 |
 |
 |
 |
 |
 |
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
Yaacob 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 Diserahkan: 6 Mac 2018/Diterima: 20 Jun 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) RUJUKAN
Beerens, A.M., Al Hadithy, A.F., Rots, M.G. & Haisma,
H.J. 2003. Protein transduction domains and
their utility in gene therapy. Curr. Gene Ther. 3: 486-494.
Bonifaci, N., Sitia, R. & Rubartelli, A. 1995. Nuclear
translocation of an exogenous fusion protein containing HIV Tat requires unfolding. AIDS 9(9):
995-1000. Brooks, A.R., Harkins, R.N., Wang, P., Qian, H.S., Liu, P. &
Rubanyi, G.M. 2004. Transcriptional
silencing is associated with extensive methylation of the CMV
promoter following adenoviral gene delivery to muscle. The
Journal of Gene Medicine 6: 395-404. Cheng, T., Xu, C.Y., Wang, Y.B., Chen, M., Wu, T., Zhang, J. &
Xia, N.S. 2004. A rapid
and efficient method to express target genes in mammalian cells
by baculovirus. World Journal of Gastroenterology:
WJG 10(11): 1612-1618. Cho, H.J., Lee, C.S., Kwon, Y.W., Paek,
J.S., Lee, S.H., Hur, J. & Kim,
Y. 2010. Induction of pluripotent stem cells from adult
somatic cells by protein-based reprogramming without genetic manipulation.
Blood 116(3): 386-395. Deng, W. 2010. Induced
pluripotent stem cells: Paths to new medicines.
EMBO Reports 11(3): 161-165. Elias, M.H., Baba, A.A., Husin, A., Sulong, S., Hassan, R., Sim, G.A., Abdul Wahid, S.F. &
Ankathil, R. 2013. HOXA4 gene promoter hypermethylation as
an epigenetic mechanism mediating resistance to imatinib
mesylate in chronic myeloid leukemia
patients. BioMed Research
International 2013: 129715. doi: 10.1155/2013/129715. Fazlina Nordin,
Zariyantey Abdul Hamid, Lucas Chan,
Farzin Farzaneh & Hamid, M.K.A.A.
2016. Transient expression of green fluorescent
protein in integrase-defective lentiviral vector-transduced 293T
cell line. In Lentiviral Vectors and
Exosomes as Gene and Protein Delivery Tools, Methods in Molecular
Biology, edited by Maurizio Federico. New York, NY:
Springer Science + Business Media. pp. 159-173. Fazlina Nordin, Noralisa
Abdul Karim & Wahid, S.F.A. 2014. Transgene
expression is transient in non-integrating lentiviral-based transduction
system: An alternative approach for safety gene therapy application.
Regenerative Research 3(1): 1-7. Flinterman, M., Farzaneh, F., Habib, N., Malik, F.,
Gaken, J. & Tavassoli,
M. 2009. Delivery of therapeutic
proteins as secretable TAT fusion products.
Molecular Therapy 17(2): 334- 342. Ford, K.G., Souberbielle, B.E., Darling,
D. & Farzaneh, F., 2001. Protein transduction: An alternative to genetic intervention? Gene
Ther. 8: 1-4. Grassi, G., Maccaroni, P., Meyer, R., Kaiser,
H., D’Ambrosio, E., Pascale, E., Grassi,
M., Kuhn, A., Di Nardo, P., Kandolf,
R. & Kupper, J.H. 2003. Inhibitors of DNA methylation and histone deacetylation activate
cytomegalovirus promoter-controlled reporter gene expression in
human glioblastoma cell line U87. Carcinogenesis 24(10):
1625-1635. Han, W., Zhao, Y. & Fu, X. 2010. Induced pluripotent stem cells: The dragon awakens. Bioscience
60(4): 278-285. Hedge, R.S. & Bernstein, H.D. 2006. The surprising complexity of signal sequences.
Trends in Biochemical Sciences 31(10): 563-571. Hsu, C.C., Li, H.P., Hung, Y.H., Leu, Y.W.,
Wu, W.H., Wang, F.S. & Huang, T.H.M. 2010. Targated methylation of CMV and E1A viral promoters. Biochemical
and Biophysical Research Communications 402(2): 228-234. Katas, H., Abdul Ghafoor, R.M. & Ee, L.C. 2014. Comparative
characterization and cytotoxicity study of TAT-peptide as potential
vectors for siRNA and Dicer-substrate siRNA. Drug Dev. Ind.
Pharm. 40(11): 1443-1450. Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S.
& Kim, K.S. 2009. Generation
of human induced pluripotent stem cells by direct delivery of
reprogramming proteins. Cell Stem Cell 4(6): 472.
Koutsokeras, A., Purkayastha, N., Rigby, A., Subang, M.C., Sclanders, M., Vessillier, S. & Gould, D. 2014. Generation of an efficiently secreted, cell penetrating
NF-κB inhibitor. The
FASEB Journal 28(1): 373. Leeper, N.J., Hunter, A.L. & Cooke, J.P. 2010. Stem cell therapy for vascular regeneration adult embryonic, and induced pluripotent stem cells. Circulation
122(5): 517-526. Okano, H., Nakamura, M., Yoshida, K., Okana,
Y., Tsuji, O., Nori, Sa. & Miura,
K. 2013. Steps toward safe cell therapy using induced pluripotent stem cell.
Circulation Research 112(3): 523-533. Mariati, M., Koh, E.Y., Yeo, J.H., Ho, S.C. &
Yang, Y. 2014. Toward stable
gene expression in CHO cells. Bioengineered
5(5): 340-345. Martinez, S.I., Nivet, E. & Belmonte,
J.C.I. 2011. The labyrinth
of nuclear reprogramming. Journal of Molecular Cell
Biology 3(6): 327-329. Michalowsky, L.A. & Jones, P.A. 1987. Differential nuclear protein binding to 5-azacytosine-containing DNA
as a potential mechanism for 5-aza-2‘-deoxycytidine resistance.
Molecular and Celullar Biology 7(9):
3076-3083. Moritz, B., Becker, P.B. & Gopfert,
U. 2015. CMV promoter mutants with a reduced propensity
to productivity loss in CHO cells. Scientific Reports
5: 16952. doi:10.1038/ srep16952. Noguchi,
H. & Matsumoto, S. 2006b. Protein transduction technology: A novel therapeutic
perspective. Acta Med. Okayama
60: 1-11. Nordin, F.,
Tye, G.J, Gaken,
J. & Farzaneh, F. 2014. TATκ
fusion protein of OCT-3/4 and KLF-4: Stable mixed population cell
lines capable of delivering fusion proteins to target cells. Journal
of Cell Science & Therapy 5(2): 158. Nordin., F., Raja Ahmad, R.N.
& Farzaneh, F. 2017. Transctivator
protein: An alternative for delivery of recombinant proteins for
safer reprogramming of induced pluripotent stem cell. Virus
Research 235: 106-114. Saunthararajah, Y. 2013. Key clinical
observations after 5-azacytidine and decitabine
treatment of myelodplastic syndromes
suggest practical solutions for better outcomes. Hematology
ASH Education Program 2013(1): 511-521. Schwarze, S.R.,
Hruska, K.A. & Dowdy, S.F. 2000. Protein transduction:
Unrestricted delivery into all cells? Trends in Cell Biology
10(7): 290-295. Stadfeld, M.
& Hochedlinger, K. 2010. Induced
pluripotency: History, mechanism, and applications. Genes
& Development 24(20): 2239-2263. Takahashi, K. & Yamanaka,
S. 2006. Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126(4):
663-676. Takahashi, K., Tanabe,
K., Ohnuki, M., Narita, M., Ichisaka,
T., Tomoda, K. & Yamanaka, S. 2007.
Induction of pluripotent stem cells from adult human fibroblast by
defined factors. Cell 131(5): 861-872. Thomson,
J.A., Itskovitz-Eldor, J., Shapiro,
S.S., Waknitz, M.A., Swiergiel, J.J.,
Marshall, V.S. & Jones, J.M. 1998. Embryonic stem cell lines derived
from human blastocyst. Science 282(5391): 1145-1147. Yamanaka, S. 2009. A fresh look at iPS cells. Cell
137(1): 13-17. Yoshida, Y. & Yamanaka,
S. 2010. Recent stem cell advances: Induced pluripotent stem cells
for disease modelling and stem cell-based regeneration. Circulation
122(1): 80-87. Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J. & Jin, Y. 2012. Reprogramming of somatic cells
via TAT-mediated protein transduction of recombinant factors.
Biomaterials 33(20): 5047-5055. Zhou,
Y.Y. & Zheng, F. 2013. Integration-free methods for generating induced pluripotent
stem cells. Genomics, Proteomics & Bioinformatics 11(5):
284-287. *Pengarang untuk surat-menyurat; email: nordinf@ppukm.ukm.edu.my
|
|||
|
