ArticleMolecular biotechnology2017
Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.
Article in Molecular biotechnology, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 26 citations in OpenAlex.
- A structure-based mechanism for initiation of AP-3 coated vesicle formation.bioRxiv : the preprint server for biology · 2024Article
- Phytochemicals: Promising Inhibitors of Human Rhinovirus Type 14 3C Protease as a Strategy to Fight the Common Cold.Current topics in medicinal chemistry · 2024Article
- Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.Research square · 2023Article
- Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron.PloS one · 2022Article
- Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron.bioRxiv : the preprint server for biology · 2021Article
- Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.Cell reports · 2020Article
- Article
- Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.bioRxiv : the preprint server for biology · 2020Article
- Design of Experiments As a Tool for Optimization in Recombinant Protein Biotechnology: From Constructs to Crystals.Molecular biotechnology · 2019Review
- Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells.Current microbiology · 2019Article
- Expression, purification, and evaluation of in vivo anti-fibrotic activity for soluble truncated TGF-β receptor II as a cleavable His-SUMO fusion protein.World journal of microbiology & biotechnology · 2018Article
- Building and Breaking Bonds via a Compact S-Propargyl-Cysteine to Chemically Control Enzymes and Modify Proteins.Angewandte Chemie (International ed. in English) · 2018Article
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Authors and funding
3 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.
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