222 11 Small Functional Peptides and Their Application in Superfunctionalizing Proteins
by Enzymes) and ID‐PRIME (Interaction‐Dependent PRobe Incorporation
Mediated by Enzymes). Meanwhile, several improvements regarding kinetics of
labeling and diversification on the derivatization of the PRIME substrates were
reported [55–58] as well as examples for their application in solving biological
questions [59].
A similar labeling strategy is based on the biotin ligase BirA from E. coli [60] or
its analogs from yeast (yBL) and Pyrococcus horikoshii (PhBL) [61]. Biotin ligases
covalently attach biotin to a lysine in a 15‐residue biotin acceptor peptide (BAP)
[62]. Like this, biotin analogs that are accepted by BirA can be attached to pro-
teins labeled with BAP [63]. Although BirA action is orthogonal to eukaryotic
biotinylation, the method is essentially restricted to protein labeling on the cell
surface as endogenous biotin still serves as a much better substrate than the cor-
responding derivatives. The BirA/BAP pair has been used for proximity studies
on cell surfaces and to image communication across cells by transsynaptic bioti-
nylation [64].
Clearly each peptide‐based labeling method has its advantages regarding
host range, complexity of the labeling approach, and optical properties of the
employed fluorescent or luminescent labels. Thus, the choice of the appropriate
labeling technique needs to be carefully considered when designing an in vivo
labeling experiment. Some excellent recent reviews elaborate in more detail on
these topics [65–67]. So far, labeling of protein at internal sites has not been
extensively explored, although it would add more flexibility in experimental
design than only focusing on terminal tagging.11.3.3 Peptides as Protease Cleavage Sites
Peptides can be used to influence the degradation kinetics of proteins for appli-
cations in basic science, synthetic biology, and biotechnology [4, 5, 68, 69].
Tuning a protein’s stability in vivo can be achieved through N‐ or C‐terminal
degradation tags [70–72]. Alternatively, the process of protein inactivation can
be rendered conditionally, for example, by the insertion of a protein cleavage site
into an internal permissive site, which is recognized by a specific (ideally host‐
orthogonal) protease. Nuclear inclusion protein a (NIa) proteases obtained from
viruses of the family Potyviridae are the most promising target proteases due to
their high activities and sequence specificity. Potyvirus proteases are responsi-
ble for processing the potyvirus polyprotein into its functional units [73]. The
best‐characterized and most commonly applied member is TEV protease, which
recognizes the consensus sequence ENLYFQG, with cleavage according between
Q (P1 positions) and G (P1*) position. TEV is relaxed toward substitutions in
the P5, P4, P2, and P1* position [74, 75]. This gives some freedom for cleavage
site design, although cleavage efficiencies vary depending on the exact residue
inserted.
The TEV consensus sequence is not found in the proteome of mammalian cells
[76], yeast [4], or E. coli [77]. Besides its application in protein purification where
it is frequently used to cleave off affinity tags [78], proteolysis by TEV protease
was used for in vivo studies as a tool to bleach essential proteins [4, 77], to
study phosphorylation‐dependent protein–protein interactions [79], to trigger