Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination.
Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.
Tyler
Robert C
RC
Department of Biochemistry, Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA.
Sreenath
Hassan K
HK
Singh
Shanteri
S
Aceti
David J
DJ
Bingman
Craig A
CA
Markley
John L
JL
Fox
Brian G
BG
D002247:Carbon Isotopes; D018929:Cell Culture Techniques / Q000379:methods; D003470:Culture Media / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; D003470:Culture Media; D009587:Nitrogen Isotopes; D011506:Proteins