crc press - E-Lib FK UWKS

Hydrophobic Membrane Translocating Sequence Peptides 125
Direct chemical synthesis
MTS
Biological synthesis
MTS
(a)
Cargo
Plasmid
Chemical ligation
Cargo
Bacterial or viral
expression
MTS Cargo
MTS
(c)
Cargo
(b)
MTS
Cargo
FIGURE 6.2 Schemes generally used to prepare MTS-cargo peptides and proteins. (a) Direct
chemical synthesis where the MTS and cargo are synthesized in tandem. (b) Biological
synthesis involves the incorporation of MTS and cargo DNA into a plasmid and expression
in a bacterial or viral host system. Often remnant enzyme cleavage site residues flank the
sequence of interest (hatched bars). (c) A general scheme for peptide ligation via thiazolidine
ring formation is shown. An MTS bearing an aldehyde is ligated with a cargo peptide or
protein containing an N-terminal cysteinyl moiety, resulting in the formation of a nonamide
bond.
To date, translocating peptides have predominantly been attached to cargo proteins
(and some peptides) using genetic recombination. 36,84,105-107 Using cell-permeable
GST-GrbSH2-MTS protein as an example, the expression plasmid was constructed
by ligating the double-stranded MTS into a BamH1 site of an expression plasmid
for GST fusion protein. 36 The design of the plasmid further allowed the insertion of
DNA fragments encoding GrbSH2 protein into the GST-MTS sequence. In this case
the protein product was used to test the effect of the GrbSH2 domain on EGF-induced
mitogenic signaling. 36 The main disadvantage associated with use of these types of
continuous synthesis strategies is that a new synthesis must be performed for production
of each vector–cargo molecule. The synthesis of peptides incorporating
vector and cargo residues may also prove difficult as peptide length increases.