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

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