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Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N. J Struct Funct Genomics 2004;5(1-2):87-93

Date

07/21/2004

Pubmed ID

15263847

DOI

10.1023/B:JSFG.0000029205.65813.42

Scopus ID

2-s2.0-3543148407   21 Citations

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.

Author List

Zhao Q, Frederick R, Seder K, Thao S, Sreenath H, Peterson F, Volkman BF, Markley JL, Fox BG

Authors

Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Arabidopsis Proteins
Carbon Isotopes
Escherichia coli
Molecular Structure
Nitrogen Isotopes
Nuclear Magnetic Resonance, Biomolecular
Polyethylene Terephthalates
Proteomics
Recombinant Proteins
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