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All Journal HAYATI Journal of Biosciences Jurnal Ilmu Pertanian Indonesia Microbiology Indonesia Biota: Jurnal Ilmiah Ilmu-Ilmu Hayati Indonesian Journal of Biotechnology Jurnal Sumberdaya Hayati Journal of Multidisciplinary Applied Natural Science Makara Journal of Science Jurnal Natural Quagga: Jurnal Pendidikan dan Biologi Bioma : Jurnal Ilmiah Biologi Berita Biologi
Rika Indri Astuti
Departemen Biologi, Fakultas Matematika Dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor, Kampus Darmaga IPB, Bogor 16680
Agriculture, Biological Sciences & Forestry Astronomy Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Earth & Planetary Sciences Education Energy Environmental Science Immunology & microbiology Materials Science & Nanotechnology Mathematics Medicine & Pharmacology Neuroscience Physics

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Modification of a Gene Cassette to Express Proglargine in Pichia pastoris: Elimination of Glu-Ala Repeats, Spacer and C-peptide Sequences Nasution, Uli Julia; Astuti, Rika Indri; Wahyudi, Aris Tri; Hardianto, Dudi; Martius, Efrida
Journal of Multidisciplinary Applied Natural Science Articles in Press
Publisher : Pandawa Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.47352/jmans.2774-3047.368

Abstract

The growing global prevalence of diabetes mellitus has sharply increased the demand for insulin and its analogues. Pichia pastoris is a well-established system for recombinant pro-insulin and its analogues production. However, conventional gene constructs often include additional sequences, such as Glu-Ala repeats, spacer peptides, and c-peptides that complicate downstream processing and reduce efficiency. This study aimed to construct and express a proglargine (PG) gene cassette lacking the Glu-Ala repeats, spacer, and c-peptide in P. pastoris GS115 to obtain a uniform PG protein. The recombinant vector propagated in Escherichia coli TOP10F’, then expressed in P. pastorisGS115. Selected transformants were cultivated in YPG medium, then induced with 1% and 2% methanol daily in BMMY. The optimum methanol concentration further evaluated in ½ BSM induction medium. The result demonstrated that optimal PG expression was achieved with 2% methanol induction in BMMY, producing higher levels than those with ½ BSM. Among the transformants, PG.c2 produced the highest PG protein in BMMY medium induced with 2% methanol. Dot-blot analysis confirmed the secretion of PG, and LC-HRMS analysis demonstrated 100% amino acid sequence coverage of PG, confirming the integrity and completeness of the expressed protein. This study presented a newly modified PG gene cassette, inserted into pPICZαA vector, to express uniform secreted PG in P. pastoris GS115. By simplifying the precursor structure, a more homogeneous precursor product can be obtained, which is expected to simplify purification and also the downstream enzymatic process of PG into mature insulin glargine.
Co-Authors A M Sabbah, Sajida Agung Priyanto, Jepri Ai Hertati, Ai Akmaliyah, Rizna Alfaridza, Annisa Nourma ALFIYAN, ACHMAD Amani, Febriyanti Nur Anja Meryandini Antonius Suwanto Apon Zaenal Mustopa Aris Tri Wahyudi Armanisa, Khafifah Chairunnisa, Sheila Chihombori, Tatenda Calvin DAROJATUL ULYA Dina Aribah DINI NURDIANI Dudi Hardianto, Dudi Efrida Martius Ekawati, Nurlaili Fathiah, Muhammad Fadhil Fatimah Firyal Noviatanti Nabilah Hartono, Faisal Diniamal Iman Rusmana Irawan, Herman Irmanida Batubara Jendri Mamangkey Jepri Agung Priyanto Jun Nomura Lenka, Audria Bayu Lisdar Idwan Sudirman Mahsunah, Anis Herliyati Manguntungi, Baso Martha, Ina Muhammad Zulfiqri, Aldy Mutiara, Ilma Nabila Tsoerayya Gustia Pudjas Nadila Fany Ashafani Nadya Nabila Qurani Nasution, Uli Julia NISA RACHMANIA MUBARIK Nurfadhilla Rahmadhani Nurhakiki Prastya, Muhammad Eka Puspitasari, Dian Japany Puspitasari, Esti Rahayu Wulan Rakhman, Ira Meylan Nafarah Refli, Redoyan Rifqiyah Nur Umami, Rifqiyah Nur RIKA RAFFIUDIN Ririn Herni Putri Rustam, Yepy Hardi Sarah Asih Faulina Sarah Asih Faulina, Sarah Asih Satya, Andreas Adhi Sidiq, Muhammad Raffel Sri Budiarti Sri Budiarti Poerwanto Sri Estuningsih Sri Listiyowati Trinugroho, Joko P Uci Cahlia Wulan Tri Wahyuni Yaya Rukayadi Yohanes Bernadino Putera Saju Zulhaerati Zulhaerati