Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.
Venters
R A
RA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Calderone
T L
TL
Spicer
L D
LD
Fierke
C A
CA
D000085:Acetates; D019342:Acetic Acid; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes
D000085:Acetates; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D002256:Carbonic Anhydrases; D019342:Acetic Acid