ArticleJournal of biomolecular NMR2010
Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.
Article in Journal of biomolecular NMR, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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9 citing papers in PubMed, 16 citations in OpenAlex.
- Arabidopsis E3 Ubiquitin Ligases PUB22 and PUB23 Negatively Regulate Drought Tolerance by Targeting ABA Receptor PYL9 for Degradation.International journal of molecular sciences · 2017Article
- Novel Arabidopsis microtubule-associated proteins track growing microtubule plus ends.BMC plant biology · 2017Article
- Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.Bioengineered · 2016Article
- Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.Frontiers in microbiology · 2016Review
- Cold shock induction of recombinant Arctic environmental genes.BMC biotechnology · 2015Article
- Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides.Chembiochem : a European journal of chemical biology · 2015Article
- The human antimicrobial peptide LL-37 binds directly to CsrS, a sensor histidine kinase of group A Streptococcus, to activate expression of virulence factors.The Journal of biological chemistry · 2014Article
- Role of the CCAAT-binding protein NFY in SCA17 pathogenesis.PloS one · 2012Article
- Optimization and stabilization of Rho small GTPase proteins for solution NMR studies: The case of Rnd1.Small GTPases · 2011Article
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Authors and funding
2 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.
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