Induced pluripotent stem cells (iPSC) is a novel technology useful for therapeutic and research applications. To date, iPSCs
is produced through genetic modification that can promote mutation; making it harmful for therapeutic use. Therefore,
application of non-genetic modification through direct delivery of recombinant proteins aided by protein transduction
domain (PTD) enable a safer production of iPSC. This study is aimed to establish a stable production of secretable
recombinant protein via recombination of green fluorescence protein (GFP) and a novel PTD peptide, namely TATκ-GFP.
293Tcell line was transfected with 20 µg/ml of TATκ-GFP plasmid and the stably transfected 293T cells were then cultured
for 54 days to determine the stability of expression and secretion of TATκ-GFP recombinant protein in prolonged culture.
Methylation at the CMV promoter of the TATκ-GFP plasmid was investigated following treatment of transfected cells with
3 µM/mL of demethylation agent, namely 5-Azacytidine for 72 h in three cycles. Flow cytometry analysis demonstrated
a transfection efficiency of 9.33% and successful secretion of TATκ-GFP proteins into the culture medium as analysed by
Western blot at 72 h post-transfection. However, the transfected cells exhibited a decreasing level of GFP expression and
secretion following prolonged culture with notable stability that only sustained for two weeks. 5-Azacytidine-treated cells
showed a slight increase of GFP expression compared to non-treated control, suggesting possible promoter methylation
which could cause instability of TATκ-GFP expression. Conclusively, promoter methylation should be considered for future
establishment of iPSCs as it could inhibit stable expression and secretion of recombinant proteins.