Abstract
Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with (13)C- and (15)N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins.