Sains Malaysiana 47(10)(2018): 2473–2480

http://dx.doi.org/10.17576/jsm-2018-4710-24

 

Establishment of Stable and Secretable Tatκ-GFP Recombinant Protein: A Preliminary Report of Promoter Methylation in 293t Cell Line

(Pembangunan Protein Rekombinan Stabil dan Terembes TATκ-GFP: Laporan Awalan Proses Pemetilan Promoter pada Sel Selanjar 293T)

 

ZARIYANTEY ABD HAMID1, FAZLINA NORDIN2*, RAJA NORAZIREEN RAJA AHMAD3, BALQIS MAT RASHID1 & UBASHINI VIJAKUMARAN2

 

1Biomedical Science Programme & Centre of Health & Applied Sciences, Universiti Kebangsaan Malaysia (UKM), Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Federal Territory, Malaysia

 

2Cell Therapy Centre (CTC), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Jalan Yaakob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

3Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan

 

Received: 6 March 2018/Accepted: 20 June 2018

 

 

ABSTRACT

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.

 

Keywords: Induced pluripotent stem cell (iPSC); methylation; protein transduction domain (PTD); trans-activator of transcription (TAT); transcription factors

 

ABSTRAK

Sel asal aruhan pluripoten (iPSC) adalah teknologi terkini yang bermanfaat bagi aplikasi perubatan dan penyelidikan. Terkini, penghasilan iPSC adalah melalui kaedah pengubahsuaian genetik yang berupaya merangsang mutasi, menjadikan ia berbahaya bagi kegunaan perubatan. Justeru, penggunaan kaedah tanpa pengubahsuaian genetik melalui penghantaran terus protein rekombinan dengan bantuan domain pentransduksi protein (PTD) membolehkan penghasilan iPSC yang lebih selamat. Kajian ini bertujuan untuk membangunkan penghasilan protein rekombinan yang stabil dan terembes melalui rekombinasi protein fluoresen hijau (GFP) dan peptida baru PTD iaitu TATκ. Sel selanjar 293T ditransfeksi dengan 20 μg/ mL plasmid TATκ-GFP dan populasi sel dengan transfeksi stabil seterusnya dikultur selama 54 hari untuk mengenal pasti kestabilan pengekspresan dan perembesan protein TATκ-GFP pada tempoh pengkulturan yang berpanjangan. Kehadiran proses pemetilan pada promoter CMV plasmid TATκ-GFP dikaji melalui rawatan ke atas sel ditransfeksi plasmid dengan 3 μM/mL agen pengenyahmetilan 5-Azasitidin selama 72 jam bagi tiga kitaran. Analisis sitometri aliran menunjukkan kecekapan transfeksi sebanyak 9.33% dan kejayaan protein TATκ-GFP dirembeskan ke dalam media pengkulturan berdasarkan analisis Western Blot selepas 72 jam transfeksi. Namun, sel ditransfeksi mempamerkan penurunan pengekspresan dan perembesan protein GFP pada tempoh pengkulturan yang berpanjangan dengan kestabilannya didapati hanya bertahan selama dua minggu. Sel terawat 5-Azasitidin menunjukkan terdapatnya sedikit peningkatan pengekspresan GFP berbanding sel kawalan tanpa rawatan yang mencadangkan kemungkinan kehadiran pemetilan pada promoter yang boleh menyebabkan ketidakstabilan pengekspresan TATκ-GFP. Kesimpulannya, pemetilan promoter perlu diberi perhatian pada masa hadapan untuk penghasilan iPSC memandangkan ia berupaya merencat kestabilan pengekspresan dan perembesan protein rekombinan.

 

Kata kunci: Faktor transkripsi; domain transduksi protein (PTD); pemetilan; sel asal aruhan pluripoten (iPSC); transkripsi pengaktif-trans(TAT)

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*Corresponding author; email: nordinf@ppukm.ukm.edu.my

 

 

 

 

 

 

 

 

 

 

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