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Resky Dwi Cahyati
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Xilanase merupakan enzim yang dapat mendegradasi xilan menjadi xilooligosakarida berbagai ukuran dengan memotong ikatan 1,4-β-D-xylosidik dan memiliki potensi tinggi dalam aplikasi industri. Pada penelitian sebelumnya, untuk produksi xilanase pendekatan teknologi DNA rekombinan telah dilakukan, yaitu dengan mengkonstruksi Pichia pastoris yang telah dimodifikasi genetik sehingga dapat memproduksi xilanase dari Bacillus halodurans CM1. Peningkatan produksi xilanase menggunakan starter Pichia pastoris telah dilakukan. Namun, karena tingginya biaya medium standar produksi dengan Pichia pastoris pada skala laboratorium dan bioreaktor, maka komposisi media standar disubtitusi dengan medium minimal. Akan tetapi aktivitas xilanase yang dihasilkan relatif rendah. Oleh karena itu dalam penelitian ini dilakukan modifikasi medium berbiaya lebih rendah, dimana gliserol murni disubtitusi dengan gliserol teknis sebagai sumber C, sedangkan sumber N organik yaitu pepton dan yeast ekstrak masing-masing disubtitusi dengan hidrolisat tepung kedelai (18% (b/v) total kandungan N) dan dedak padi (bekatul) (14,63% (b/v) total kandungan N), dan amonium sulfat sebagai tambahan sumber N anorganik yang dioptimasi untuk menghasilkan xilanase dengan harapan biaya yang digunakan lebih murah. Penggunaan gliserol teknis 1% (v/v) dan campuran hidrolisat tepung kedelai 15 g/100 mL, hidrolisat dedak padi 30 g/100 mL,dan amonium sulfat 2,5% (b/v) ditemukan sebagai media sesuai yang menghasilkan tingkat tertinggi aktivitas xilanase (1383,898 U/mL), aktivitas spesifik (861,705 U/mg), kadar protein (1,606 mg/mL), dan berat sel kering (43,300 g/L). Penambahan konsentrasi amonium sulfat meningkatkan produksi xilanase rekombinan sekitar hampir dua kali lipat, dengan persen peningkatan sebesar 97,46%


Xylanase is an enzyme that can degrade xylan into xylooligosaccharides of various sizes using 1,4-β-D-xylosidik bonds and has high potential in industrial applications. In the previous study, to produce xylanase using recombinant DNA technology was done, namely by constructing Pichia pastoris which has facilitated genetics so that it can produce xylanase from Bacillus halodurans CM1. The increase in xylanase production using starter has been done by Pichia pastoris. However, due to the high cost of standard production with Pichia pastoris on a laboratory scale and bioreactor, the composition of standard media is substituted with a minimum medium. However, the xylanase activity produced is relatively low. Therefore in this study modification of the lower cost medium, pure glycerol was substituted with technical glycerol as karbon source, while the organic nitrogen source is peptone and yeast extract substituted with soybean hydrolyzate (18% (b/v) total N content) and rice bran (14.63% (b/v) total N content), respectively,and ammonium sulfate as an additional source of nitrogen inorganic which was optimized to produce xylanase in the hope that costs are cheaper. The use of technical glycerol 1% (v/v) and a mixture of 15 g/100 mL soybean hydrolyzate, rice bran hydrolyzate 30 g/100 mL, and 2.5% (b/v) ammonium sulfate were found to be suitable media that yielded profit high xylanase activity (1383,898 U/mL), specific activity (861.705 U/mg), protein content (1,606 mg/mL, based bradford method), and dry cell weight (43,300g/L). The addition of ammonium sulfate concentrations increased the production of recombinant xylanase by almost double, with a percent increase of 97,46%

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Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam, 2020
T55461
UI - Tesis Membership  Universitas Indonesia Library
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Sembiring, Enny Rimita
"Granulocyte Colony Stimulating Factor (G-CSF) merupakan faktor pertumbuhan hematopoetik yang berfungsi merangsang proliferasi dan diferensiasi neutrofil. Protein G-CSF rekombinan yang dikembangkan dan diproduksi menggunakan sel inang Escherichia coli dan Chinese Hamster Ovary (CHO) masih memiliki kelemahan, sehingga pada penelitian ini dikembangkan suatu produk biosimilar G-CSF rekombinan menggunakan sel inang Pichia pastoris. Fokus penelitian ini adalah memproduksi dan mempurifikasi protein G-CSF rekombinan. Produksi protein rekombinan dilakukan dengan menginduksi kultur menggunakan metanol konsentrasi 0,5% tiap 12 jam dan dilakukan sampling terhadap kultur pada jam ke-0, 12, 24, 36 dan 48. Hasil analisis western blot menunjukkan adanya peningkatan produksi protein rekombinan tiap 12 jam. Protein G-CSF rekombinan dipresipitasi menggunakan amonium sulfat konsentrasi 80%, kemudian didialisis. Konsentrasi protein total diukur dengan spektrofotometer menggunakan metoda Bicinchoninic Acid (BCA). Hasil pengukuran menunjukkan konsentrasi protein total tertinggi adalah sampel protein yang dipresipitasi dengan 80% amonium sulfat. Selanjutnya, purifikasi dilakukan menggunakan teknik kromatografi afinitas dengan resin Ni-NTA. Hasil analisis SDS PAGE menunjukkan protein GCSF rekombinan berukuran 18,5 kDa dan dengan analisis slot blot terdeteksi berwarna ungu.

Granulocyte Colony Stimulating Factor (G-CSF) is a hematopoietic growth factor that acts to stimulate neutrophilic proliferation and differentiation. Recombinant protein G-CSF developed and produced using cellular host Escherichia coli and Chinese hamster ovary (CHO) still has a weakness, so that in this study we developed a bio similar product of recombinant G-CSF using cellular host Pichia pastoris. The aim of this research was to produce and purify recombinant protein G-CSF. Production of recombinant protein was done by inducing culture with methanol 0.5% every 12 hours and sampling was carried out at 0, 12, 24, 36 and 48 hours. The results of western blot analysis showed an increase the production of recombinant protein every 12 hours. Recombinant protein G-CSF was precipitated using ammonium sulfate 80% of concentration, and then dialyzed. Concentration of total protein was measured by a spectrophotometer using the Bicinchoninic Acid (BCA) method. The measurement results showed the highest concentrations of total protein was present in samples that precipitated with 80% ammonium sulfate. Furthermore, purification performed using affinity chromatography techniques with Ni-NTA resin. The results of SDS PAGE analysis showed the recombinant protein G-CSF sized 18.5 kDa and with a slot blot analysis detected a purple color."
Depok: Universitas Indonesia, 2012
S1689
UI - Skripsi Open  Universitas Indonesia Library
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Dian Fairuza
"Lignoselulosa dapat dijadikan sebagai biomassa untuk menghasilkan produk bahan bakar. Hidrolisis biomassa lignoselulosa menggunakan enzim selulase. Selulase mengandung dari 3 komplek enzim yaitu, eksoglukanase, endoglukanase dan betaglukosidase Namun, betaglukosidase memiliki jumlah lebih sedikit daripada eksoglukanase dan endoglukanase. Semakin sedikit betaglukosidase dapat memicu proses hidrolisis selulosa terhambat, oleh karena itu pengembangan betaglukosida perlu dilakukan dengan diekpresikan ke dalam Pichia pastoris. Transformasi plasmid pLIPI-TnBgl1A dilakukan dengan metode elektroporasi, sedangkan ekspresi gen dan hasil purifikasi protein rekombinan dianalisis menggunakan SDS-PAGE dan Western blot. Gen betaglukosidase dari Thermotoga neapolitana berhasil ditransformasikan kedalam Pichia pastoris. Transforman yang telah diseleksi menghasilkan 2 koloni positif. Berat molekuler protein diperkirakan sekitar 53 kDa dan jumlah protein estimasi 1 mg/mL dan 1,4 mg/mL. Hasil analisis kemurnian protein rekombinan melalui SDS PAGE dan western blot memperlihatkan pita tepat di 53 kDa. Jumlah yield protein yang terpurifikasi didapatkan sekitar 21,4 % dan 24,1%. Hasil menunjukkan bahwa gen TnBgl1A telah berhasil ditransformasi dan terekspresikan dengan baik di Pichia pastoris dan protein rekombinan berhasil dipurifikasi dengan kemurnian yang cukup baik.

Lignocellulose can be used as biomass to produce fuel products. Hydrolysis of lignocellulosic biomass using the cellulase enzyme. Cellulase contains 3 enzyme complexes, there are exoglucanase, endoglucanase and betaglucosidase. However, betaglukosidase has less amount than exoglucanase and endoglucanase. The less betaglucosidase can trigger the cellulose hydrolysis process is inhibited, therefore the development of betaglucoside needs to be done by expressing it into Pichia pastoris. Transformation of the pLIPI-TnBgl1A plasmid was performed by electroporation method, while gene expression and recombinant protein purification results were analyzed using SDS-PAGE and Western blot. The betaglucosidase gene from Thermotoga neapolitana was successfully transformed into Pichia pastoris. Transformants that have been selected produce 2 positive colonies. The molecular weight of protein is estimated to be around 53 kDa and the estimated protein amount is 1 mg/mL and 1.4 mg/mL. The results of the analysis of recombinant protein purity through SDS PAGE and western blot show the right band at 53 kDa. The amount of purified protein yield was around 21.4% and 24.1%. The results showed that the TnBgl1A gene was successfully transformed and well expressed in Pichia pastoris and the recombinant protein was purified with good purity.
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Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2019
T54890
UI - Tesis Membership  Universitas Indonesia Library
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Emilia Rahmadaniah Utami
"Epidermal growth factor receptor variant III (EGFRvIII) adalah salah satu varian mutan dari protein human EGFR. Mutasi yang terjadi pada EGFR menyebabkan terjadinya kanker. Berbagai mutan EGFR, termasuk EGFRvIII, telah banyak dipelajari karena potensinya sebagai molekul target dalam terapi kanker. Gen penyandi domain ekstraselular EGFRvIII telah berhasil dikonstruksi pada penelitian sebelumnya untuk studi ekspresi protein sebagai molekul target dalam terapi kanker. Penelitian bertujuan untuk subkloning gen EGFRvIII ke dalam plasmid ekspresi pPICZ? dan transformasi plasmid rekombinan ke dalam sel Pichia patoris SMD1168H. Fragmen gen EGFRvIII diperoleh melalui amplifikasi secara in vitro dengan teknik PCR plasmid pJ404-EGFFRvIII-bfp. Gen EGFRvIII tersebut telah terfusi dengan gen bfp penyandi blue fluorescent protein BFP pada ujung-C. Fusi gen tersebut disubklon ke dalam plasmid pPICZ? pada situs XhoI untuk memperoleh plasmid pPICZa-EGFRvIII-bfp. Plasmid rekombinan ditransformasikan ke dalam sel E. coli TOP10 F dengan metode kejut panas. Plasmid rekombinan diseleksi dan dikarakterisasi dengan analisis PCR dan sequencing. Plasmid yang telah dikonfirmasi susunan basa dan ukurannya ditransformasikan ke dalam sel P. pastoris SMD1168H dengan metode elektroporasi untuk ekspresi protein rekombinan. Hasil penelitian menunjukkan bahwa fusi gen EGFRvIII-bfp 1317 bp telah berhasil disubklon ke dalam plasmid pPICZ? dan plasmid rekombinan pPICZ?-EGFRvIII-bfp berhasil ditransformasikan ke dalam P. patoris SMD1168H dengan efisiensi transformasi sebesar 90 CFU/ g DNA plasmid.

Epidermal growth factor receptor variant III EGFRvIII is one of the mutant variant of the EGFR protein. Mutations that occur in EGFR cause cancer. Various mutants of EGFR, including the mutant variant III have been widely studied because of their potential as target molecules in cancer therapy. The extracellular domain coding EGFRvIII gene has been successfully constructed in previous studies for the study of protein expression as a molecule target in cancer therapy. The objective of research are to subclone the EGFRvIII gene into the pPICZ expression plasmid then recombinant plasmid transformation into Pichia patoris SMD1168H cells. Epidermal growth factor receptor variant III EGFRvIII gene fragment was obtained with in vitro amplification by PCR of pJ404 EGFFRvIII bfp plasmid. Epidermal growth factor receptor variant III EGFRvIII gene has been fused with the bfp gene encoding blue fluorescent protein BFP at the C end. The gene fusion was subcloned into the pPICZ at the XhoI site to obtain the pPICZa EGFRvIII bfp plasmid. Recombinant plasmid was transformed into E. coli TOP10 F 39 cells by heat shock method. Recombinant plasmids were selected and characterized by PCR analysis and sequencing. The confirmed plasmid of the base structure and size is transformed into the P. pastoris SMD1168H cell by an electroporation method for the expression of the recombinant protein. Results showed that the fusion of the EGFRvIII bfp 1317 bp gene was successfully subcloned into the pPICZ and the recombinant plasmid pPICZ EGFRvIII bfp was successfully transformed into P. patoris SMD1168H with a transformation efficiency of 90 CFU g DNA plasmid.
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Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2017
S68714
UI - Skripsi Membership  Universitas Indonesia Library
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Yora Permata Dewi
"Infeksi DENV masih menjadi masalah kesehatan masyarakat di Indonesia karena dapat menyebabkan penyakit berat dan bahkan mungkin berakibat fatal. Pengembangan vaksin rekombinan dengan antigen yang mampu dengan efektif menginduksi respon imun perlu untuk dikembangkan. Kesesuaian genotipe yang digunakan di vaksin dan genotipe yang beredar di suatu wilayah berimplikasi terhadap keberhasilan pengembangan vaksin. Plasmid rekombinan yang dirancang berdasarkan gen prM-E DENV-2 strain 151 diekspresikan di Pichia pastoris strain X-33. Telah dilakukan optimasi ekspresi dan antigenisitas protein rekombinan prM-E. Diperoleh 4 koloni P. pastoris rekombinan dengan fenotipe Mut . Hasil SDS-PAGE dan Western blot menunjukkan protein telah berhasil diekspresikan pada ukuran 50 kDa. Kondisi ekspresi optimum protein rekombinan prM-E DENV-2 yaitu pada konsentrasi metanol 1 dengan waktu inkubasi 48 jam. Protein rekombinan prM-E DENV-2 dikenali oleh antibodi anti- prM-E DENV-2 dan bereaksi silang dengan antibodi anti-prM-E DENV-1, DENV-3, serta DENV-4. Protein rekombinan prM-E DENV-2 yang diperoleh dapat digunakan sebagai antigen dalam pengembangan vaksin protein rekombinan dengue strain Indonesia.

DENV infection is still a public health problem in Indonesia because it can cause severe illness and may even be fatal. Development of recombinant vaccine with antigens capable of effectively inducing an immune response needs to be developed. The suitability of genotypes used in vaccines and genotypes circulating in a region has implications for the successful development of vaccines. Construction of a recombinant plasmid based on prM E gene of DENV 2 strain 151 was used for expression in Pichia pastoris strain X 33. Optimization and antigenicity of DENV 2 prM E recombinant protein were tested. Four Mut phenotypes were generated. SDS PAGE and Western blot analysis showed that the protein was expressed with a molecular weight of 50 kDa. Optimal protein expression level occurred at concentration of 1 methanol with 48 hours incubation time. DENV 2 prM E recombinant protein was recognized by anti prM E DENV 2 and also showed cross reaction with anti prM E DENV 1, DENV 3, and DENV 4 antibodies. Thus, the DENV 2 prM E recombinant protein can be used as an antigen in the development of the recombinant protein vaccine of the dengue strains of Indonesia. "
Jakarta: Fakultas Kedokteran Universitas Indonesia, 2017
T-Pdf
UI - Tesis Membership  Universitas Indonesia Library
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Anggia Oktaviani Dwi Putri
"Gen CSF3syn adalah gen sintetis yang dibangun secara in vitro menggunakan teknik PCR, yang mengkode Human Granulocyte Colony-Stimulating Factor (hG-CSF) yang termasuk dalam hematopoiesis sitokin. Protein hG-CSF berperan dalam merangsang proliferasi, diferensiasi, dan pematangan sel progenitor granulosit. Penelitian sebelumnya telah berhasil memasukkan kodon metionin ke ujung 5 'dari gen CSF3syn, menghasilkan gen metCSF3syn. Penelitian ini bertujuan untuk mengubah vektor rekombinan pGAPZα-metCSF3syn menjadi strain Pichia pastoris SMD1168H, untuk mengekspresikan protein met-hG-CSF sebagai biosimilar dari filgastrim. Vektor rekombinan pGAPZα-metCSF3syn diisolasi dari Escherichia coli DH5α, kemudian dilinierisasi menggunakan enzim restriksi BamHI. Vektor rekombinan diubah menjadi P. pastoris menggunakan teknik elektroporasi. Hasil penelitian menunjukkan bahwa koloni P. pastoris transforman tumbuh pada media YPD yang mengandung 100 μg / mL zeosin. Koloni transforman kemudian diseleksi pada medium yang sama dengan konsentrasi zeosin 500 μg / mL. Semua koloni yang diuji tumbuh dengan baik pada media seleksi, menunjukkan bahwa sel transforman secara genetik stabil dan resisten terhadap zeosin. Verifikasi gen metCSF3syn dilakukan dengan teknik koloni PCR, diperoleh 7 dari 8 klon positif yang menunjukkan pita gen metCSF3syn sebesar 532 bp yang menunjukkan klon tersebut mengandung gen target dalam genomnya. Analisis SDS-PAGE menunjukkan klon yang membawa gen metCSF3syn berhasil mengekspresikan protein met-hG-CSF dengan berat molekul 18,8 kDa, sesuai dengan bobot molekul teoritisnya. Hasil kuantifikasi protein pada analisis Western blot menunjukkan bahwa klon nomor 1 memiliki tingkat ekspresi tertinggi. Peningkatan ekspresi protein met-hG-CSF dan jumlah sel P. pastoris transforman berbanding lurus dengan waktu kultur menggunakan metode kultur sistem fed-batch.

CSF3syn gene is a synthetic gene constructed in vitro using PCR technique, which encodes Human Granulocyte Colony-Stimulating Factor (hG-CSF) which is included in cytokine hematopoiesis. The hG-CSF protein plays a role in stimulating the proliferation, differentiation, and maturation of granulocyte progenitor cells. Previous research has succeeded in inserting a methionine codon into the 5 'end of the CSF3syn gene, resulting in the metCSF3syn gene. This study aims to convert the recombinant vector pGAPZα-metCSF3syn into a strain of Pichia pastoris SMD1168H, to express the met-hG-CSF protein as a biosimilar from filgastrim. The recombinant vector pGAPZα-metCSF3syn was isolated from Escherichia coli DH5α, then linearized using BamHI restriction enzyme. The recombinant vector was converted into P. pastoris using electroporation techniques. The results showed that P. pastoris transforman colonies grew on YPD media containing 100 μg / mL zeosin. Transformant colonies were then selected on the same medium with a zeosin concentration of 500 μg / mL. All the colonies tested grew well on the selection media, indicating that the transformant cells were genetically stable and resistant to zeosin. Verification of the metCSF3syn gene was carried out using PCR colony technique, obtained 7 out of 8 positive clones which showed the metCSF3syn gene band of 532 bp, indicating that the clone contained the target gene in its genome. SDS-PAGE analysis showed a clone carrying the metCSF3syn gene was successful in expressing the met-hG-CSF protein with a molecular weight of 18.8 kDa, according to its theoretical molecular weight. The results of protein quantification in Western blot analysis showed that clone number 1 had the highest expression level. The increase of met-hG-CSF protein expression and the number of transforman P. pastoris cells were directly proportional to the culture time using the fed-batch system culture method."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2019
S-Pdf
UI - Skripsi Membership  Universitas Indonesia Library
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Putty Alifa Melyani
"Penelitian bertujuan untuk memperoleh Pichia pastoris transforman yang mengandung gen anti-TfR-scFv dan mengekspresikan protein rekombinan anti-TfR-scFv. Protein tersebut merupakan fragmen antibodi untai tunggal (singlechain-variable-domain, scFv) yang mengenali protein reseptor transferin yang dijumpai pada permukaan sel manusia. Gen anti-TfR-scFv telah diklon ke dalam vektor ekspresi pPICZ di bawah kontrol promoter terinduksi PAOX1 dan di fusi dengan sinyal sekresi MF- (mating factor-) dari Saccharomyces cerevisiae. Gen anti-TfR-scFv juga di fusi dengan gen EGFP (enhanced green fluorescent protein) pada ujung-C. Vektor rekombinan pPICZα_TfR_EGFP ditransformasi ke dalam P. pastoris SMD1168H menggunakan teknik elektroporasi.
Hasil penelitian menunjukkan bahwa vektor rekombinan berhasil ditransformasikan ke dalam genom P. pastoris. Sebanyak 22 koloni transforman berhasil diperoleh dengan tingkat efisiensi transformasi sebesar 0,062x103 cfu/μg DNA. Proses seleksi transforman dilakukan pada medium seleksi YPD agar yang mengandung zeocin. Uji fenotipe Mut (methanol utilization) terhadap tujuh klona transforman memperlihatkan dua klona termasuk Mut+, empat klona MutS dan satu klona Mut-. Protein rekombinan telah berhasil diekspresikan secara ekstraselular. Visualisasi menggunakan mikroskop flouresen menunjukkan adanya pendaran fluoresen dari protein EGFP pada P. pastoris transforman yang mengindikasikan bahwa protein rekombinan telah terekspresi dengan benar. Analisis SDS-PAGE dan Western blotting menunjukkan protein rekombinan (±50kDa) berhasil dideteksi.

This research aimed to obtain Pichia pastoris transformant containing anti-TfRscFv gene which express anti-TfR-scFv recombinant protein. This recombinant protein consist of a single-chain-variable-domain (scFv) recognizing the extracellular domain of human_transferrin_receptor found on the surface of human cell. The anti-TfR-scFv gene was cloned into pPICZ expression vector under the control of inducible promoter PAOX1 and fused with MF- (mating factor-) secretion signal from Saccharomyces cerevisiae. The gene was also fused at the C-terminal with EGFP (enhanced-green-fluorescent-protein) reporter gene. The recombinant vector pPICZα_TfR_EGFP has been transformed into P. pastoris SMD1168H using electroporation technique.
The results showed that the recombinant vector has been successfully transformed into P. pastoris genome. A number of 22 transformant colonies has been obtained with a transformation efficiency number of 0,062x103 cfu/μg DNA. Screening process of transformants was carried out on YPD agar medium containing zeocin. Assay on the Mut (methanol utilization) phenotype of seven transformant clones showed that two of them are Mut+, four are MutS and one is Mut-. The recombinant protein was successfully expressed and secreted from the cell. Visualization using fluorescence microscopy showed fluorescent light coming out from the transformant cells, proving that the recombinant protein has been correctly expressed. The SDS-PAGE and Western blotting analyses showed that the recombinant protein (±50kDa) has been detected.
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Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2014
S56462
UI - Skripsi Membership  Universitas Indonesia Library
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Prety Ihda Arfia
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2010
S31655
UI - Skripsi Open  Universitas Indonesia Library
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Andri Wardiana
"ABSTRACT
For clinical purposes, pure protein and identification of carbohydrate structure from recombinant erythropoietin are needed. Purification was done by Immobilized Metal Affinity Chromatography (IMAC) column charged with Ni2+(His-Trap affinity chromatography) and continued with gel filtration chromatogram phy column to get purer protein. The carbohydrate group which is o ligosaccharide from the resulting pure protein then can be recognized by using N- and O-glycosidase. Pure oligosaccharide was hydrolyzed to produce various monosaccharide through incubation with 4 N HCl in 100oC temperature for 6 hours and the result was applied on High Performance Liquid Chromatography (HPLC) column to learn the composition of its monosaccharide."
[Direktorat Riset dan Pengabdian Masyarakat UI;Lembaga Ilmu Pengetahuan Indonesia. Pusat Penelitian Bioteknologi;Lembaga Ilmu Pengetahuan Indonesia. Pusat Penelitian Bioteknologi, Lembaga Ilmu Pengetahuan Indonesia. Pusat Penelitian Bioteknologi], 2011
J-Pdf
Artikel Jurnal  Universitas Indonesia Library
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"Eritropoietin (EPO) adalah hormon glikoprotein yang terdiri dari 165 asam amino dan memiliki berat molekul sebesar 30.400 Daltons. Sebagian besar kebutuhan EPO didapatkan dari hasil sintesis pada sel
mamalia Chinese hamster ovary (CHO). Pichia pastoris adalah sejenis khamir yang populer digunakan untuk menggantikan sistem ekspresi pada sel mamalia. P. pastoris dapat menggunakan metanol sebagai satu-satunya sumber energi karbon. Pada studi ini, protein rekombinan EPO (rhEPO) disintesis dengan cara mengekspresikan gen hEPO pada khamir metilotropik P. pastoris strain X33. Studi ini dilakukan untuk mengetahui konsentrasi metanol dan waktu
inkubasi yang optimal untuk mensintesis rhEPO. Pada studi ini konsentrasi metanol yang digunakan adalah 0%, 0.5%, 1%, 2.5%, 5%, 10%, dan 20%. Sedangkan waktu inkubasi yang digunakan adalah 0 jam, 24 jam, 48 jam, 72 jam, 96 jam, 120 jam, and 144 jam. Hasil penelitian ini menunjukkan bahwa ekspresi protein yang tertinggi terjadi pada
konsentrasi metanol sebesar 2.5% dan waktu inkubasi selama 48 jam.

Abstract
Erythropoietin (EPO) is a glycoprotein hormone consists of 165 amino acids and has molecular mass of 30,400 Daltons. The large quantities of these hormone required to satisfy clinical demand are currently met by recombinant expression in mammalian cell, namely chinese hamster ovary (CHO). Pichia pastoris has become popular yeast based protein
production systems to substitute mammalian expression systems.
P. pastoris is capable to use methanol as sole carbon and energy source. In this study, recombinant human EPO (rhEPO) protein obtained by expressing the hEPO gene in methylotropic yeast
P. pastoris, strain X33. The present work was carried out to study the optimal methanol concentration for induction and the incubation time to obtain rhEPO protein. To perform this study, the transformed
P. pastoris was induced with various concentrations of methanol (0%, 0.5%, 1%, 2.5%, 5%, 10%, and 20%) and incubation times (0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours). The results demonstrate that the highest protein expression level occurred at concentration of 2.5% methanol induction, while the optimal incubation time was at 48 hrs. "
[Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia, Lembaga Ilmu Pengetahuan Indonesia (LIPI). Pusat Penelitian Bioteknologi], 2012
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Artikel Jurnal  Universitas Indonesia Library
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