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

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 de...

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Main Authors:	Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran
Format:	Article
Language:	English
Published:	Penerbit Universiti Kebangsaan Malaysia 2018
Online Access:	http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf

id	ukm-12518
recordtype	eprints
spelling	ukm-125182019-01-28T21:32:47Z http://journalarticle.ukm.my/12518/ Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran, 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. Penerbit Universiti Kebangsaan Malaysia 2018-10 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf Zariyantey Abd Hamid, and Fazlina Nordin, and Rajaa Norazireen Raja Ahmad, and Balqis Mat Rashid, and Ubashini Vijakumaran, (2018) Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line. Sains Malaysiana, 47 (10). pp. 2473-2480. ISSN 0126-6039 http://www.ukm.my/jsm/malay_journals/jilid47bil10_2018/KandunganJilid47Bil10_2018.htm
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	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.
format	Article
author	Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran,
spellingShingle	Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran, Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line
author_facet	Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran,
author_sort	Zariyantey Abd Hamid,
title	Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line
title_short	Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line
title_full	Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line
title_fullStr	Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line
title_full_unstemmed	Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line
title_sort	establishment of stable and secretable tatκ-gfp recombinant protein: a preliminary report of promoter methylation in 293t cell line
publisher	Penerbit Universiti Kebangsaan Malaysia
publishDate	2018
url	http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf
first_indexed	2023-09-18T20:02:47Z
last_indexed	2023-09-18T20:02:47Z
_version_	1777406960219455488

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

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