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"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."

"Gen Epidermal Growth Factor Receptor Varian III (EGFRvIII) merupakan mutan dari gen EGFR yang terbentuk akibat mutasi delesi ekson 2 sampai 7. Gen EGFRvIII mengkode ekspresi protein EGFRvIII yang hanya diekspresikan pada sel kanker, sehingga protein ini berpotensi untuk digunakan sebagai molekul target dalam terapi kanker yang ditargetkan. dengan antibodi monoklonal. Fragmen anti-EGFRvIII untai tunggal (scFv) adalah antibodi monoklonal yang secara khusus mengenali epitop unik EGFRvIII. Antibodi ini perlu diuji aktivitasnya dengan protein EGFRvIII. Protein EGFRvIII dapat diproduksi dengan menggunakan plasmid pPICZα-EGFRvIII-bfp. pPICZα-EGFRvIII-bfp adalah plasmid rekombinan dengan promotor AOX1 yang tidak dapat digunakan, yang dirancang untuk diubah menjadi P. pastoris sehingga protein EGFRvIII dapat diekspresikan secara ekstraseluler. Penelitian ini bertujuan untuk mendapatkan klon P. pastoris transforman yang mengandung gen EGFRvIII dan dapat mengekspresikan protein EGFRvIII. Metode transformasi yang digunakan adalah elektroporasi. Seleksi P. pastoris transforman dilakukan dengan antibiotik zeosin. Gen target pada transforman diverifikasi menggunakan PCR koloni. Ekspresi protein target dilakukan dengan menggunakan teknik induksi metanol. Ekspresi protein dilihat di bawah mikroskop fluoresensi dan dianalisis dengan SDS-PAGE. Hasil penelitian menunjukkan klon P. pastoris transforman yang mengandung gen EGFRvIII-bfp (750 bp) berhasil diperoleh, dengan efisiensi transformasi plasmid 0,003 x 103 cfu/μg. Protein EGFRvIII berhasil diekspresikan berdasarkan pendaran biru protein BFP, tetapi diduga mengalami glikosilasi dan masih menyatu dengan sinyal sekresi karena ukuran protein SDS-PAGE (sekitar ~ 66,2 kDa dan ~ 116 kDa) lebih besar dari ukuran target (~ 41,9 kDa).

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Epidermal Growth Factor Receptor Variant III (EGFRvIII) gene is a mutant of the EGFR gene which is formed as a result of deletion mutations of exons 2 to 7. The EGFRvIII gene encodes the expression of the EGFRvIII protein which is only expressed in cancer cells, so that this protein has the potential to be used as a target molecule in cancer therapy. targeted. with monoclonal antibodies. The single-stranded anti-EGFRvIII (scFv) fragment is a monoclonal antibody that specifically recognizes the unique epitope of EGFRvIII. These antibodies need to be tested for their activity with the EGFRvIII protein. The EGFRvIII protein can be produced using the plasmid pPICZα-EGFRvIII-bfp. pPICZα-EGFRvIII-bfp is a recombinant plasmid with the unusable AOX1 promoter designed to be converted into P. pastoris so that the EGFRvIII protein can be expressed extracellularly. This study aims to obtain clones of P. pastoris transforman that contain the EGFRvIII gene and can express the EGFRvIII protein. The transformation method used is electroporation. P. pastoris transforman selection was carried out with zeosin antibiotics. The target genes in the transformants were verified using colony PCR. The target protein expression was carried out using methanol induction technique. Protein expression was viewed under a fluorescence microscope and analyzed by SDS-PAGE. The results showed that P. pastoris transforman clones containing the EGFRvIII-bfp (750 bp) gene were successfully obtained, with a plasmid transformation efficiency of 0.003 x 103 cfu/μg. EGFRvIII protein was successfully expressed based on the blue luminescence of BFP protein, but it was suspected to undergo glycosylation and still fuse with the secretion signal because the size of the SDS-PAGE protein (approximately ~ 66.2 kDa and ~ 116 kDa) was larger than the target size (~ 41.9 kDa) ."

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Epidermal growth factor receptor variant III (EGFRvIII) telah ditemukan sebagai protein reseptor spesifik pada beberapa jenis sel kanker. Protein tersebut merupakan hasil ekspresi dari salah satu jenis mutan gen EGFR, yaitu gen EGFRvIII. Mutasi pada gen EGFRvIII terjadi karena adanya delesi ekson  2  hingga 7 yang merupakan bagian dari domain ekstraselular yang berinteraksi dengan ligan alaminya. Keberadaan EGFRvIII yang spesifik hanya pada sel kanker memberikan potensi bagi protein tersebut untuk dijadikan sebagai molekul target pada terapi penargetan kanker dengan antibodi monoklonal. Fragmen antibodi untai tunggal (ScFv) anti-EGFRvIII merupakan antibodi monoklonal yang dapat digunakan sebagai ligan spesifik terhadap EGFRvIII dalam simulasi terapi penargetan kanker. Antibodi ScFv-anti-EGFRvIII dapat diproduksi menggunakan plasmid pPICZα-ScFv-anti-EGFRvIII-egfp. Plasmid rekombinan ini memiliki promotor indusibel AOX1 dan sinyal sekresi matting factor-a yang dapat ditransformasikan pada Komagataella phaffii sehingga antibodi ScFv-anti-EGFRvIII dapat disekresikan oleh sel inang. Tujuan penelitian ini adalah mendapatkan klona K. phaffii transforman yang membawa gen ScFv-anti-EGFRvIII. Plasmid diisolasi dari Escherichia coli TOP10F` dan dipurifikasi dengan pelarut organik. Plasmid kemudian dipotong dengan enzim SacI sehingga diperoleh plasmid linear yang berukuran 5.150 bp. Metode transformasi yang digunakan adalah elektroporasi. Seleksi K. phaffii transforman dilakukan pada medium seleksi yang mengandung antibiotik zeocin. Hasil penelitian menunjukkan klona K. phaffii transforman yang mengandung plasmid rekombinan  pPICZα-ScFv-anti-EGFRvIII-egfp (5150 bp) berhasil diperoleh. Hasil seleksi K. phaffii transforman dengan replica platting menunjukkan, bahwa seluruh koloni tunggal transforman yang terpilih dapat tumbuh dengan baik pada konsentrasi zeocin 100 μg/mL. Hal tersebut mengindikasikan, bahwa plasmid rekombinan pPICZα-ScFv-anti-EGFRvIII-egfp dapat berintegrasi ke dalam genom K. phaffii secara stabil.

 

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Cancer is a disease caused by abnormal proliferation of cell which can produce fatal effects for an organ system. Epidermal growth factor receptor variant III (EGFRvIII) has been found to be a spesific receptor protein in several tyoes of cancer cells. The protein is expressed by one of many types of mutant from EGFR gene, namely EGFRvIII. Mutation in EGFRvIII gene occur because of deletion on exon 2 to exon 7 that are part of the extracellular domain that interacts with their natural ligand. The specificity of EGFRvIII on cancer cells EGFRvIII provides a potential for the protein to be used as target molecule in cancer-targeting therapy with monoclonal antibodies. Single-chain variable fragment (ScFv) of anti-EGFRvIII is a monoclonal antibody that can be used as specific ligands against EGFRvIII in simulating cancer-targeting theraphy. The ScFv-anti-EGFRvIII antibody can be produced using the pPICZα-ScFv-anti-EGFRvIII-egfp plasmid. This recombinant plasmid has an inducible AOX1 promoter and matting factor-a secretion signal that can be transformed into Komagataella phaffii so that the ScFv-anti-EGFRvIII antibody can be expressed extracellularly. The purpose of this study is to obtain a K. phaffii transformant clone that carries the ScFv-anti-EGFRvIII gene. The plasmid were isolated from Escherichia coli TOP10F` and purified. Plasmid were then cut with the SacI enzym so that a 5.150 bp linear plasmid was obtained. The method of transformation in this study is electroporation. The selection of K. phaffii transformant was carried out on a selection medium containing zeocin antibiotic. The results showed that K. phaffii transformant clone containing pPICZα-ScFv-anti-EGFRvIII-egfp (5150 bp) recombinant plasmid was succesfully obtained. The selection of K. phaffii transforman with replica platting shows that all selected single colony transformants can grow well at zeocin concentration of 100 μg/mL. The result indicate that the pPICZα-ScFv-anti-EGFRvIII-egfp recombinant plasmid can be integrated into K. phaffii genome stably.

 

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"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 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."

"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."

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Protein Granulocyte Colony Stimulating Factor (G-CSF) memiliki peran spesifik dalam menstimulasi poliferasi dan pematangan sel neutrofil. Penelitian sebelumnya, gen CSF3 telah berhasil dikontruksi secara in vitro di dalam vektor pPICZα dengan penyisipan kodon metionin pada ujung-5’ dan kodon stop pada ujung-3’ dari gen CSF3. Kontruksi tersebut menghasilkan pPICZα-metCSF3. Tujuan penelitian ini adalah transformasi plasmid pPICZα-metCSF3 pada sel inang Komagataella phaffii dan melakukan pengujian fenotipe Mut pada sel transforman yang diperoleh. Plasmid pPICZα-metCSF3 diisolasi dari Escherichia coli DH5α dan dilinierisasi sehingga menghasilkan plasmid linier berukuran 4.131 pb. Plasmid rekombinan dipurifikasi dan dikuantifikasi, selanjutnya ditransformasikan ke dalam sel inang K. phaffii dengan metode elektroporasi. Jumlah koloni yang terbentuk berkisar 214 koloni transforman dan nilai efisiesi transformasi mencapai 0,18 x 10³ cfu/µg plasmid DNA. Seleksi koloni transforman dilakukan pada medium YPD dengan konsentrasi zeosin yang bertingkat. Sebanyak 15 dari 23 koloni transfoman memiliki resistensi terhadap seluruh tingkatan konsentrasi zeosin. Pengujian fenotipe Mut pada 23 koloni transforman memiliki fenotipe Mut⁺. Berdasarkan data pengamatan yang diperoleh bahwa K. phaffii berhasil membawa plasmid pPICZα-metCSF3 dan koloni transforman memiliki keberadaan gen aktif AOX1 dan AOX2 (fenotipe Mut⁺).

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Granulocyte Colony Stimulating Factor (G-CSF) protein has a specific role to stimulate proliferation and maturation of neutrophils. Previous research has succeeded an in vitro construction of CSF3 gene on pPICZα vector, while inserting a methionine codon at 5’-end and two stop codons at 3’-end of CSF3 gene sequences. The metCSF3 gene has been formed in recombinant plasmid named pPICZα-metCSF3. The purposes of this research are to transform the pPICZα-metCSF3 recombinant plasmid into Komagataella phaffii host and conducting the phenotype Mut analysis on the transformant colonies. The pPICZα-metCSF3 plasmid was isolated from Escherichia coli DH5α and was linearized to 4.131 bp of plasmid in size. pPICZα-metCSF3 plasmid was purified and quantified, then it was transformed into K. phaffii through electroporation method. Approximately 214 transformant colonies successfully produced and the transformation efficiency reached up to 0,18 x 10³ cfu/µg of DNA plasmid. The transformant was selected on YPD medium with increasing zeocin concentration. Approximately 15 out of 23 transformant were resistant against all stage of zeocin concentration. The determination of Mut phenotype on 23 transformant colonies categorized as Mut⁺ phenotype. In brief, K. phaffii has been succeded to bring pPICZα-metCSF3 plasmid and transformant colonies have active AOX1 and AOX2 gene (Mut⁺ phenotype).

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"Human Epidermal Growth Factor (hEGF) merupakan polipeptida yang terdiri atas 53 asam amino. Protein hEGF berfungsi untuk proliferasi sel epitel dan epidermis secara in vitro dan in vivo. Protein hEGF dikode oleh gen EGF. Gen EGFsyn telah dikonstruksi secara sintetik untuk optimasi kodon pada Escherichia coli. Optimasi kodon berfungsi untuk meningkatkan ekspresi protein rekombinan pada E. coli. Subkloning gen EGFsyn ke plasmid pJ404 yang mengandung gen peptida sinyal endoxylanase sangat diperlukan dalam ekspresi protein hEGF rekombinan pada E. coli. Kendala ekspresi protein rekombinan pada E. coli yaitu terbentuknya badan inklusi di sitoplasma. Peptida sinyal endoxylanase digunakan untuk mentranslokasi protein ke periplasma. Digesti gen EGFsyn dan vektor pJ404 dengan enzim NheI dan BamHI diperlukan untuk persiapan subkloning dan ekspresi protein hEGF ke periplasma. Hasil penelitian menunjukkan bahwa gen EGFsyn dan plasmid pJ404 berhasil dipotong dan siap digunakan untuk subkloning.

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Human Epidermal Growth Factor (hEGF) is a polypeptide consisted of 53 amino acids. Its function is to promote epithel and epidermis cells proliferation in vitro and in vivo. It is encoded by EGF gene. An EGFsyn has been constructed to optimize codon usage in Escherichia coli. Codon optimization enhances recombinant protein expression in E. coli. Subcloning EGFsyn gene to a plasmid carrying endoxylanase signal peptide gene is important for recombinant hEGF expression in E. coli. One of the problem expressing recombinant protein in E. coli is the accumulation of inclusion bodies in cytoplasm. Endoxylanase signal peptide is used to translocate protein to periplasm. Digestion of EGFsyn gene and plasmid pJ404 by enzyme NheI and BamHI is needed for preparation of subcloning and hEGF expression to periplasm. The results showed that EGFsyn gene and plasmid pJ404 was digested and can be used for subcloning."

"Epidermal growth factor receptor variant III (EGFRvIII) is a mutant EGFR lacking 267 amino acids (exon-2 throught 7) within its extracellular domain, resulting in the formation of a new epitope as a tumor-Antibodies ..."