Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination.
A simplified approach developed recently for the production of heterologous proteins in Escherichia coli uses 2-liter polyethylene terephthalate beverage bottles as disposable culture vessels [Sanville Millard, C. et al. 2003. Protein Expr. Purif. 29, 311-320]. The method greatly reduces the time and effort needed to produce native proteins for structural or functional studies. We now demonstrate that the approach is also well suited for production of proteins in defined media with incorporation of selenomethionine to facilitate structure determination by multiwavelength anomalous diffraction. Induction of a random set of Bacillus stearothermophilus target genes under the new protocols generated soluble selenomethionyl proteins in good yield. Several selenomethionyl proteins were purified in good yields and three were subjected to amino acid analysis. Incorporation of selenomethionine was determined to be greater than 95% in one protein and greater than 98% in the other two. In the preceding paper [Zhao et al., this issue, pp. 87-93], the approach is further extended to production of [U-15N]- or [U-13C, U-15N]-labeled proteins. The approach thus appears suitable for high-throughput production of proteins for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy.
Stols
Lucy
L
Environmental Research Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Millard
Cynthia Sanville
CS
Dementieva
Irina
I
Donnelly
Mark I
MI
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D011093:Polyethylene Terephthalates; D040901:Proteomics / Q000295:instrumentation*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012645:Selenomethionine
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D012645:Selenomethionine