Biomedical Engineering Reference
In-Depth Information
TABLE 26.1
(Continued)
Protein
CPP
Application
References
p53
a,d
VP22
Cancer therapy
[53-55]
Peroxiredoxin 5 and 6
(PRDX5&6)
a
Tat
Protection against high-glucose-induced cytotoxicity
in retinal pericytes
[56]
Rac
a
Tat
Treatment of inflammatory diseases and cancer
[22-24]
Red fluorescent protein (RFP)
a
R9, Tat
Fluorescent protein delivery via arginine-rich
peptides to plant cells
[41]
Rho
a
Tat
Treatment of inflammatory diseases and cancer
[22-24]
RNAse A
b
Tat
Protein transduction of active enzyme
[29]
Single chain variable fragment
(scFV)
a
Penetratin, Tat
Intracellular delivery of scFV fragments targeting
various proteins
[13,57,58]
Suppressor of cytokine signaling
3 (SOCS3)
a
MTM
Suppression of apoptosis and inflammation
[15]
T7 RNA polymerase
a
VP22
Delivery of active enzyme
[35]
b
-galactosidase
a
Penetratin, Tat, VP22
Protein delivery
[29,43]
a
Recombinant expression, C-terminal.
b
Maleimide-cysteine bond. Illustrated in Figure 26.1.
c
Disulfide bond (Cys-Cys).
d
Recombinant expression, N-terminal.
or probes across biological barriers are required. CPPs have
already contributed greatly to improve drug delivery in vivo
to treat diseases such as cancer, ischemia, inflammation, and
autoimmune diseases [7].
Protein transduction is an emerging technology with the
potential to overcome many of the limitations of small
molecule pharmaceuticals and recombinant genetic
approaches, the latter often suffering from difficulties
achieving sustained and appropriately regulated expression.
Currently, proteins exceeding 100 kDa have successfully
been delivered into cells in culture and mammalian model
systems. The sheer size of macromolecules possible to
transport across the cell membrane makes CPP-mediated
delivery very promising, potentially enabling delivery of a
large repertoire of proteins.
A solution to the problem of tissue nonspecificity of CPPs
is the development of so-called activatable CPPs. Constructs
have been described that are “activated” either outside or
inside the cells [8-10].
Cell-penetrating fusion proteins have been used for a
large variety of applications and the list of successfully
delivered proteins is ever expanding. To date, several groups
have reported successful delivery of CPP-fusion proteins
in vitro [11-14] and a growing number of transducible
proteins covering a wide range of sizes and functional
classes have been delivered in vivo [15,16]. Table 26.1
presents an extensive selection of proteins that have been
successfully delivered into cells for various applications as
well as the CPP that mediated delivery.
Many of the fusion proteins presented have applications
associated with cancer therapeutics, which can range from
proapoptotic and anti-proliferative agents to tumor imaging
and migration inhibitors. Other common applications
include immunosuppression, apoptosis protection, diagnos-
tics, and vaccines.
26.3 TECHNOLOGICAL ASPECTS
26.3.1 Creating CPP-Fusion Proteins
Production of CPP-fusion proteins is fairly straightforward
and relies mostly on traditional means of cloning, creating an
expression vector, transfection to and subsequent expression
in Escherichia coli, resulting in either a N- or C-terminal
attachment of the peptide, N-terminal attachment being the
most common. Here, we give three examples of different
vectors expressing different proteins in fusionwithCPPs. Two
of the examples utilize the TAT peptide and the last example is
a fusion between the VP22 peptide and enhanced green
fluorescent protein (eGFP). In this section, a brief general
description of the methods used for purification of the fusion
proteins and for synthesis of peptides is included.
We also give an example of expressed protein ligation as
an alternative method for production of CPP-fusion proteins,
and describe how this method may have advantages over the
much more common cloning method. In addition, there is
a short section about the utilization of cysteine to make
disulfide bonds between peptides and proteins. A selection
of common methods for creating CPP-fusion peptides are
listed in Table 26.1, and illustrated in Figure 26.1.
26.3.2 Recombinant Fusion Protein
Expression Systems
The most common method by far, as can be seen in
Table 26.1, for generating fusion proteins is by recombinant
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