Pinka Alisa Diena Shafa, - (2018) OVERPRODUKSI, KARAKTERISASI DAN ANALISIS EFISIENSI PURIFIKASI PROTEIN FUSI HUMAN INTERFERON-α2a HUMAN SERUM ALBUMIN. S1 thesis, Universitas Pendidikan Indonesia.
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Abstract
Kanker dan hepatitis merupakan penyakit yang sangat berbahaya, pengobatannya terus berkembang salah satunya dengan protein Human Interferon alfa 2a (IFN-α2a). IFN-α2a merupakan protein yang sering digunakan untuk keperluan klinis karena memiliki sifat sebagai antiviral, antiproliferatif dan immunomodulator. Tag fusi HSA pada IFN-α2a bertujuan untuk menambah paruh waktu IFN α-2a di dalam tubuh. IFN-α2a salah satunya dihasilkan oleh kelompok Yeast metilotropik yaitu Pichia pastoris. Purifikasi dilakukan untuk mendapatkan protein yang lebih murni, tetapi dengan purifikasi terdapat proses kehilangan protein. Penelitian ini bertujuan untuk karakterisasi, overproduksi dan analisis efisiensi purifikasi HSA IFN-α2a pada Yeast Pichia pastoris. Pichia pastoris HSA-IFN SMD1168 klon no1 diinokulasi pada media YPD dan YPDS yang mengandung zeocin. Kultur kemudian dioverproduksi skala 3 ml pada media BMMY dan BMGY. Screening tahap awal dilakukan dengan metode Dot Blot dan Western Blot untuk memastikan bahwa protein pada saat overproduksi skala 3 ml berhasil diekspresikan, hasil Dot Blot menunjukan hasil yang positif dengan tervisualisasikannya dot pada membran nitroselusose, hasil Western Blot menunjukan hasil yang positif dengan tervisualisasikannya pita berukuran 81,2 kDa. Selanjutnya overproduksi dilakukan pada skala 50 ml pada media BMMY dan BMGY. Proses filtrasi dilakukan menggunakan membran Molecular Weight Cut off (MWCO) yang berukuran 10 kDa, dan diambil 1 ml untuk sampel TFF. Purifikasi dilakukan dengan menggunakan blue sepharose dan diambil 1 ml untuk sampel ELUAT. Sampel TFF dan ELUAT kemudian dikarakterisasi menggunakan metode Western Blot dan didapatkan pita berukuran sama. Analisis efisiensi dilakukan dengan metode ELISA sandwich dan didapatkan rendemen bernilai 74,07% dan bersifat efisien. .... Cancer and hepatitis are diseases with high level of lethallity, the treatment is still being developed with the human interferon protein alpha 2a (IFN-α2a) as one of them. IFN-α2a is a protein that are widely used for the medical necessity for having properties such as antiviral, anti proliferative, and immunomodulatory. HSA on IFN-α2a aims to increase the half-life of IFN-α2a in the body IFN-α2a, one of which is produced by the methylotropic Yeast group of Pichia pastoris. Purification is performed to obtain a more pure protein, but with purification there being a protein loss process. This study was aimed for characterization, overproduction and efficiency analysis purification fusion protein of IFN-α2a HSA on Yeast Pichia pastoris. Pichia pastoris HSA-IFN SMD1168 clone number 1 inoculated on YPD and YPDS media containing zeocin. Culture then overproduced 3 ml scale on BMMY and BMGY media. Screening the initial stage is done by the method of Dot Blot and Western Blot to ensure protein at the time of 3 ml scale overproduction was successfully expressed. Dot Blot showed positive result with dot visualization on nitroselusose membrane. Western Blot showed positive result with visualized band measuring 81,2 kDa. Furthermore, overproduction is done on a 50 ml scale on BMMY and BMGY media. The filtration process was carried out using a 10 kDa Molecular Weight Cut Off (MWCO) membrane, and 1 ml was taken for the TFF sampel. Purification was performed using blue sepharose and taken 1 ml for the ELUAT sampel. TFF and ELUAT samples were then characterized using Western Blot method and obtained the same sized bands. The efficiency analysis was done by ELISA sandwich method and the yield was 74.07% and was efficient.
| Item Type: | Thesis (S1) |
|---|---|
| Additional Information: | No. Panggil : S BIO PIN o-2018 ; Pembimbing I. Didik Priyandoko, II. Ratih Asmana N. ; NIM: 1404352 |
| Uncontrolled Keywords: | recombinant protein, hIFN-α2a protein, protein expression, Pichia pastoris, protein rekombinan, protein hIFN-α2a, ekspresi protein, Pichia pastoris |
| Subjects: | Q Science > Q Science (General) Q Science > QH Natural history > QH301 Biology Q Science > QK Botany |
| Divisions: | Fakultas Pendidikan Matematika dan Ilmu Pengetahuan Alam > Jurusan Pendidikan Biologi |
| Depositing User: | Mr Tatang Saja |
| Date Deposited: | 13 Aug 2019 07:01 |
| Last Modified: | 13 Aug 2019 07:01 |
| URI: | http://repository.upi.edu/id/eprint/37705 |
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