledge. It is also not clear whether all proteins
and enzymes are similarly tolerant to insertions. Traditional ways to explore
the structural flexibility of a protein and to identify permissive sites have there-
fore been random library approaches. A few studies suggest that permissive
sites can be identified more rationally through comparative sequence analysis
[11, 21, 22].
Two main library approaches have been applied for the identification of per-
missive sites in various proteins: the first is to generate insertions by limited
digestion of a plasmid‐encoded target gene using different restriction enzymes
and religating it with a resistance cassette to be able to select for successful inser-
tions. The resistance cassette needs to be flanked by unique restriction sites to
subsequently excise the cassette and leave the gene of interest with a defined
oligonucleotide insertion. The method does not enable to completely query the
possible insertion space, but depending on the number of enzymes chosen for
digestion, a sufficient degree of coverage can be reached [23, 24]. The second
approach is insertion mutagenesis mediated by transposons, also known as
linker insertion mutagenesis. Transposons are mobile genetic elements, which
quasi‐randomly insert in any DNA sequence mediated by the action of its
corresponding transposase. Like this, any sequence can be delivered, ideally
randomly, into a gene of interest as long as it is placed between the two trans-
posase‐specific recognition sites. In the simplest case, the transposon consists of
a resistance marker, which is flanked by unique restriction sites as well as the
transposase recognition sites. Subsequent excision of the resistance marker
results in a characteristic in‐frame fingerprint, which is composed of sequences
from the restriction sites, the transposon ends, and target site nucleotides
that were duplicated during the primary transposition event [18]. In addition,