Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N.
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.
Zhao
Qin
Q
Center for Eukaryotic Structural Genomics and Department of Biochemistry, University of Wisconsin--Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Frederick
Ronnie
R
Seder
Kory
K
Thao
Sandy
S
Sreenath
Hassan
H
Peterson
Francis
F
Volkman
Brian F
BF
Markley
John L
JL
Fox
Brian G
BG
D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011093:Polyethylene Terephthalates; D040901:Proteomics / Q000295:instrumentation*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D029681:Arabidopsis Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins