KARAKTERISASI DAN PENETAPAN KADAR PREKURSOR INSULIN YANG DIPRODUKSI OLEH REKOMBINAN Pichia pastoris

  • Dudi Hardianto Pusat Riset Vaksin dan Obat, Organisasi Riset Kesehatan, BRIN, Laptiab G610-614 Puspiptek, Tangerang Selatan, Indonesia
  • Fahroziah Assyifa Laboratorium Bioteknologi, Deputi Bidang Fasilitas Riset dan Inovasi, BRIN, Laboratorium Bioteknologi G630 Puspiptek, Tangerang Selatan, Indonesia
  • Efrida Martius Pusat Riset Hortikultura dan Perkebunan, Organisasi Riset Pertanian, BRIN, Laptiab G610-614 Puspiptek, Tangerang Selatan, Indonesia
  • Dihan Laziba Laboratorium Bioteknologi, Deputi Bidang Fasilitas Riset dan Inovasi, BRIN, Laboratorium Bioteknologi G630 Puspiptek, Tangerang Selatan, Indonesia
  • Juwartina Ida Royani Pusat Riset Hortikultura dan Perkebunan, Organisasi Riset Pertanian, BRIN, Laptiab G610-614 Puspiptek, Tangerang Selatan, Indonesia
  • Triyani Sumiati Sekolah Tinggi Teknologi Industri dan Farmasi Bogor
  • Anna Safarrida Pusat Riset Rekayasa Genetika, Organisasi Riset Hayati dan Lingkungan, BRIN, Bioteknologi G630 Puspiptek, Tangerang Selatan, Indonesia
Keywords: Diabetes melitus, Pichia pastoris, prekursor insulin

Abstract

Jumlah penderita diabetes melitus meningkatkan setiap tahunnya sehingga kebutuhan insulin meningkat. Insulin merupakan obat utama untuk pasien diabetes mellitus tipe 1 (DMT1). Dalam beberapa kasus, diabetes mellitus tipe 2 (DMT2) memerlukan insulin untuk pengobatannya. Saat ini, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, dan Hansenula polymorpha merupakan sistem ekspresi yang digunakan secara luas untuk memproduksi prekursor insulin. Prekursor insulin diubah menjadi insulin secara enzimatik. Pada penelitian ini, digunakan Pichia pastoris untuk memproduksi prekursor insulin karena mampu memproduksi prekursor insulin dalam jumlah besar dan disekresikan ke luar sel Pichia pastoris sehingga mempermudah proses pemurnian. Penelitian diawali dengan regenerasi dan seleksi Pichia pastoris. Pichia pastoris terpilih ditumbuhkan dalam media fermentasi kemudian prekursor insulin yang dihasilkan dikarakterisasi dengan elektroforesis SDS-PAGE dan kadar insulin diukur dengan Elisa. Hasil karakterisasi menunjukkan prekursor insulin berupa pita berukuran sekitar 7 KDa dengan kadar prekursor insulin 6,88 µUI/mL.

References

[1] Hardianto, D. 2020. Telaah komprehensif Diabetes Melitus: Klasifikasi, Gejala, Diagnosis, Pencegahan, dan Pengobatan. Jurnal Biotechnologi dan Biosains Indonesia. 7(2): 304-317.
[2] Mane, K., Chaluvaraju K.C., Niranjan, M.S., Zaranappa, Manjuthej, T.R. 2012. Review of Insulin and Its Analogues in Diabetes Mellitus. Journal of Basic and Clinical Pharmacy. 003(002): 283-293.
[3] Rachdaoui, N. 2020. Insulin: the Friend and the Foe in the Development of Type 2 Diabetes Mellitus, International Journal of Molecular Sciences. 21: 1-21.
[4] Demir, T., Serap, T., Kursad, U., Oya, T., Tufan, T., Dilek, G.Y. 2021. Use of Insulin Degludec/Insulin Aspart in the Management of Diabetes Mellitus: Expert Panel Recommendations on Appropriate Practice Patterns. Frontiers Endocrinolology. 12: 1-11.
[5] Ghosh, S., Romik, G. 2020. Glargine-300: an Updated Literature Review on Randomized Controlled Trials and Real-world Studies. World Journal of Diabetes. 11(4): 100-114.
[6] Govender, K., Tricia, N., Johnson, L., Sooraj, B., Anil, A.C., Naeem, S.A., Taskeen, D., Hendrik, G.K., Thavendran G. 2020. A Novel and more Efficient Biosynthesis Approach from Human Insulin Production in Escherichia coli (E. coli). AMB Express. 10(43): 1-9.
[7] Tibaldi, J.M. 2014. Evolution of Insulin: from Human to Analog. The American Journal of. Medicine. 127(10A): S25-S38.
[8] Alkhatib, A.J., Amer, M.S., Eman, H.A. 2021. Insulin as a Predictor of Diabetes Type 2: a New Medical Hypothesis. Advances in Obesity, Weight Management and Control. 11(1): 1-3.
[9] Cheng, AYY., Dhiren, K.P., Timothy, S.R., Kathleen, W. 2019. Differentiating Basal Insulin Preparations: Understanding How They Work Explains Why They are Different. Advances in Therapy. 36: 1018-1030.
[10] Gotham, D., Melissa, J.B., Andrew, H. 2018. Production Costs and Potential Prices for Biosimilars of Human Insulin and Insulin Analogues. BMJ Global Health. 1-10.
[11] Kasavadev, J., Banshi, S., Meera, B.K., Gopika, K. 2020. Evolution of Insulin Delivery Devices: from Syringes, Pens, and Pumps to DIY Artificial Pancreas. Diabetes Therapy. 11: 1251-269.
[12] Scholten, B.J., Frederik, F.K., Stephen CLG., Matthias, H. 2021. Current and Future Therapies for Type 1 Diabetes. Diabetologia. 64: 1037-1048.
[13] Ahmad, K. 2014. Insulin Sources and Types: a Review of Insulin in Terms of Its Mode on Diabetes Mellitus. Journal of Traditional Chinese Medicine. 34(2): 234-237.
[14] Shen, Y., Witoon, P., Zhimin, X. 2019. Insulin: a Review of Analytical Methods. The Analyst. 144: 4139-4148.
[15] Allen, D., Ruan, C., King, B., Ruan, K. 2019. Recent Advances and Near Future of Insulin Production and Therapy. Future Medicinal Chemistry. 11(13): 1513-1517.
[16] Baeshen, N.A., Mohammad, N.B., Abdullah, S., Roop, S.B., Mohamed, MMA., Hassan, AIR., Kulvinder, S.S., Elrashdy, M.R. 2014. Cell Factories for Insulin Production. Microbial Cell Factory. 13(141): 1-9.
[17] Fu, Z., Elizabeth, R.G., Dongmin, L. 2013. Regulation of Insulin Synthesis and Secretion and Pancreatic Beta-cell Dysfunction in Diabetes. Current Diabetes Reviews. 9(1): 25-53
[18] Kuhlmann, M., Andrea S. 2014. Production and Manufacturing of Biosimilar Insulin: Implications for Patients, Physicians, and Health Care System. Biosimilars. 4: 45-58.
[19] Hardianto, D. 2021. Insulin: produksi, jenis, analisis, dan rute pemberian. Jurnal Biotechnologi dan Biosains Indonesia. 8(2): 321-331.
[20] Pathak, V., Pathak, N.M., O’Neill, C.L., Guduric-Fuchs, J., Medina, RJ. 2019. Therapies for Type 1 Diabetes: Current Scenario and Future Perspectives. Clinical Medicine Insights: Endocrinology and Diabetes, 12: 1-13.
[21] Sandow, J., Wolfgang, L., Reinhard, B., Gerhard, S. 2015. Equivalent Recombinant Human Insulin Preparations and their Place in Therapy. European Endocrinology, 11(1): 10-16.
[22] Riggs, A.D. 2021. Making, Cloning, and the Expression of Human Insulin Genes in Bacteria: the Path to Humulin. Endocrine Reviews. 42(3): 374-380.
[23] Stubbs, D.J., Levy, N., Dhatariya, K, 2017. Diabetes Medication Pharmacology. BJA Education. 17(6): 198-207.
[24] Sekaringtyas, F.C., Dudi H., Karimah, N., Nida, V., Zahra, A. 2021. Transformation and Characterization of Human Insulin Precursor Gene in Pichia pastoris X-33. IOP Conference Series: Earth and Environmental Science. 948 012084.
[25] Jacob, S., Mohamed, A.M., Anroop, N. 2018. An Overview on the Insulin Preparation and Devices. Indian Journal of Pharmaceutical Education and Research. 52(4): 550-557.
[26] Mansur, M., Cecilia, C., Lester, H., Jose, P., Laura, V., Jorge, V., Yanet, T., Abdel, H., Liuba, P., Vladimir, B., Liudys, G., Emilio, L., Lila, C., Eduardo, M. 2005. Multiple Gene Copy Number Enhances Insulin Precursor Secretion in the Yeast Pichia pastoris. Biotechnology Letters. 27: 339-345.
[27] Gurramkonda, C., Sulena, P., Natasa, S., Ahmad, A., Thomas, G., Dipti, C., Sathyamangalam, S., Navin, K., Sergio, T., Ursula, R. 2010. Application of Simple Fed-batch Technique to High-level Secretory Production of Insulin Precursor Using Pichia pastoris with Subsequent Purification and Conversion to Human Insulin, Microbial Cell Factories. 9: 31.
[28] Wang, Y., Zhen-He, L., You-Shang, Z., Shi-Yin, Y., Yin-Gao, X., Yue-Hua, T., Shang-Quan, Z., Da-Fu, C., You-Min, F. 2001. Human Insulin from a Precursor Overexpressed in the Methylotrophic Yeast Pichia pastoris and a Simple Procedure for Purifying the Expression Product. Biotechnology and Bioengineering. 73(1): 74-79.
[29] Zhu, T., Guo, M., Tang, Z., Zhang, M., Zhuang, Y., Chu, J., Zhang, S. 2008. Efficient Generation of Multi-copy Strains for Optimizing Secretory Expression of Porcine Insulin Precursor in Yeast Pichia pastoris. Journal of Applied Microbiology. 107: 954-963.
[30] T, Trastoy, M.N., Martin, D., Florence, L. 2005. Resistance to the Antibiotic Zeocin by Stable Expression of the sh ble Gene Does Not Fully Suppress Zeocin-induced DNA Clevage in Human Cells. Mutagenesis. 20(2): 111-114.
[31] Knopp, J.L., Lui, H., Geoffrey, C. 2019. Insulin Units and Conversion Factors: A Story of Truth, Boots, and Faster Half-Truths. Journal of Diabetes Science and Technology. 13(3): 597-600.
Published
2022-12-01
How to Cite
Hardianto, D., Assyifa , F., Martius, E., Laziba , D., Royani, J. I., Sumiati, T., & Safarrida, A. (2022). KARAKTERISASI DAN PENETAPAN KADAR PREKURSOR INSULIN YANG DIPRODUKSI OLEH REKOMBINAN Pichia pastoris . Jurnal Farmamedika (Pharmamedika Journal), 7(2), 136-142. https://doi.org/10.47219/ath.v7i2.178