EXPRESSION AND LOADING OF RECOMBINANT HEAVY AND LIGHT-CHAIN HOMOPOLYMERS OF RAT-LIVER FERRITIN

The full-length genes for the heavy (H) and light (L) chains of ferrit in isolated from a rat liver cDNA library were amplified using polymer ase chain reaction. Each was inserted at the unique BglII site downstr eam of the p10 promoter of the baculovirus transfer vector pAcUW21. Th e genes were transferred separately to infectious Autographa californi ca nuclear polyhedrosis virus (AcNPV) expression vectors after in vivo homologous recombination. Ferritin homopolymers of either H or L chai n were expressed up to approximately 1.5 mg per 100 ml of infected cul tures (2.0 x 10(6) cells/ml) of Spodoptera frugiperda, Sf-21, 4 days p ostinfection. Both recombinant H chain ferritin (rH-Ft) and recombinan t L chain ferritin (rL-Ft) assembled as multi-subunit complexes with p redicted electrophoretic mobility. Neither rH-Ft nor rL-Ft homopolymer s had ferroxidase activity in 50 mM NaCl, as we have reported previous ly for native ferritin [D. DeSilva, D. M. Miller, D. W. Reif, and S. D . Aust (1992) Arch. Biochem. Biophys. 293, 409-415]. When ceruloplasmi n, a copper-containing protein, was used as a ferroxidase, rH-Ft loade d iron at rates comparable those obtained with native rat liver apofer ritin, but rL-Ft failed to load any iron. The initial rate of Fe(II) o xidation catalyzed by ceruloplasmin was increased in the presence of r H-Ft or rat liver ferritin but not in the presence of rL-Ft. A maximum of about 2500 atoms of iron were incorporated into both rH-Ft and rat liver ferritin, These results demonstrate that both rat liver rH-Ft a nd rL-Ft homopolymer can be properly produced by the baculovirus expre ssion system and ceruloplasmin can only load iron into H chain ferriti n. The physiological significance of these results is discussed. (C) 1 996 Academic Press, Inc.