An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli.
A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.
Cai
M
M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Huang
Y
Y
Sakaguchi
K
K
Clore
G M
GM
Gronenborn
A M
AM
Craigie
R
R
D002247:Carbon Isotopes; D003362:Cost-Benefit Analysis; D004926:Escherichia coli / Q000235:genetics; D019427:HIV Integrase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D019427:HIV Integrase