A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.
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http://dx.doi.org/10.1016/j.ab.2017.01.008 | DOI Listing |
Protein Expr Purif
December 2024
Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms.
View Article and Find Full Text PDFAvicenna J Med Biotechnol
January 2024
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Background: The low solubility of Tobacco Etch Virus (TEV) protease, a functional enzyme that cleaves protein tags without significant modification in its sequence, is one of the most important limitations of this enzyme. In this study, the aim was to increase the solubility of TEV by changing the expression conditions and designing lysis buffer with various solubilizing agents to improve its solubility.
Methods: (.
Bioengineering (Basel)
October 2024
Department of Marine Biopharmacology, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
As an essential thrombolytic agent, the tissue plasminogen activator receives increasing attention due to its longer half-life, lower immunogenicity, and easier administration, which are superior to other thrombolytic agents. In this study, the isolated and purified plasminogen activator from the sandworm () was expressed in . () to investigate its potential for simplifying the development process.
View Article and Find Full Text PDFBiochimie
October 2024
Department of Chemistry, Umeå University, 901 87, Umeå, Sweden. Electronic address:
Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers.
View Article and Find Full Text PDFCells
October 2024
Faculty of Biology and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79104 Freiburg, Germany.
Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand.
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