Stabilitas Ekspresi Kerangka Baca Terbuka Penyandi Interferon Alfa-2a pada Yeast Pichia pastoris

Try Kurniawan Mutaqien, - (2017) Stabilitas Ekspresi Kerangka Baca Terbuka Penyandi Interferon Alfa-2a pada Yeast Pichia pastoris. S1 thesis, Universitas Pendidikan Indonesia.

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Abstract

Recombinant human Interferon Alfa-2a (rhIFNα-2a) saat ini dipakai dalam pengobatan untuk beberapa penyakit terlebih pada penyakit kanker dan hepatitis. Dalam penelitian ini. Untuk produksi berlebihan protein, budidaya dilakukan di medium kompleks Buffered mengandung gliserol (BMGY) selama 24 jam sedangkan dalam proses produksi untuk menentukan stabilitas protein tersebut harus menggunakan media kompleks Buffer yang mengandung 0.5% metanol (BMMY) yang dapat dalam ekspresi protein selama 48 jam. Penelitian ini meliputi overproduksi, karakterisasi protein, pengukuran densitas sel. Untuk overproduction protein, kultur dilakukan di medium kompleks Buffered mengandung gliserol (BMGY) selama 24 jam dan produksi dilakukan dalam medium kompleks Buffered mengandung metanol (BMMY) selama 72 jam pada 30 °C. Hasil analisis protein hIFNa-2a oleh dot-Blotting dan Western Blotting bahwa pita protein non fusi yang diamati sekitar 16 kDa, dan pita protein fusi yang diamati sekitar 84 kDa tersebut terkonfirmasi merupakan protein rekombinan hIFNα2a. Hasil penelitian yang dilakukan waktu generasi P. pastoris yang mengekspresikan protein Fusi dan Non Fusi memiliki waktu yang berbeda yaitu 1,5 generasi/jam pada protein fusi sedangkan 1,39 pada protein non fusi. Untuk melihat tingkat kestabilan ekspresi dari P. pastoris tersebut dapat ditentukan berdasarkan hasil pengukuran densitas sel dan hasil dot-Blotting. Setiap sampel protein tersebut memiliki peningkatan nilai densitas sel nya dan kemudian dilakukan karakterisasi protein yang diindikasikan perubahan warna pada membrane nitroselulosa. Pada Hasil dot-Blotting tersebut menginformasikan bahwa ekspresi ORF berhasil dilakukan. Pada hasil tersebut, kedua protein tersebut masih stabil sampai Generasi ke-48 untuk Protein Fusi maupun Non-Fusi, tetapi tahap pengkespresian yang dilakukan ragi Yeast P. pastoris tersebut berbeda, dimana protein Interferon α-2a yang lebih baik dan ekspresi yang lebih banyak terlihat pada ragi yang difusi menggunakan protein Human Serum Albumin daripada yang tidak dilakukan fusi. ------ Recombinant human Interferon Alfa-2a (rhIFNα-2a) is currently used in the treatment of some diseases especially in cancer and hepatitis. In this research, for overproduction of protein, the cultivation is carried out in the medium of the Buffered complex containing glycerol (BMGY) for 24 hours while in the production process to determine the protein stability should use a buffer complex media containing 0.5% methanol (BMMY), which can be in protein expression for 48 hours. This research included overproduction, protein characterization, and cell density measurement. For protein overproduction, culture was carried out in a buffered complex medium containing glycerol (BMGY) for 24 hours and the production was carried out in a buffered complex medium containing methanol (BMMY) for 72 hours at 30 °C. Results of analysis of hIFNa-2a proteins by dot-blotting and Western blotting that the non-fusion protein bands were observed were about 16 kDa, and the observed fusion protein band of about 84 kDa was confirmed as hIFNα2a recombinant protein. The results of a study conducted by P. pastoris generation who expressed Fusion and Non Fusion proteins had different time, for 1.5 generations / hour in fusion protein while 1.39 in non-fusion protein. To see the level of stability of expression of P. pastoris can be determined based on the results of cell density measurements and dot-blotting results. Each protein sample had an increase in its cell density value and then characterization of the protein indicated discoloration on the nitrocellulose membrane. The dot-blotting results inform that the ORF expression is successful. In these results, both proteins are still stable until the 48th Generation for both Fusion and Non-Fusion Proteins, but the yeasting Yeast P. pastoris yeast expression stage is different, where the better Interferon α-2a protein and more visible expression in diffuse yeast using the Human Serum Albumin protein rather than non-fusion

Item Type:	Thesis (S1)
Uncontrolled Keywords:	Interferon alfa-2a manusia, Pichia pastoris, Kerangka Baca Terbuka, Stabilitas protein, Protein Fusi-Non Fusi, Human Serum Albumin, Human Interferon alpha-2a, P. pastoris, Open Reading Frame, Protein Stability, Fusion and Non Fusion Protein, Human Serum Albumin
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Fakultas Pendidikan Matematika dan Ilmu Pengetahuan Alam > Jurusan Pendidikan Biologi > Program Studi Biologi (non kependidikan)
Depositing User:	Mrs. Santi Santika
Date Deposited:	04 May 2021 07:34
Last Modified:	04 May 2021 07:34
URI:	http://repository.upi.edu/id/eprint/60566

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