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All Journal HAYATI Journal of Biosciences BERITA BIOLOGI ANNALES BOGORIENSES Makara Journal of Technology Annales Bogorienses
Neng Herawati, Neng
Research Center for Biotechnology, Indonesian Institute of Science, Cibinong, Bogor 16911, Indonesia
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Civil Engineering, Building, Construction & Architecture Earth & Planetary Sciences Electrical & Electronics Engineering Engineering Immunology & microbiology Materials Science & Nanotechnology Mechanical Engineering Medicine & Pharmacology

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Expression of No Affinity Tagged Recombinant Human Interferon Alpha-2a in Methilotropic Yeast Pichia pastoris Herawati, Neng; Wardiana, Andri; Ningrum, Ratih Asmana
ANNALES BOGORIENSES Vol 19, No 2 (2015): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v19i2.254

Abstract

Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for clinical therapy as antiviral, anticancer as well as immunomodulator. In this study, the open reading frame (ORF) encoding synthetic hIFNα-2a was constructed to be in framed with N-terminal alpha factor secretion system in methylotropic yeast Pichia pastoris. This research aimed to construct, express and analyse the non-affinity tagged recombinant human interferon alpha-2a in the methilotropic yeast P. pastoris. We used pPICZαB plasmid for cloning and expression vector. The confirmed recombinant plasmid containing the correct DNA sequence of hIFNα-2a was linearized by SacI restriction enzyme, then transformed into P. pastoris genome using electroporation. We screened two multi-copy recombinants in YPDS plates containing Zeocin™. Buffered complex medium containing 0.5 % methanol (BMMY) was used for protein expression for 48 hours in the culture condition. The recombinant protein was purified by blue sepharose affinity chromatography. Analyses of hIFNα-2a protein by SDS-PAGE and Western blot confirmed that protein band in which was observed around 19.2 kDa, was recombinant hIFNα-2a. The quantification of purified rhIFNα-2a using colorimetric binichoninic assay (BCA) informed that the yield was 44 mg/L culture (OD600= 2-3).
Pichia pastoris: SEL RAGI UNTUK PRODUKSI PROTEIN REKOMBINAN Herawati, Neng; Kusumawati, Arizah; Santoso, Adi
BERITA BIOLOGI Vol 17, No 2 (2018)
Publisher : Research Center for Biology-Indonesian Institute of Sciences

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (5218.527 KB) | DOI: 10.14203/beritabiologi.v17i2.3644

Abstract

Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viruses so it is safer and simplifies the downstream processes in bioproduction. The level of endogenous protein in the low supernatant allows Pichia to cultivate with a high volumetric productivity, therefore the process of protein production becomes very economical. This review provides an overview of several things that must be considered in utilizing P. pastoris as an expression system including the selection of vectors, strains, vector integration mechanisms into the genome, glycosylation processes, and applications in industry.
Effect of Methanol Induction and Incubation Time on Expression of Human Erythropoietin in Methylotropic Yeast Pichia Pastoris Santoso, Adi; Herawati, Neng; Rubiana, Yana
Makara Journal of Technology Vol. 16, No. 1
Publisher : UI Scholars Hub

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Abstract

Erythropoietin (EPO) is a glycoprotein hormone consists of 165 amino acids and has molecular mass of 30,400 Daltons. The large quantities of these hormone required to satisfy clinical demand are currently met by recombinant expression in mammalian cell, namely chinese hamster ovary (CHO). Pichia pastoris has become popular yeast based protein production systems to substitute mammalian expression systems. P. pastoris is capable to use methanol as sole carbon and energy source. In this study, recombinant human EPO (rhEPO) protein obtained by expressing the hEPO gene in methylotropic yeast P. pastoris, strain X33. The present work was carried out to study the optimal methanol concentration for induction and the incubation time to obtain rhEPO protein. To perform this study, the transformed P. pastoris was induced with various concentrations of methanol (0%, 0.5%, 1%, 2.5%, 5%, 10%, and 20%) and incubation times (0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours). The results demonstrate that the highest protein expression level occurred at concentration of 2.5% methanol induction, while the optimal incubation time was at 48 hrs.
Naringin Effect on SARS-CoV-2 Pseudovirus Entry and Spike Mediated Syncytia Formation in hACE2-overexpressing Cells Septisetyani, Endah Puji; Prasetyaningrum, Pekik Wiji; Paramitasari, Komang Alit; Suyoko, Ahmad; Himawan, Alayna Lillahida Indri; Azzahra, Salsabila; Wisnuwardhani, Popi Hadi; Anam, Khairul; Ramadani, Ratna Dwi; Santoso, Adi; Ningrum, Ratih Asmana; Herawati, Neng; Rubiyana, Yana
HAYATI Journal of Biosciences Vol. 31 No. 2 (2024): March 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.2.336-347

Abstract

A molecular docking study demonstrates the interaction between naringin, a citrus flavonoid, with SARS-CoV-2 spike RBD. Nevertheless, in vitro investigation of the inhibitory effect of naringin on SARS-CoV-2 entry and syncytia models has yet to be carried out. We synthesized VSV∆G-GFP/Spike* pseudovirus (PSV) as a SARS-CoV-2 model by pseudotyping VSV∆G-GFP/S* in BHK-21 cells overexpressing the SARS-CoV-2 spike glycoprotein. In the SARS-CoV-2 PSV entry assay, we utilized CHO-K1 cells transfected with hACE2 plasmid, which were then treated with naringin and SARS-CoV-2 PSV/naringin. After 16-18 h incubation, PSV internalization represented by the GFP signal was observed under a fluorescence microscope. Immunofluorescence staining was also performed to probe the SARS-CoV-2 spike and confirm the PSV entry. We performed a syncytia assay using 293T cells co-transfected with SARS-CoV-2 spike/hACE2. Six hours after transfection, the cells were treated with naringin and incubated for another 16-18 hours. Then, we observed syncytia using a phase contrast microscope. Based on fluorescence foci quantification, the results indicated that naringin might inhibit SARS-CoV-2 PSV entry at a concentration of 100 µM (P<0.05). However, naringin did not prevent syncytia formation compared to solvent control. These PSV entry and syncytia assay results suggested that naringin potentially inhibited SARS-CoV-2 viral infection but not cell-to-cell viral transmission.
Expression of No Affinity Tagged Recombinant Human Interferon Alpha-2a in Methylotrophic Yeast Pichia pastoris Herawati, Neng; Wardiana, Andri; Ningrum, Ratih Asmana
Annales Bogorienses Vol. 19 No. 2 (2015): Annales Bogorienses
Publisher : BRIN

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

Recombinant human interferon alpha-2a (rhIFN-2a) has been widely used for clinical therapy as antiviral, anticancer as well as immunomodulator. In this study, the open reading frame (ORF) encoding synthetic hIFN-2a was constructed to be in framed with N-terminal alpha factor secretion system in methylotrophic yeast Pichia pastoris. This research aimed to construct, express and analyse the non-affinity tagged recombinant human interferon alpha-2a in the methylotrophic yeast P. pastoris. We used pPICZB plasmid for cloning and expression vector. The confirmed recombinant plasmid containing the correct DNA sequence of hIFN-2a was linearized by SacI restriction enzyme, then transformed into P. pastoris genome using electroporation. We screened two multi-copy recombinants in YPDS plates containing Zeocin™. Buffered complex medium containing 0.5 % methanol (BMMY) was used for protein expression for 48 hours in the culture condition. The recombinant protein was purified by blue sepharose affinity chromatography. Analyses of hIFN-2a protein by SDS-PAGE and Western blot confirmed that protein band in which was observed around 19.2 kDa, was recombinant hIFN-2a. The quantification of purified rhIFN-2a using colorimetric binichoninic assay (BCA) informed that the yield was 44 mg/L culture (OD600= 2-3). 
Heterologous Expression of Recombinant Human Insulin Glargine (hIG) in Methylotrophic Yeast Pichia pastoris Herawati, Neng; Rubiyana, Yana; Desriani, Desriani; Santoso, Adi
Annales Bogorienses Vol. 26 No. 1 (2022): Annales Bogorienses
Publisher : 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) Atikana, Akhirta; Prasetyoputri, Anggia; Rubiyana, Yana; Herawati, Neng; Desriani, Desriani; Pratiwi, Riyona Desvy; Wulandari, Dwi; Sukmarini, Linda; Kusharyoto, Wien; Santoso, Adi; Putra, Masteria Yunovilsa; Lisdiyanti, Puspita
Annales Bogorienses Vol. 26 No. 1 (2022): Annales Bogorienses
Publisher : 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 A.A. Ketut Agung Cahyawan W Adi Santoso Anam, M. Khairul Andri Wardiana, Andri Atikana, Akhirta Azzahra, Salsabila Desriani Desriani Dwi Wulandari Endah Puji Septisetyani, Endah Puji Himawan, Alayna Lillahida Indri Komang Alit Paramitasari Kusharyoto, Wien Kusumawati, Arizah LINDA SUKMARINI Popi Hadi Wisnuwardhani, Popi Hadi Prasetyaningrum, Pekik Wiji Prasetyoputri, Anggia PUSPITA LISDIYANTI Putra, Masteria Yunovilsa Ramadani, Ratna Dwi RATIH ASMANA NINGRUM Riyona Desvy Pratiwi, Riyona Desvy Suyoko, Ahmad Yana Rubiana, Yana Yana Rubiyana, Yana