|Journal of Structural and Functional Genomics (2009) 10:219-25|
|Northeast Structural Genomics Consortium|
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The ability to produce isotope-enriched proteins is fundamental to the success of modern protein NMR, and is particularly essential for NMR activities in structural genomics projects. ...
Conventional methods of protein production often prove to be cost prohibitive for obtaining samples, particularly perdeuterated and site-specifically labeled proteins. The condensed single protein production system (cSPP), providing protein expression following condensation of cells 10-40 fold, allows for the production of such samples at a fraction of the cost. The previously described cSPP system is a two plasmid system where both the MazF toxin and ACA-less target gene are coinduced with IPTG. Coinduction results in 10-20% of the target protein produced without isotopic enrichment. Though the unlabeled protein is generally not visible in isotope-filtered NMR experiments, it results in an effective reduction in yield of the observable sample. By altering the cSPP system and separating the induction of the MazF toxin, required to convert cells into a semiquiescent state prior to condensation, from the expression of the target gene, we are now able to eliminate the unlabeled protein fraction and improve the isotope incorporation. Here we describe a series of pCold(tet) vectors with various features that can be used in the dual inducible cSPP(tet) system to obtain high-quality isotopically enriched protein at as little as 2.5% the cost of traditional methods.
|metabolism genetics |
|Molecular Sequence Data Proteins Escherichia coli Cloning, Molecular Nuclear Magnetic Resonance, Biomolecular Base Sequence Gene Expression Isotope Labeling Genetic Vectors Isopropyl Thiogalactoside |
|11 (Last update: 02/16/2019 4:28:07pm)|
Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system