ArticleAnalytical biochemistry2016
A dual protease approach for expression and affinity purification of recombinant proteins.
Article in Analytical biochemistry, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 22 citations in OpenAlex.
- Differential Assembly of Native Epithelial Sodium Channel Complexes Across Mouse Epithelial Tissues.Kidney360 · 2026Article
- Protocol to purify human and mouse cytokines using an endotoxin-free Escherichia coli-based platform.STAR protocols · 2026Article
- Efficient in vivo removal of solubility tag to improve the bacterial production of soluble, bioactive human IGF-1.World journal of microbiology & biotechnology · 2026Article
- Efficient production of human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli using a polyprotein strategy.Protein expression and purification · 2025Article
- Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain.Communications biology · 2025Article
- Molecular mechanisms controlling the biogenesis of the TGF-β signal Vg1.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
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- Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.PloS one · 2020Article
- Design of a Human Rhinovirus-14 3C Protease-Inducible Caspase-3.Molecules (Basel, Switzerland) · 2019Article
- Biofunctionalized Materials Featuring Feedforward and Feedback Circuits Exemplified by the Detection of Botulinum Toxin A.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2019Article
- Single-Input Regulatory Cascade forFrontiers in bioengineering and biotechnology · 2019Article
- Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.Molecular biotechnology · 2017Article
Corrections and comments
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Authors and funding
2 authors at 2 institutions in 1 country.
Funding
Abstract
We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.