LABELING OF RECOMBINANT PROTEIN FOR NMR-SPECTROSCOPY - GLOBAL AND SPECIFIC LABELING OF THE RAT-LIVER FRUCTOSE 2,6-BISPHOSPHATASE DOMAIN

Methods for the efficient use of the C-13-labeled nutrients, glucose a nd histidine, in the production of recombinant protein were developed to provide the large amount of sample required for NMR studies. The nu trient requirements were reduced by determining the minimum amount of these metabolites needed during both the growth and the induction phas es of the BL21(DE3) and newly constructed BL21(DE3) histidine auxotrop hic Escherichia coli cultures. These methods were developed using the separate bisphosphatase domain of rat liver hosphofructo-2-kinase/fruc tose-2,6-bisphosphatase, which is expressed to high levels in the pET3 a/BL21(DE3) bacterial system. Use of the optimized expression methods reduced the requirements for the labeled nutrients, glucose and histid ine, by 90 and 93.8%, respectively. The savings realized by use of the minimized media and modified induction protocols were obtained withou t significant reduction of the yield of purified protein. Comprehensiv e study of the bisphosphatase domain by NMR spectroscopy requires larg e amounts of protein because of its low solubility and the short lifet ime (2-3 days) of the NMR samples. The significant reduction in the co sts of labeled protein samples realized by the optimized expression me thods can meet these sample requirements in a cost-effective way, and thereby, allow NMR studies of the bisphosphatase domain to proceed. (C ) 1997 Academic Press.