An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli.

Important note: Information in this article was accurate in 1998. The state of the art may have changed since the publication date.

An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli.

J Biomol NMR. 1998 Jan;11(1):97-102. Unique Identifier : AIDSLINE MED/98227443
Cai M; Huang Y; Sakaguchi K; Clore GM; Gronenborn AM; Craigie R; Laboratory of Chemical Physics, National Institute of Diabetes and; Digestive and Kidney Diseases, National Institutes of Health,; Bethesda, MD 20892-0520, USA.

Abstract: 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.

Keywords: *Recombinant Proteins/CHEMISTRY
980830
M9881172

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