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All Journal Annales Bogorienses
Riyona Desvy Pratiwi
National Research and Inovation Agency (BRIN)

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Factors Affecting Expression Level of Recombinant Human Epidermal Growth Factor in Escherichia coli BL21(DE3) and Size Exclusion Purification Thereof Riyona Desvy Pratiwi; Nabella Agustina; Aminah Aminah; Asrul Muhamad Fuad
Annales Bogorienses Vol. 25 No. 2 (2021): Annales Bogorienses
Publisher : National Research and Innovation Agency (BRIN)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2021.v25.n2.66-72

Abstract

Recombinant human epidermal growth factor (rhEGF) has been developed to provide the protein for therapeutic uses. Isopropyl β-d-1-thiogalactopyranoside (IPTG)-induced expression in Escherichia coli BL21(DE3) has shown the most effective system among other inducers systems in a similar host. However, suitable conditions related to IPTG concentration, incubation time, and incubation temperature are different depending on the amino acid content of the recombinant protein. This study aimed to evaluate the effects of various IPTG concentration, incubation time, and incubation temperature on rhEGF concentration. According to each analysis of those factors, induction with 0.05 mM IPTG for 2 h at 23°C was the most appropriate condition to obtain the highest concentration of rhEGF. The rhEGF was positively confirmed with a monoclonal antihEGF antibody and purified in high purity reaching 95.2%, yet recovery was low (1.44%) due to loss in fractions containing endogenous proteins. Therefore, further studies related to type of matrix, column length, and sample concentration in applying size exclusion chromatography are requested for higher recovery
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 Aminah Aminah Anggia Prasetyoputri Asrul Muhamad Fuad Desriani Desriani Dwi Wulandari Linda Sukmarini Masteria Yunovilsa Putra Nabella Agustina Neng Herawati Puspita Lisdiyanti Wien Kusharyoto Yana Rubiyana