Biosynthesis, isolation and characterization of 57Fe-enriched Phaseolus vulgaris ferritin after heterologous expression in Escherichia coli.
Ferritin is the major iron storage protein in the biosphere. Iron stores of an organism are commonly assessed by measuring the concentration of the protein shell of the molecule in fluids and tissues. The amount of ferritin-bound iron, the more desirable information, still remains inaccessible owing to the lack of suitable techniques. Iron saturation of ferritin is highly variable, with a maximum capacity of 4,500 iron atoms per molecule. This study describes the direct isotopic labeling of a complex metalloprotein in vivo by biosynthesis, in order to measure ferritin-bound iron by isotope dilution mass spectrometry. [(57)Fe]ferritin was produced by cloning and overexpressing the Phaseolus vulgaris ferritin gene pfe in Escherichia coli in the presence of (57)FeCl(2). Recombinant ferritin was purified in a fully assembled form and contained approximately 1,000 iron atoms per molecule at an isotopic enrichment of more than 95% (57)Fe. We did not find any evidence of species conversion of the isotopic label for at least 5 months of storage at -20 degrees C. Transfer efficiency of enriched iron into [(57)Fe]ferritin of 20% was sufficient to be economically feasible. Negligible amounts of non-ferritin-bound iron in the purified [(57)Fe]ferritin solution allows for use of this spike for quantification of ferritin-bound iron by isotope dilution mass spectrometry.
Hoppler
Matthias
M
Laboratory of Human Nutrition, ETH Zurich, Schmelzbergstrasse 7, 8092, Zurich, Switzerland.
Meile
Leo
L
Walczyk
Thomas
T
D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007887:Fabaceae / Q000378:metabolism; D005293:Ferritins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D007503:Iron Isotopes / Q000737:chemistry; D027805:Phaseolus / Q000235:genetics / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D007503:Iron Isotopes; D010936:Plant Extracts; D011994:Recombinant Proteins; D005293:Ferritins