nalog insulin adalah sub-group insulin manusia yang dimodifikasi secara genetik dengan mengubah a... more nalog insulin adalah sub-group insulin manusia yang dimodifikasi secara genetik dengan mengubah atau menambahkan urutan asam-asam amino tertentu agar dapat digunakan lebih cepat oleh tubuh jika dibandingkan insulin biasa serta menciptakan jenis insulin dengan kerja yang lebih cepat atau serentak. Analog insulin dimodifikasi agar mempunyai kemiripan dengan insulin manusia yang memiliki fungsi dalam mengontrol kadar glukosa secara normal. Kata ‘analog’ bermakna ‘mirip’, yaitu mirip dengan insulin dalam tubuh manusia, namun tidak sama persis (Steele dkk, 2008). Penggunaan analog insulin secara tepat memungkinkan penderita diabetes lebih fleksibel dalam mengatur waktu makan, cemilan, dan olahraga, sehingga meningkatkan kemampuan mereka untuk menjalani kehidupan normal (Hirsch, 2005).
Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for clinical therapy as an... more Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for clinical therapy as antiviral, anticancer as well as immunomodulator. In this study, the open reading frame (ORF) encoding synthetic hIFNα-2a was constructed to be in framed with N-terminal alpha factor secretion system in methylotropic yeast Pichia pastoris. This research aimed to construct, express and analyse the non-affinity tagged recombinant human interferon alpha-2a in the methilotropic yeast P. pastoris . We used pPICZαB plasmid for cloning and expression vector. The confirmed recombinant plasmid containing the correct DNA sequence of hIFNα-2a was linearized by SacI restriction enzyme, then transformed into P. pastoris genome using electroporation. We screened two multi-copy recombinants in YPDS plates containing Zeocin™. Buffered complex medium containing 0.5 % methanol (BMMY) was used for protein expression for 48 hours in the culture condition. The recombinant protein was purified by blue sepharose...
Human interferon alpha-2b (hIFNα-2b) is a cytokine that used as therapeutic protein in hepatitis ... more Human interferon alpha-2b (hIFNα-2b) is a cytokine that used as therapeutic protein in hepatitis and several cancer treatments. In our previous study, the open reading frame encoding hIFNα-2b was cloned to pPICZαB vector and expressed in methylotropic yeast Pichia pastoris (P. pastoris) under the control of alcohol oxidase 1 (AOX1) promoter. This study was aimed to overproduce, purify and characterize the recombinant human IFN-α2b protein (rhIFNα-2b). The protein was overproduced in BMMY media at 30 °C for 48 hour by using 0.5% methanol as inducer. The rhIFN-α2b purification was performed by immobilized affinity chromatography column using AKTA purifier system. The quantification of purified rhIFN-α2b by using colorimetric bicinchoninic acid assay informed that the yield was 22.43 mg/L culture (OD600 = 2.3). SDS-PAGE and Western blot analyses on protein characterization confirmed that the protein was rhIFN-α2b with 24.050 kDa in size. The rhIFN-α2b was containing c-myc epitope and p...
The choice of culture condition on Pichia pastoris expression system is an important factor not o... more The choice of culture condition on Pichia pastoris expression system is an important factor not only to improve the recombinant protein production but also to avoid proteolysis as well. In our previous work we constructed the human interferon alpha 2b (hIFN-α2b) open reading frame which was overexpressed in P. pastoris. Without any further study of culture condition, the yield was 22 mg in 1 L culture (OD600= 2.3). This research was carried out to study the effect of nonnutritional factors on recombinant human interferon alpha 2b (rhIFNα-2b) production. There were three factors to be studied: induction time (24h, 48h, 72h, 96h and 120h), methanol concentration (0%, 0.25%, 0.5%, 1%, 1.5%, 2% and 2.5%) and temperature of induction (25, 30 and 37°C). Expression level was monitored by using SDS PAGE and relatively quantified by using ImageJ software. The results showed that temperature of induction more than 48 h result in protein degradation and the best expression level was achieved a...
IOP Conference Series: Earth and Environmental Science, 2020
As a workhorse for manufacturing recombinant proteins, ChineseHamster Ovary(CHO)cells are becomin... more As a workhorse for manufacturing recombinant proteins, ChineseHamster Ovary(CHO)cells are becoming an extremely valuable cell for producing recombinant proteins having glycosylation patterns like in human system. Development of cell line, basically, focused on achieving the maximum amount of active proteins by screening the cell line to select higher producing clone. Currently, one of mammalian cell line development technologies used by most biopharmaceutical companies is based on gene amplification technology with methotrexate (MTX) to increase protein expression. With that in mind, the aim of this study is part of an effort to develop cell line capable of producing erythropoietin with 2 additional N-links. Gene amplification was performed on CHO-DG44 cell pools expressing EPO through addition of different concentrations of MTX to medium. The cells were cultured using orbital shaker at the speed of 130 rpm at 37°C and 5% CO2 condition. Stepwise increasing MTX concentrations from 20...
Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein produ... more Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viru...
Roti, keju dan minuman beralkohol seperti bir dan wine adalah jenis makanan dan minuman yang mema... more Roti, keju dan minuman beralkohol seperti bir dan wine adalah jenis makanan dan minuman yang memanfaatkan yeast (ragi) dalam proses pembuatannya. Sejak zaman peradaban Mesir, yeast sudah dikenal dan dimanfaatkan dalam industri roti. Jenis yeast yang digunakan dalam industry tersebut adalah Saccharomyces cereviseae.Ilmu pengetahuan modern telah mengidentifikasi lebih dari 1000 varietas wild-yeast dan tersebar di alam. Mereka ditemukan hidup berasosiasi dengan mikroorganisme sebagai bagian dari habitat normal di perairan, tanah, dan lingkungan air lainnya. Bahkan di udara yang kita hirup untuk bernafas, pada batang batang pohon dan di atas daun-daun tanaman yeast juga bisa ditemukan.
Banyak orang berasumsi, bahwa anemia merupakan penyakit kurang darah, 5L (lesu, lemah, letih, lun... more Banyak orang berasumsi, bahwa anemia merupakan penyakit kurang darah, 5L (lesu, lemah, letih, lunglai dan loyo) adalah serangkaian gejalanya. Untuk mengatasi gejala ini di televisi ataupun media-media cetak sering kita lihat berbagai iklan produkproduk antianemia yang dapat dengan mudah dibeli di pasaran. Masalahnya, apakah benar anemia yang dipahami secara medis cukup hanya diatasi dengan mengkonsumsi obat antianemia saja. Tentu jawabannya tidak sesederhana itu. Secara ilmiah anemia adalah suatu keadaan dimana jumlah sel darah merah atau jumlah Hemoglobin (protein pembawa oksigen) dalam sel darah merah berada di bawah normal (kadar Hb<10g/dl). Sel darah merah membawa oksigen (O2) dari paruparu ke jaringan dan organ-organ tubuh yang akan digunakan sebagai energi.
nalog insulin adalah sub-group insulin manusia yang dimodifikasi secara genetik dengan mengubah a... more nalog insulin adalah sub-group insulin manusia yang dimodifikasi secara genetik dengan mengubah atau menambahkan urutan asam-asam amino tertentu agar dapat digunakan lebih cepat oleh tubuh jika dibandingkan insulin biasa serta menciptakan jenis insulin dengan kerja yang lebih cepat atau serentak. Analog insulin dimodifikasi agar mempunyai kemiripan dengan insulin manusia yang memiliki fungsi dalam mengontrol kadar glukosa secara normal. Kata ‘analog’ bermakna ‘mirip’, yaitu mirip dengan insulin dalam tubuh manusia, namun tidak sama persis (Steele dkk, 2008). Penggunaan analog insulin secara tepat memungkinkan penderita diabetes lebih fleksibel dalam mengatur waktu makan, cemilan, dan olahraga, sehingga meningkatkan kemampuan mereka untuk menjalani kehidupan normal (Hirsch, 2005).
Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for clinical therapy as an... more Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for clinical therapy as antiviral, anticancer as well as immunomodulator. In this study, the open reading frame (ORF) encoding synthetic hIFNα-2a was constructed to be in framed with N-terminal alpha factor secretion system in methylotropic yeast Pichia pastoris. This research aimed to construct, express and analyse the non-affinity tagged recombinant human interferon alpha-2a in the methilotropic yeast P. pastoris . We used pPICZαB plasmid for cloning and expression vector. The confirmed recombinant plasmid containing the correct DNA sequence of hIFNα-2a was linearized by SacI restriction enzyme, then transformed into P. pastoris genome using electroporation. We screened two multi-copy recombinants in YPDS plates containing Zeocin™. Buffered complex medium containing 0.5 % methanol (BMMY) was used for protein expression for 48 hours in the culture condition. The recombinant protein was purified by blue sepharose...
Human interferon alpha-2b (hIFNα-2b) is a cytokine that used as therapeutic protein in hepatitis ... more Human interferon alpha-2b (hIFNα-2b) is a cytokine that used as therapeutic protein in hepatitis and several cancer treatments. In our previous study, the open reading frame encoding hIFNα-2b was cloned to pPICZαB vector and expressed in methylotropic yeast Pichia pastoris (P. pastoris) under the control of alcohol oxidase 1 (AOX1) promoter. This study was aimed to overproduce, purify and characterize the recombinant human IFN-α2b protein (rhIFNα-2b). The protein was overproduced in BMMY media at 30 °C for 48 hour by using 0.5% methanol as inducer. The rhIFN-α2b purification was performed by immobilized affinity chromatography column using AKTA purifier system. The quantification of purified rhIFN-α2b by using colorimetric bicinchoninic acid assay informed that the yield was 22.43 mg/L culture (OD600 = 2.3). SDS-PAGE and Western blot analyses on protein characterization confirmed that the protein was rhIFN-α2b with 24.050 kDa in size. The rhIFN-α2b was containing c-myc epitope and p...
The choice of culture condition on Pichia pastoris expression system is an important factor not o... more The choice of culture condition on Pichia pastoris expression system is an important factor not only to improve the recombinant protein production but also to avoid proteolysis as well. In our previous work we constructed the human interferon alpha 2b (hIFN-α2b) open reading frame which was overexpressed in P. pastoris. Without any further study of culture condition, the yield was 22 mg in 1 L culture (OD600= 2.3). This research was carried out to study the effect of nonnutritional factors on recombinant human interferon alpha 2b (rhIFNα-2b) production. There were three factors to be studied: induction time (24h, 48h, 72h, 96h and 120h), methanol concentration (0%, 0.25%, 0.5%, 1%, 1.5%, 2% and 2.5%) and temperature of induction (25, 30 and 37°C). Expression level was monitored by using SDS PAGE and relatively quantified by using ImageJ software. The results showed that temperature of induction more than 48 h result in protein degradation and the best expression level was achieved a...
IOP Conference Series: Earth and Environmental Science, 2020
As a workhorse for manufacturing recombinant proteins, ChineseHamster Ovary(CHO)cells are becomin... more As a workhorse for manufacturing recombinant proteins, ChineseHamster Ovary(CHO)cells are becoming an extremely valuable cell for producing recombinant proteins having glycosylation patterns like in human system. Development of cell line, basically, focused on achieving the maximum amount of active proteins by screening the cell line to select higher producing clone. Currently, one of mammalian cell line development technologies used by most biopharmaceutical companies is based on gene amplification technology with methotrexate (MTX) to increase protein expression. With that in mind, the aim of this study is part of an effort to develop cell line capable of producing erythropoietin with 2 additional N-links. Gene amplification was performed on CHO-DG44 cell pools expressing EPO through addition of different concentrations of MTX to medium. The cells were cultured using orbital shaker at the speed of 130 rpm at 37°C and 5% CO2 condition. Stepwise increasing MTX concentrations from 20...
Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein produ... more Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viru...
Roti, keju dan minuman beralkohol seperti bir dan wine adalah jenis makanan dan minuman yang mema... more Roti, keju dan minuman beralkohol seperti bir dan wine adalah jenis makanan dan minuman yang memanfaatkan yeast (ragi) dalam proses pembuatannya. Sejak zaman peradaban Mesir, yeast sudah dikenal dan dimanfaatkan dalam industri roti. Jenis yeast yang digunakan dalam industry tersebut adalah Saccharomyces cereviseae.Ilmu pengetahuan modern telah mengidentifikasi lebih dari 1000 varietas wild-yeast dan tersebar di alam. Mereka ditemukan hidup berasosiasi dengan mikroorganisme sebagai bagian dari habitat normal di perairan, tanah, dan lingkungan air lainnya. Bahkan di udara yang kita hirup untuk bernafas, pada batang batang pohon dan di atas daun-daun tanaman yeast juga bisa ditemukan.
Banyak orang berasumsi, bahwa anemia merupakan penyakit kurang darah, 5L (lesu, lemah, letih, lun... more Banyak orang berasumsi, bahwa anemia merupakan penyakit kurang darah, 5L (lesu, lemah, letih, lunglai dan loyo) adalah serangkaian gejalanya. Untuk mengatasi gejala ini di televisi ataupun media-media cetak sering kita lihat berbagai iklan produkproduk antianemia yang dapat dengan mudah dibeli di pasaran. Masalahnya, apakah benar anemia yang dipahami secara medis cukup hanya diatasi dengan mengkonsumsi obat antianemia saja. Tentu jawabannya tidak sesederhana itu. Secara ilmiah anemia adalah suatu keadaan dimana jumlah sel darah merah atau jumlah Hemoglobin (protein pembawa oksigen) dalam sel darah merah berada di bawah normal (kadar Hb<10g/dl). Sel darah merah membawa oksigen (O2) dari paruparu ke jaringan dan organ-organ tubuh yang akan digunakan sebagai energi.
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