Cover Image

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...

Full description

Saved in:
Bibliographic Details
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/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf
http://journalarticle.ukm.my/12518/
http://www.ukm.my/jsm/malay_journals/jilid47bil10_2018/KandunganJilid47Bil10_2018.htm
Tags: Add Tag
No Tags, Be the first to tag this record!
id my-ukm.journal.12518
record_format eprints
spelling my-ukm.journal.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
institution Universiti Kebangsaan Malaysia
building Perpustakaan Tun Sri Lanang Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Kebangsaan Malaysia
content_source UKM Journal Article Repository
url_provider http://journalarticle.ukm.my/
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/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf
http://journalarticle.ukm.my/12518/
http://www.ukm.my/jsm/malay_journals/jilid47bil10_2018/KandunganJilid47Bil10_2018.htm
_version_ 1643738811745697792
score 13.223943

Similar Items