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368 Cell-Penetrating Peptides: Processes and Applications
presence of a common, undefined internalization mechanism likely dependent on
an interaction between the charged groups of the basic residues and lipid phosphates
on the cell surface. 11,12
To date, fusions created with PTDs consisting of Tat-(48–57) show markedly
better cellular uptake than similar fusions using the 16-amino acid sequence from
antennapedia or VP22. However, recently devised peptoid transducers such as the
retro-inverso form of Tat-(57–48) or homopolymers of arginine appear to increase
cellular uptake several-fold. 11,12 Moreover, although the antennapedia PTD can transduce
into cells when associated with chemically synthesized peptides, 17,18 efficiency
dramatically decreases with incorporation of larger proteins.
VP22 transduction is somewhat different from Tat or Antp. In this system, DNA
encoding the entire VP22 protein is genetically fused to the gene of interest and first
transfected into cells. The fusion transgene is transcribed and the translated protein
transduces from the primary transfected cells into the surrounding cells to varying
levels. 10,19 Exogenously added VP22 fusion proteins have been reported to be internalized,
but little data about the efficiency of this protein delivery mode are available.
17.3.1 CLONING AND PURIFICATION OF TAT FUSION PROTEINS
In an effort to exploit Tat-mediated protein delivery as a powerful tool to study
cellular biology and as a potential system for therapeutic delivery of full-length
proteins, we have developed a bacterial expression system that permits rapid cloning
and expression of in-frame fusion proteins using an N-terminal 11 amino acid
sequence corresponding to the region 47–57 of Tat (YGRKKRRARRR). In addition,
purification protocols were devised that appeared to increase the biological uptake
and activity of these chimeric proteins. 20-22 In general, the cDNA encoding the open
reading frame of the gene of interest is cloned in-frame downstream of the N-terminal
6xHis-Tat-HA (hemagglutinin-tagged) sequence in the pTat-HA expression vector.
Although, recombinant Tat fusion proteins may be expressed in soluble form
within Escherichia coli, the majority of these proteins are insoluble and present in
the bacterial inclusion bodies which, although more difficult to extract, are resistant
to proteolytic degradation within the cell. Consequently, inclusion bodies generally
contain full length, undegraded protein. Soluble proteins are purified from the bacterial
pellet by sonication in a denaturant, such as in 8 M urea, which serves to
solubilize proteins stored in inclusion bodies and also to denature those not folded
correctly by bacteria. In this case, denaturation serves two purposes: (1) it aids in
isolation of insoluble recombinant proteins and (2) unfolding complex tertiary protein
structure has been observed to increase transduction efficiency over proteins
purified under native conditions. 22
Interestingly, this latter observation is in keeping with earlier findings that
supported a role for protein unfolding in the increased cellular uptake of the Tat
fusion protein Tat-DHFR. 23 It is possible that the higher energy (∆G) of partial or
fully denatured proteins increases efficiency of transduction compared to lowerenergy
correctly folded ones, in part, due to relief of steric hindrance surrounding
the Tat domain. Once inside the cells, these denatured proteins are believed to be