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 13C-labeled nutrients, glucose and histidine, in the production of recombinant protein were developed to provide the large amount of sample required for NMR studies. The nutrient requirements were reduced by determining the minimum amount of these metabolites needed during both the growth and the induction phases of the BL21(DE3) and newly constructed BL21(DE3) histidine auxotrophic Escherichia coli cultures. These methods were developed using the separate bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase, which is expressed to high levels in the pET3a/BL21 (DE3) bacterial system. Use of the optimized expression methods reduced the requirements for the labeled nutrients, glucose and histidine, by 90 and 93.8%, respectively. The savings realized by use of the minimized media and modified induction protocols were obtained without significant reduction of the yield of purified protein. Comprehensive study of the bisphosphatase domain by NMR spectroscopy requires large amounts of protein because of its low solubility and the short lifetime (2-3 days) of the NMR samples. The significant reduction in the costs of labeled protein samples realized by the optimized expression methods can meet these sample requirements in a cost-effective way, and thereby, allow NMR studies of the bisphosphatase domain to proceed.
Okar
D A
DA
Department of Biochemistry, School of Medicine, University of Minnesota, Minneapolis 55455, USA.
Felicia
N D
ND
Gui
L
L
Lange
A J
AJ
D000818:Animals; D004926:Escherichia coli; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013113:Spin Labels
D011994:Recombinant Proteins; D013113:Spin Labels; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases