CPG2 toxic levels of MTX. Glucarpidase as

CPG2 as
a recombinant enzyme rapidly hydrolyses extracellular MTX to its non-toxic
metabolites thus is used as a novel drug for reducing elevated plasma MTX
concentrations in patients with renal delayed MTX clearance (Goda et al., 2009; Mitrovic et al., 2016). Therefore, intracellular delivery of CPG2
can convert MTX to its non-toxic metabolites and prevents the accumulation of
toxic levels of MTX. Glucarpidase as a dimeric
protein with two 41 KDa subunits is not able to enter the cells.  Therefore,
for intracellular delivery of CPG2, we have evaluated the possibility of
protein transduction using the TAT peptide as an effective CPP. To produce TAT-fusion
protein, cpg2 gene was fused to the HIV-1 TAT peptide encoding gene. The
production of purified recombinant TAT-
CPG2 fusion protein was confirmed by Western blot analysis. To determine the transduction efficiency of the TAT-CPG2,
Western blot analysis and fluorescence staining have been used. Results showed that
both native and denatured TAT-CPG2 were successfully
transduced into the HepG2 cells in a concentration and time-dependent manner. Stability
of proteins is very important in the host for therapeutic applications.
Significant intracellular stability of TAT-CPG2
protein continued for up to 36 h after initial transduction into the cells. Fluorescence
staining results indicated that TAT-CPG2 protein transduced into approximately
100% of the cells.   A direct comparison
of transduction efficiency of native and denatured TAT-CPG2 protein by Western blot
analysis and fluorescence
microscopy showed that denatured TAT-CPG2 transduced into the cells more efficiently than the native TAT-CPG2. Similar observations
have been reported in other studies. Jin et al. has reported that denatured Tat-CAT
and 9Arg-CAT efficiently transduced into HeLa and PC12 cells (Jin et al., 2001). Kim et al. showed that in contrast to the native
TAT-SOD, denatured TAT-SOD has been successfully delivered into the cells
(Kim et al.,
2006). They have demonstrated that unfolding
of protein is needed for efficient transduction of TAT-SOD into HeLa
cells. Nagahara et al. showed that transduction of denatured TAT-p27
protein into HepG2 cells induced cell migration
at low concentrations; while correctly folded TAT-p27
protein could not induce cell migration at even higher concentrations. This might be due to the low efficiency of
correctly folded TAT-p27 to achieve the required intracellular threshold
level to elicit cell migration, not the complete disability to
enter the cell (Nagahara et al.,
1998). Lower