Functional and Immunological Analysis of Recombinant Mouse H- and L-Ferritins from Escherichia coli
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文摘
The production and characterization of recombinant mouse H- and L-ferritin chains from Escherichia coli are described. The proteins were efficiently expressed and purified with yields of 7–40 mg per liter of cell culture. They had the expected molecular mass and showed a physical stability analogous to that of the corresponding human ferritins. Mouse H- and L-ferritins had a very similar mobility on denaturing SDS–PAGE, but could be readily separated on nondenaturing PAGE because of the distinct slow mobility of mouse L-ferritin. Direct comparative experiments showed that mouse and human H-ferritins had the same iron incorporation activity, whereas mouse L-ferritin incorporated iron less efficiently than human L-ferritin. The difference was attributed to the substitution of a residue exposed on the cavity surface (Glu140 → Lys) in mouse L-ferritin, a hypothesis confirmed by the finding that the mouse L-ferritin mutant Lys140–Glu incorporated iron as efficiently as human L-ferritin. Rabbit antisera elicited by the recombinant mouse ferritins were specific for the H- and L-chains and did not cross-react with the human ferritins. The antibodies and the derived specific ELISA assays allow the determination of H- and L-ferritins in mouse tissues.