Whole-genome sequencing (WGS) has played a significant role in understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a genomic surveillance during the COVID-19 pandemic. Nottingham–Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in the Research Center for Biotechnology, National Research and Innovation Agency (BRIN), to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the importance of clinical and demographic metadata collection and the importance of open access and data sharing. Lineage and phylogenetic analyses of two periods defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the most predominant in Indonesia before the Delta variant outbreak, having a unique spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-lineage took over after June 2021, and (3) the highest rate of virus transmissions between Indonesia and other countries was through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.
A year after the World Health Organisation declared COVID-19 as a pandemic, much has been learned with respect to SARS-CoV-2 epidemiology, vaccine production and disease treatment. Whole-genome sequencing (WGS) has played a significant role in contributing to our understanding of the epidemiology and biology of this virus. In this paper, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asia and the impact of technological development, access to resources, and demography of individual countries on its uptake. Facilitated by the Nottingham-Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative, we showcased a bilateral collaboration between the University of Nottingham and the Indonesian Institute of Sciences (LIPI/Lembaga Ilmu Pengetahuan Indonesia) to establish WGS of SARS-CoV-2 using Oxford Nanopore Technology® in Indonesia. Analyses of SARS-CoV-2 genomes deposited on GISAID from Southeast and East Asian countries reveals the importance of collecting clinical and demographic metadata and the importance of open access and data sharing. Lineage and phylogenetic analyses per 1 June 2021 found that: 1) B.1.466.2 variants were the most predominant in Indonesia, with mutations in the spike protein including D614G at 100%, N439K at 99.1%, and P681R at 69.7% frequency, 2) The variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) were first detected in Indonesia in January 2021, 2) B.1.470 was first detected in Indonesia and spread to the neighbouring regions, and 3) The highest rate of virus transmissions between Indonesia and the rest of the world appears to be through interactions with Singapore and Japan, two neighbouring countries with high degree of access and travels to and from Indonesia. Overall, we conclude that WGS of SARS-CoV-2 using Oxford Nanopore Technology® platforms fits well with the Indonesian context and can catalyse the increase of sequencing rates in the country.
Production of sufficient insulin at a more affordable price is necessary. The increase in the number of people living with diabetes puts more burden on healthcare and the economy. P. pastoris is a promising host to produce human insulin precursors at a high yield in minimal medium and secretes low levels of endogenous protein impurities. Production of the precursor involves several parameters, including glycerol concentration, culture density, methanol concentration, and medium composition. This study evaluated the effect of those parameters on protein expression in the flask culture. Subsequently, fermentation in the bioreactor was carried on according to the information obtained from flask culture. Methanol feeding to induce protein expression was undertaken by pulses and fed-batch modes. The fed-batch method was modified from a standard technique by incorporating constant flow rates with variable feed concentrations. Cell density was determined based on optical density measurement at 600 nm and dry cell weight. Tricine SDS-PAGE and reversed-phase HPLC conducted protein analysis. The pulse feeding produced higher precursor concentrations at ~445 mg/L than modified fed-batch feeding at ~267 mg/L. However, the modified fed-batch feeding can be an alternative to producing human insulin precursors when a standard fed-batch feeding with variable flow rates and 100% (v/v) methanol feed is difficult to apply.
Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.
The methylotrophic yeast, Pichia pastoris, is one of the preferred yeast hosts for recombinant protein expression. It has been developed as a potential host to express a high level of recombinant proteins, and to achieve efficient secretion as well as growth to very high cell densities. Previously, we have obtained 19 P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. To select P. pastoris recombinant clones which exhibit high levels of protein expression, we conducted secreted expressions of IP protein in shake flasks. The secretion of IP into the culture supernatants was verified by SDS-PAGE. IP protein concentrations were estimated using ImageJ by applying lysozyme as standard. All of the 19 P. pastoris recombinant clones were confirmed to secrete the IP protein into their culture supernatants, and a single protein band with a molecular size of approximately 7 kDa was found in the SDS-PAGE gel. The six highest IP-expressing clones were selected for second screening in shake flasks. We selected three recombinant clones (CL-3, CL-4, and CL-18), which secreted the highest levels of IP proteins compared to the other clones. The secreted IP concentrations estimated by ImageJ for clones CL-3, CL-4, and CL-18 were 1230, 1143, and 1010 mg/L, respectively.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.