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Protein Transport 367
cellular uptake. These chimeric proteins are found within the cytoplasm and the
nucleus and, remarkably, retain their enzymatic activity, thus indicating that these
proteins remain functionally active. Transduction of these proteins occurs in a rapid
process independent of receptors, occurs at 37 and 4˚C, targets 100% of cells, and
results in a concentration-dependent, uniform intracellular loading. These peptide
domains have since been found to induce transduction of a wide variety of molecules
in addition to proteins, such as DNA, drugs, and even inorganic 40-nm iron particles.
The advantages and versatility of protein transduction over viral transgene delivery
were recently illustrated in a pair of papers from van der Noen’s group. In a
continuation of their previous studies that investigated transduction of β-galactosidase
into rat salivary gland cells in vivo using retroviral vectors, 14,15 they more
recently compared this to cellular uptake and activity of recombinant Tat-β-galactosidase
protein into the same tissue by retrograde injection. 16
In contrast to viral delivery, which has limited capacity to infect nondividing
cells, all cell types were susceptible to Tat-mediated protein transduction. With Tat
transduction it was possible to target 100% of the cells in a concentration-dependent
manner, whereas viral delivery could achieve only 30 to 50% transduction efficiency
with variable levels of expression within those cells. Furthermore, β-galactosidase
activity could be detected intracellularly within ascinar cells from 10 min to 6 h
following injection, while viral delivery was associated with a significantly delayed
onset of enzyme activity, likely due to the added cellular requirement of transcription
and translation of the β-galactosidase.
The direct delivery and efficient cellular uptake of transducing proteins offers
several advantages over traditional DNA-based methods of manipulating the cellular
phenotype. Consequently, a vast increase in use of PTD fusion to address biological
questions and for the introduction of pharmacologically relevant proteins in vitro
and in vivo has now begun.
17.3 DEVELOPMENT OF PTD FUSION PROTEINS
At present two methods are employed to generate chimeric proteins capable of
cellular transduction: chemical conjugation of protein transduction domains to proteins
or in vivo expression of in-frame recombinant fusions of transduction domains
with full length proteins. In both cases the resulting chimeric proteins are able to
be taken up by cells similar to the Tat protein or the antennapedia homeoprotein.
Using this approach, it is possible to target virtually 100% of primary and transformed
cells. Each cell contains nearly identical intracellular concentrations of
biologically active protein readily manipulated by titrating the amount of exogenous
protein. Furthermore, PTD fusion proteins are rapidly internalized and reach maximal
intracellular loading in less than 5 min, suggesting that timing of treatment can
be precisely controlled.
While the different protein transduction domains show similar characteristics
for cellular uptake, it is clear that they vary in efficacy in transporting their cargo
into the cell. Although no homology between the primary and secondary structure
of these PTDs exists, the rate of cellular uptake has been found to correlate strongly
to the number of basic residues present, specifically arginine. This indicates the