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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Penerbit Universiti Kebangsaan Malaysia
2018
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| Online Access: | http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf |
| _version_ | 1848813024335364096 |
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| author | Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran, |
| author_facet | Zariyantey Abd Hamid, Fazlina Nordin, Rajaa Norazireen Raja Ahmad, Balqis Mat Rashid, Ubashini Vijakumaran, |
| author_sort | Zariyantey Abd Hamid, |
| building | UKM Institutional Repository |
| collection | Online Access |
| 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. |
| first_indexed | 2025-11-15T00:11:37Z |
| format | Article |
| id | oai:generic.eprints.org:12518 |
| institution | Universiti Kebangasaan Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T00:11:37Z |
| publishDate | 2018 |
| publisher | Penerbit Universiti Kebangsaan Malaysia |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | oai:generic.eprints.org: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 |
| 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 |
| title | 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_short | 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 |
| url | http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/ http://journalarticle.ukm.my/12518/1/24%20Zariyantey%20Abd%20Hamid.pdf |