A rapid and efficient approach for preparing isotopically labeled recombina
nt proteins is presented. The method is demonstrated for C-13 labeling of t
he C-terminal domain of angiopoietin-2, N-15 labeling of ubiquitin and for
H-2/C-13/N-15 labeling of the Escherichia coli outer-membrane lipoprotein L
pp-56. The production method generates cell mass using unlabeled rich media
followed by exchange into a small volume of labeled media at high cell den
sity. Following a short period for growth recovery and unlabeled metabolite
clearance, the cells are induced. The expression yields obtained provide a
fourfold to eightfold reduction in isotope costs using simple shake flask
growths.