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All Journal Annales Bogorienses
Yana Rubiyana
National Research and Inovation Agency (BRIN)

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Heterologous Expression of Recombinant Human Insulin Glargine (hIG) in Methylotrophic Yeast Pichia pastoris Neng Herawati; Yana Rubiyana; Desriani Desriani; Adi Santoso
Annales Bogorienses Vol. 26 No. 1 (2022): Annales Bogorienses
Publisher : National Research and Innovation Agency (BRIN)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2022.v26.n1.13-20

Abstract

World health organization (WHO) announced that diabetic patients increased significantly yearly worldwide. Consequently, the need for insulin becomes very large. Pichia pastoris, defined as methylotrophic yeast and a well-known expression and protein production host, is widely used for biopharmaceutical-based drug production. This research aims to synthesize human insulin glargine (hIG) protein in yeast P. pastoris. Human insulin glargine is a group of long-acting analogue insulin.We used a synthetic hIG-encoding gene constructed in frame with the truncated α-factor secretion signal in a pD902 expression vector. Recombinant plasmid pD902-hIG was linearized using Sac1 enzyme and transformed into P. pastoris genome. Multicopy clones were selected on YPDS plates containing Zeocin™ with concentrations of 100; 500; 1,000; and 2,000 µg//mL. Analyses using SDS-PAGE, slot blot, and Western blot showed that recombinant hIG protein had been obtained with a molecular weight of approximately 6,000 Daltons.
A Preliminary Report on The Syntheses of Oligonucleotide Primers in The National Research and Innovation Agency (NRIA) Akhirta Atikana; Anggia Prasetyoputri; Yana Rubiyana; Neng Herawati; Desriani Desriani; Riyona Desvy Pratiwi; Dwi Wulandari; Linda Sukmarini; Wien Kusharyoto; Adi Santoso; Masteria Yunovilsa Putra; Puspita Lisdiyanti
Annales Bogorienses Vol. 26 No. 1 (2022): Annales Bogorienses
Publisher : National Research and Innovation Agency (BRIN)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2022.v26.n1.21-27

Abstract

A PolyGen DNA Synthesizer is equipment that is used for synthesizing oligonucleotide primers for any amplification targets. Oligonucleotide primers are indispensable components for any Polymerase Chain Reaction (PCR)-based detections. In the present study a number of oligonucleotide primer sets were synthesized to target (1) the Human Insuline Glargin (HIG) and (2) the Human Erythropoietin (EPO), as well as (3) the RNA-dependent RNA Polymerase (RdRp) and (4) the Nucleocapsid (N) genes of the severe acute respiratory syndrome virus 2 (SARS-CoV-2). A solid-phase oligonucleotide synthesis method was used according to the default protocol of the Polygen’s instrument to synthesize primers at a 40 nmol scale. The synthesized primers in this study were compared to commercially produced primers in their ability to amplify the gene target(s) in PCR and quantitative real-time PCR (qPCR) reactions. The first two sets of primers showed similar results in PCR compared to commercial primers; however, these primers were not tested for qPCR due to sample limitation. In contrast, the primer sets 3 and 4 were not able to produce amplicons in PCR reactions and only the primer set 4 successfully amplified the gene target in qPCR. These results indicate that the crude primers synthesized in this study are promising candidates for molecular detection and diagnostics, but these primers would benefit from further optimization for routine applications.
Co-Authors Adi Santoso Akhirta Atikana Anggia Prasetyoputri Desriani Desriani Dwi Wulandari Linda Sukmarini Masteria Yunovilsa Putra Neng Herawati Puspita Lisdiyanti Riyona Desvy Pratiwi Wien Kusharyoto