|Journal of Structural and Functional Genomics (2010) 11:143-54|
|Northeast Structural Genomics Consortium|
(click to unfold)
Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. ...
Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality (2)H, (13)C, (15)N-enriched protein samples at 2.5-10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced (2)H, (13)C, (15)N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40x condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce (2)H, (13)C, (15)N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein's structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10-40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies.
|metabolism chemistry genetics |
|Escherichia coli Escherichia coli Proteins Nuclear Magnetic Resonance, Biomolecular Heat-Shock Proteins Multienzyme Complexes Membrane Proteins Bacterial Outer Membrane Proteins Gene Expression Genetic Vectors Deuterium Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization |
|12 (Last update: 03/16/2019 5:38:51pm)|