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J Struct Funct Genomics. 2004;5(1-2):87-93.

Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N.

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1
Center for Eukaryotic Structural Genomics and Department of Biochemistry, University of Wisconsin--Madison, 433 Babcock Drive, Madison, WI 53706, USA.

Abstract

The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue, pp. 95-102], this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.

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