The prevalence of Insulin Dependent Diabetes Mellitus (IDDM) has increased significantly in the last decades, resulting in an increased demand for insulin. In addition, new routes of oral and inhaled insulin require higher doses thus increasing insulin requirements. Insulin is the primary drug in patients with type 1 diabetes mellitus (T1DM). In some cases, type 2 diabetes mellitus (T2DM) requires insulin for treatment. The researchers have widely developed another expression system to meet the ever-increasing demand for insulin with higher production capacities. Human insulin precursor (HIP) production can use several microorganisms such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. In this study, P. pastoris X-33 was used to produce human insulin precursor. Pichia pastoris becomes the promising yeast host for recombinant protein expression because of its ability to reach high cell densities by its robust methanolinducible alcohol oxidase 1 (AOX1) promoter and simple development process, which provide to high quality and a high percentage of recombinant proteins, both intracellular and secretory. In this study, several zeocin-resistant clones were characterized by PCR and sequencing using a specific human insulin precursor gene to detect plasmid integration into the P. pastoris genome. In addition, a test of the effect of zeocin concentration on the growth of the transformation was carried out. The expression of HIP protein in P. pastoris X-33 was characterized by SDS-PAGE and Elisa. The result of PCR and sequencing showed that the HIP gene was successfully integrated into selected colonies of P. pastoris X-33. All of 20 transformant colonies were able to grow at 100 to 2000 μg per mL and selected colonies of P. pastoris X-33 can produce HIP protein.
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.
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