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"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.

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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."

"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.

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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."

"Gen CSF3syn adalah gen sintetik yang menyandi protein G-CSF. Protein G-CSF dapat diproduksi secara rekombinan. Sel inang alternatif yang dapat digunakan yaitu Pichia pastoris. Penelitian bertujuan untuk menyeleksi P. pastoris transforman yang stabil, mendapatkan P. pastoris transforman yang terintegrasi dengan gen CSF3syn, dan menganalisis ekspresi protein G-CSF pada P. pastoris transforman dengan promotor konstitutif GAP. Sebanyak 47 transforman berhasil diseleksi pada konsentrasi zeosin 1000 µg/ml. Analisis PCR menunjukkan gen CSF3syn sebesar 567 pb berhasil terintegrasi dalam genom P. pastoris. Analisis SDS PAGE, slot blot, dan western blot menunjukkan protein G-CSF berhasil diekspresikan. Analisis western blot menunjukkan G-CSF terglikosilasi ~20 kDa dan tidak terglikosilasi ~18 kDa. Selain itu, terdapat protein dengan berat molekul lebih dari protein target yaitu protein fusi terglikosilasi ~40--60 kDa.

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CSF3syn gene is a synthetic gene that encodes G-CSF protein. G-CSF protein can be produced by recombinant technique. Pichia pastoris can be used as an alternative host. The objectives of this study were to select stability of the P. pastoris transformant, to obtain P. pastoris transformants which were integrated with CSF3syn gene, and expressed G-CSF recombinant in P. pastoris using the constitutive GAP promoter. A total of 47 transformants were selected in YEPD medium with 1000 µg/ml zeocin.

Analyses by PCR confirmed the inserted CSF3syn gene in P. pastoris genome of 567 bp. Analyses of SDS PAGE, western blot, and slot blot showed that the G-CSF protein was expressed successfully. Western blot analyses showed that the bands of ~20 kDa as glycosylated G-CSF and ~18 kDa as non glycosylated G-CSF. The result also showed that the band with higher molecular mass ~40--60 kDa which was probably glycosylated fusion protein."

"ABSTRAKEpidermal 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 rsquo; 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.

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ABSTRACTEpidermal 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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Chikungunya merupakan penyakit menular yang bersifat re-emerging atau penyakit lama yang dapat tersebar kembali. Penyakit yang disebabkan virus chikungunya ini memiliki manifestasi klinis non-spesifik sehingga dibutuhkan metode diagnosis yang cepat dan akurat. Protein E2 yang dikode oleh gen E2 pada virus chikungunya berperan penting sebagai pengikatan reseptor sehingga berpotensi untuk digunakan dalam proses diagnosis penyakit chikungunya. Penelitian ini bertujuan menghasilkan protein rekombinan E2 sebagai bahan dasar produksi antibodi monoklonal yang akan digunakan dalam pengembangan perangkat diagnostik penyakit chikungunya. Metode penelitian yang dilakukan mencakup pembentukan plasmid rekombinan pPICZaA-E2, transformasi plasmid rekombinan pada sel inang Escherichia coli, analisis koloni transforman, transformasi plasmid rekombinan pada sel inang ekspresi Pichia pastoris X-33, analisis fenotipe, hingga ekspresi protein rekombinan E2. Hasil penelitian menunjukkan 281 koloni E. coli transforman pPICZaA-E2 dapat tumbuh pada medium yang mengandung antibiotik zeocin. Hasil analisis PCR koloni transforman menunjukkan gen E2 dengan ukuran 1.260 bp berhasil ditransformasikan ke sel inang E. coli menggunakan vektor pICZaA dengan ukuran 3.569 bp. Hasil analisis PCR genom berhasil mengamplifikasi gen AOX1 berukuran 2,2 kb dan 1,8 kb yang menunjukkan plasmid rekombinan berhasil terintegrasi pada genom P. pastoris dan menghasilkan fenotipe Mut⁺. Pita protein berukuran 40 kDa pada hasil SDS-PAGE menunjukkan protein E2 berhasil terekspresi.

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Chikungunya is known as an infectious, re-emerging disease. Because of the non-specific clinical manifestation, chikungunya needs a rapid and accurate diagnostic method for its detection. Envelope 2 (E2) protein coded by E2 gene in the genome of the virus has an important role as an attachment receptor to a cell that made it potential to be used for diagnosis. This research is aimed to obtain E2 recombinant protein as a basic material for monoclonal antibody production in chikungunya rapid diagnostic test kit development. Chikungunya E2 gene is amplified and ligated with pPICZaA vector to make recombinant DNA clones from E. coli. The clones then isolated and used for protein expression in P. pastoris. The result shows 281 transformants of E. coli colonies can grow on a selection medium that contains zeocin. Analysis of direct colony PCR show E2 gene was transformed and cloned using pPICZaA vector. Analysis of genomic PCR shows 2,2 kb and 1,8 kb bands formed that indicate AOX1 gene was amplified and the integration of recombinant plasmid pPICZaA to P. pastoris genome was successful. Visualization of protein electrophoresis shows protein band was formed at the size of40 kDa that indicate the protein expression was successful.

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"Lipase merupakan salah satu enzim yang penting di industri enzim, dan banyak digunakan dalam pembuatan zat aditif makanan, kosmetik, dan industri farmasi. Penelitian sebelumnya yang dilakukan di PTB-Laptiab BPPT, pengklonaan gen sintetik T. lanuginosus lipase pada B. substilis memiliki aktivitas lipase yang rendah yaitu sebesar 1,488 U/mg. Penelitian ini bertujuan untuk mengklona gen sintetik T. lanuginosus lipase TLL ke dalam vektor ekspresi Pichia pastoris menggunakan sinyal peptida asli TTL. Gen TLL yang mengandung sinyal peptida asli diamplifikasi dengan PCR, dan disisipkan ke dalam pPICZ? A di antara situs XhoI dan XbaI, kemudian ditransformasikan ke dalam sel kompeten E. coli DH5?. Hasil transformasi dipilih dua rekombinan positif untuk dilakukan analisis sekuensing. Hasil sekuensing, kedua rekombinan mengandung gen target lipase. Plasmid yang telah dikonfirmasi kemudian dilinearisasi dan ditransformasikan ke dalam P. pastoris X-33 dengan menggunakan metoda elektroporasi. Gen T. lanuginosus lipase berhasil diintegrasi ke dalam kromosom P. pastoris X-33, yang ditunjukkan dengan terbentuknya zona bening pada media Yeast extract Peptone Dextrose Tributyrin YPD.TB agar yang mengandung zeocin. Thermomyces lanuginosus lipase memiliki daerah open reading frame ORF 916 bp yang mengkode 291 asam amino dengan massa molekul teoritis 35 kDa. Enzim rekombinan T. lanuginosus memiliki suhu optimum 80 C dan pH optimum 8.

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Lipase is one of the most important industrial enzymes, which is widely used in the preparation of food additives, cosmetics, and pharmaceutical industries. In the previous study, we have cloned synthetic Thermomyces lanuginosus lipase gene into Bacillus subtilis and Escherichia coli and resulting low expression of enzyme activity.

The aim of this research was to construct the T. lanuginosus lipase TLL gene into P. pastoris vector expression with TLL original signal peptide. Thermomyces lanuginosus lipase gene was amplified by PCR and contained original signal peptide and then inserted into pPICZ A between XhoI and XbaI site, and transformed into competent cell E. coli DH5. From the transformant, two of positive recombinants were analyzed by sequencing analysis.

As the result, both of two recombinant have a positive target gene which has lipase gene. The correct plasmid was linearized and then was transformed into P. pastoris X 33 by electroporation method. Thermomyces lanuginosus synthetic gene lipase has been successfully integrated into chromosome of P. pastoris X 33, which revealed by clear zones around the colony on Yeast extract Peptone Dextrose Tributyrin YPD.TB plate with zeocin.

The Thermomyces lanuginosus lipase had an open reading frame of 916 bp encoding TLL of 291 amino acids with theoretical molecular mass of 35 kDa. The recombinant enzyme, T. lanuginosus lipase had optimal temperature at 80 C and optimal pH at pH 8.0."

"Munculnya varian virus influenza yang berpotensi menimbulkan pandemi merupakan kejadian yang tidak terduga dan jarang terjadi. Penanganan dalam situasi seperti ini membutuhkan produksi vaksin skala besar dalam waktu singkat seperti pada kasus virus influenza H1N1 pdm09. Salah satu strategi yang dapat dilakukan adalah produksi vaksin subunit dengan memanfaatkan sistem ekspresi ragi untuk memproduksi protein antigen rekombinan. Tujuan penelitian ini adalah mengekspresi protein fusi HA1-MA2-NS1 rekombinan untuk pengembangan vaksin subunit. Plasmid pPICZαA-HA1-MA2-NS1 linear diintegrasikan ke dalam genom Pischia pastoris strain GS115 melalui rekombinasi homolog. Proses transformasi ini dilakukan dengan metode elektroporasi dan menghasilkan 13 klon ragi yang mengandung multiple integran gen fusi HA1-MA2-NS1. Analisis bioinformatika menduga protein fusi memiliki berat molekul 75-85 kDa. Visualisasi protein pada supernatan dan pelet dengan SDS-PAGE memperlihatkan adanya pita protein dengan ukuran yang diharapkan. Hasil western blot pada pellet mendeteksi ukuran protein target antara 42-135 kDa. Pemurnian protein fusi dengan NI-NTA gagal dilakukan dan diduga karena sedikitnya jumlah protein fusi yang terbentuk sehingga tidak terdeteksi pada visualisasi SDS-PAGE.

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The emergence of influenza virus variants that could potentially cause a pandemic is an unforeseen occurrence and rare. Handling a situation like this requires a large-scale vaccine production in a short time as in the case of the H1N1 virus pdm09. One strategy that can be done is a subunit vaccine production by utilizing a yeast expression system to produce recombinant protein antigens.

The purpose of this study was to express a recombinant proteins of fusion HA1- MA2-NS1 for subunit vaccine development. PPICZαA HA1-MA2-NS1 linear plasmid integrated into the genome Pichia pastoris strain GS115 through homologous recombination. This transformation process is carried out by electroporation method and generating 13 yeast clones containing multiple genes HA1-MA2-NS1 integrant. Bioinformatics analysis of protein suspect fusion protein has a molecular weight of 75-85 kDa. Visualization of proteins in the supernatant and pellet by SDS-PAGE showed protein bands with sizes expected.

But western blot results in the pellet detect the target protein size between 42-135 kDa. Purification of fusion proteins with NI-NTA were failed due to small amount of fusion protein that undetected on SDS-PAGE visualization."

"Chikungunya merupakan penyakit menular yang bersifat re-emerging dan ditransmisikan melalui gigitan nyamuk Aedes aegypti & Aedes albopictus. Penyakit yang disebabkan virus chikungunya ini memiliki manifestasi klinis non-spesifik sehingga dibutuhkan metode diagnosis yang cepat dan akurat. Protein E2 yang dikode oleh gen E2 virus chikungunya berpotensi digunakan dalam proses diagnosis penyakit chikungunya. Protein rekombinan E2 diekspresikan pada inang Pichia pastoris-X33 Mut+ koloni B4, F, dan N. Penelitian ini bertujuan menghasilkan protein rekombinan E2 serta didapatkan waktu inkubasi dan konsentrasi induksi metanol optimum pada sistem ekspresi Pichia pastoris. Hasil penelitian ini digunakan sebagai bahan dasar produksi antibodi monoklonal yang akan digunakan dalam pengembangan perangkat diagnostik penyakit chikungunya. Metode yang digunakan pada adalah inokulasi Pichia pastoris pada medium MM dan MD. Induksi koloni pada medium BMGY dan BMMY selama 24, 48, dan 72 jam inkubasi. Koloni diinduksi dengan metanol murni dengan variasi konsentrasi akhir 0,5%, 1%, dan 2%. Verifikasi ekspresi protein melalui SDS-PAGE dan Western blot. Hasil penelitian menunjukkan koloni Pichia pastoris yang membawa gen pPICZaA-E2 dapat mengekspresikan protein E2 berukuran 35-38 pada koloni pada setiap variasi induksi dan waktu inkubasi.Waktu inkubasi terbaik adalah 72 jam dan induksi metnaol akhir terbaik adalah 0,5%

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Chikungunya is known as an infectious disease that re-emerging and transmitted by Aedes aegypti & Aedes albopictus mosquitoes. This chikungunya infectious has non-specific clinical manifestation so it requires a rapid and accurate diagnostic method for its detection. Envelope 2 (E2) protein coded by E2 gene in the genome of the virus has potential to be used for diagnosis. B4, F and N colonies of Pichia pastoris X33 Mut+ used as the expression system. This research is aimed to obtain E2 recombinant protein, then to obtain the optimum of incubation times and methanol induction. This result used as a basic material for monoclonal antibody production in chikungunya rapid diagnostic test kit development. The method used in the expression of E2 recombinant protein in Pichia pastoris host cells was Pichia pastoris inoculation on MM and MD medium. Colony induction on BMGY and BMMY medium for 24, 48, and 72 hours of incubation. Colonies were induced with pure methanol with various final concentrations of 0,5%, 1%, and 2%. Verification of protein expression through SDS-PAGE and Western blot. The results showed that Pichia pastoris colonies carrying the pPICZaA-E2 gene were able to express 35—38 kDa. Protein E2 on SDS-PAGE results indicating that E2 protein was successfully expressed on each induction and times. The optimum incubation time is 72 hours and 0,5 % methanol induction"

"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.

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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."

"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.

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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."