Establishment of stable and secretable tatκ-GFP recombinant protein: A preliminary report of promoter methylation in 293t cell line

Zariyantey Abd Hamid, Fazlina Nordin, Raja Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)2473-2480
Number of pages8
JournalSains Malaysiana
Volume47
Issue number10
DOIs
Publication statusPublished - 1 Oct 2018

Fingerprint

HEK293 Cells
Staphylococcal Protein A
Recombinant Proteins
Methylation
Fluorescence
Cell Line
Proteins
Azacitidine
Induced Pluripotent Stem Cells
Transfection
Plasmids
Therapeutic Human Experimentation
Therapeutic Uses
Genetic Recombination
Culture Media
Flow Cytometry
Western Blotting
Technology
Peptides
Mutation

Keywords

  • Induced pluripotent stem cell (iPSC)
  • Methylation
  • Protein transduction domain (PTD)
  • Trans-activator of transcription (TAT)
  • Transcription factors

ASJC Scopus subject areas

  • General

Cite this

Establishment of stable and secretable tatκ-GFP recombinant protein : A preliminary report of promoter methylation in 293t cell line. / Abd Hamid, Zariyantey; Nordin, Fazlina; Raja Ahmad, Raja Norazireen; Rashid, Balqis Mat; Vijakumaran, Ubashini.

In: Sains Malaysiana, Vol. 47, No. 10, 01.10.2018, p. 2473-2480.

Research output: Contribution to journalArticle

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