His-CPP5-Cre-Purification

The cDNA sequences of VPTLK and KLPVM replaced the TAT sequence of pET28.2 TAT-Cre plasmid developed by Dr. Dowdy [11]. Using the human codons from Ku70, the cDNA sequences of the CPP5s VPTLK (CAT GGA TGT GAC TTC) and KLPVM (TTC GAC GGA CAT TAC) were inserted into the plasmid. The restriction enzyme used were NcoI and NdeI. The DNA sequence in the plasmid was otherwise kept as the original 11. To generate Cre protein without TAT or CPP5s, Cre cDNA was subcloned into the pET32a+ vector. EcoRI and NotI were used to cut and subclone the Cre cDNA into the pET32a+ vector. After verification of the DNA sequence, E. coli BL21 rossetta was transformed by these plasmids. Protein production was induced by IPTG (500 μM). His-tagged VPTLK-Cre, KLPVM-Cre, TAT-Cre and Cre were purified using Ni-column chromatography. Purified recombinant Cre proteins were dissolved in a high-salt buffer [10 mM phosphate, 500 mM NaCl, 5% glycerol, 10 mM beta mercaptoethanol (BME), pH 6.8]. BME and glycerol were added prior to storing the solutions at -80 C, and after measuring the protein concentration. The frozen stocks were taken from -80 C and kept on ice until they were added to the Tex lox P EG cells

First, cells were cultured for 3 hrs in the serum free medium in the presence of 5
or 10 μM Cre, TAT-Cre, KLPVM-Cre, and VPTLK-Cre. GFP expression was measured
48 hrs after the incubation with CPP-Cre proteins. As shown in Fig. 4.5 B to D, KLPVM
and VPTLK were able to induce GFP expression as TAT did, whereas Cre itself did not.
In serum free containing medium, KLPVM and TAT showed similar protein transduction
activity, and VPTLK showed lower GFP expression than other TAT and KLPVM.
Second, Cre fusion proteins (5 μM) were added to the culture medium containing serum,
and GFP expression was examined 48 hrs after the treatment (Fig. 4.5 E and F). VPTLK-
Cre was able to induce GFP expression, however, TAT-Cre and KLPVM-Cre were not.
This result suggests that VPTLK has the best protein transduction activity in the presence
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of serum among three protein transduction sequences tested. The addition of 10 μM Cre-
fusion proteins was tested too, however, at this condition it was difficult to evaluate the
results due to the substantial precipitation of serum proteins probably because of the
addition of high salt buffer used to dissolve 10 μM Cre proteins.
VPTLK was able to maintain protein transduction activity
in the presence of serum whereas TAT-dependent Cre delivery was significantly
interfered by the presence of serum
VPTLK localizes mostly in the cytosol.
VPTLK-Cre was able to switch on GFP gene expression indicating that VPTLK-Cre was
able to reach chromosomal DNA in the nucleus.
VPTLK
reached 6 μM in the cytosol when culture medium contains 800 μM VPTLK (Fig. 4.4 E),
however, intracellular VPTLK concentration did not significantly increase even medium
VPTLK concentration increased to 1.6 mM.
TAT at higher doses induced apoptosis since apoptosis is the
energy-dependent cellular response activating specific proteases (caspase) to induce
programmed cell death
Tex lox P EG cells (500,000 cells/ml) were incubated with 5, and 10 μM
recombinant Cre, VPTLK-Cre, KLPVM-Cre and TAT-Cre for 3 hours in media without
serum (serum free condition). Other samples were incubated in the presence of serum.
The Tex-loxP-EG cells were incubated with purified recombinant Cre, VPTLK-Cre,
KLPVM-Cre, and TAT-Cre (5 and 10 μM) for 3 h in serum free or 10% serum
containing media. In the case of experiments using serum-free media, serum (final 10%)
was added to the medium after 3 hrs incubation. Forty eight hours after the incubation,
the transduction of Cre by VPTLK, KLPVM and TAT was determine by the expression
of GFP. Expression of GFP was analyzed by fluorescence microscopy and by FACS