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All Journal Jurnal Gizi dan Pangan Jurnal Akuakultur Indonesia ACTA VETERINARIA INDONESIANA Hemera Zoa Jurnal Sain Veteriner Jurnal Veteriner Statistika Jurnal Natur Indonesia Jurnal Ilmu Ternak Veteriner Journal of Innovation and Applied Technology EduChemia: Jurnal Kimia dan Pendidikan ANNALES BOGORIENSES Makara Journal of Science JUARA: JURNAL WAHANA ABDIMAS SEJAHTERA Jurnal Seni dan Reka Rancang: Jurnal Ilmiah Magister Desain E-JRM Jurnal Kedokteran Hewan Kohesi: Jurnal Sains dan Teknologi Annales Bogorienses
Retno Damayanti Soejoedono
Department Of Animal Disease & Veterinary Public Health, Faculty Of Veterinary Medicine, IPB University, Bogor 16680, Indonesia
Agriculture, Biological Sciences & Forestry Arts Humanities Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Computer Science & IT Decision Sciences, Operations Research & Management Economics, Econometrics & Finance Education Energy Engineering Environmental Science Health Professions Immunology & microbiology Industrial & Manufacturing Engineering Materials Science & Nanotechnology Mathematics Medicine & Pharmacology Social Sciences Veterinary Other

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Journal : Annales Bogorienses

 1 
Construction and Expression of Immunotoxin Anti EGFRvIII scFv-HPR Conjugate in Pichia pastoris as A Targeted Drug Candidate for Cancer Therapy Yuliawati, Yuliawati; Soejoedono, Retno Damayanti; Fuad, Asrul Muhamad
Annales Bogorienses Vol. 18 No. 1 (2014): Annales Bogorienses
Publisher : BRIN

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Abstract

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant of EGFR lacking 267 amino acids (exon-2 through 7) within its extracellular domain, resulting in the formation of a new epitope as a tumor specific target. EGFRvIII is commonly found in GBM, breast, ovarian, prostate, and lung carcinomas. Antibodies or part of antibodies (e.g. single chain variable fragment or scFv) with specific activity against this receptor have been developed. These antibodies are internalized into the cell after receptor binding. Ribonucleases can be cytotoxic due to their inherent ability to degrade RNA, therefore causing cell death via inhibition of protein synthesis. The aim of this research is to construct, clone and express an immunotoxin-based conjugate combining an anti-EGFRvIII scFv with a HPRmut (human pancreatic ribonuclease mutant variant) in Pichia pastoris. The anti-EGFRvIII scFv gene was cloned into yeast expression vector pPICZαA and fused with EGFP gene as a marker. The HPRmut gene was subsequently cloned at the C-terminal of the scFv::EGFP fusion, resulting in the scFv::EGFP::HPR fusion construct. The fusion construct was successfully obtained and nucleotide sequence of plasmid was verified. This construct was used to transform P. pastoris SMD 1168H. The gene fusion was successfully transformed and expressed in P. pastoris with a transformation efficiency of 102 cfu/μg DNA. The transformed yeasts were screened on agar media containing up to 1000 μg/ml zeocin. Yeast transformants showed green fluorescent due to the expression of EGFP gene. The recombinant proteins have been expressed and secreted from P. pastoris as showed by immunoblotting and SDS-PAGE analyses, then purified by affinity chromatography method. The selected yeast transformant produced at least 15.85 mg of purified protein per liter of yeast culture. 
Co-Authors . Darmawi Achmad Agus Priyono Adji, Rahmat Setya Agus Setiyono Agus Wahyudi Agustin Indrawati Anneke Anggraeni Anugrah Nur Rahmanto Ardiansyah, Romadhony Asrul Muhamad Fuad Asrul Muhamad Fuad, Asrul Muhamad Assaat, Lusiani Dewi Ayi Santika Bagas Setya Dian Nugraha Bagus Nanang Luwito Bambang Susilo BIBIANA W LAY Darmawi D Dewi Elfidasari Dwi Desmiyeni Putri Ekowati Handharyani Emilia A Emilia A, Emilia Erick Teguh Leksono Etih Sudarnika Evy Damayanthi Fachriyan Hasmi Pasaribu Fachriyan Hasmi Pasaribu Fachriyan Hasymi Pasaribu Fachriyan Hasymi Pasaribu Fajriah, Sofa Fitrianti Rahayu Fitriyana, Intan Nur Hadri Latif Hana Fitria Navratilova Handika Martu Kurniawan Hapsari Mahatmi Harry Tetra Antono I Nyoman Suartha I wayan Teguh Wibawan Irvan Faizal Iskandar, Layla Nurina Kartika Ivandini Tribidasari Anggraningrum Ketut Karuni Nyanakumari Natih Komar Sumantadinata Lilis Suryani M. Lutfi Maggy Thenawidjaja Suhartono Makmur iknu wijaya Mangaraja Pidoli Tampubolon Melani Wahyu Adiningsih Mirnawati Sudarwanto Monika Danaparamitha Andriani Muhammad Hambal Natih, Ketut Karuni Nyanakumari Ni Luh Putu Ika Mayasari Nova Dilla Yanthi Novera Nirmalasanti Nurhidayah Nurhidayah Okti Nadia Poetri R.A. Heryani Wahyuningrum Rahmat Setya Adji Ramlah Ramlah Razali Daud Razali Daud Retno Wijayanti Risa Tiuria Risma Juniarti Silitonga Rizky Alviodinasyari Rudy Ariyanto Saepudin, Endang Sandra Sandra Siti Gusti Ningrum, Siti Gusti sri murtini . Sri Nuryati Suherman . Sukenda . Surachmi Setiyaningsih Sutanti, , Syafril Daulay Syahruddin Said TARUNI SRI PRAWAST MIEN KAOMINI ANY ARYANI DEDY DURYADI SOLIHIN Trioso Purnawarman Ummu Balqis Usman Nurhasan Wijaya, Makmur Yuliawati Yuliawati Yuliawati Yus Rusila Noor Yus Rusila Noor Yusuf Hendrawan