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 ferritin isolated from a rat liver cDNA library were amplified using polymerase chain reaction. Each was inserted at the unique BglII site downstream of the p10 promoter of the baculovirus transfer vector pAcUW21. The genes were transferred separately to infectious Autographa californica nuclear polyhedrosis virus (AcNPV) expression vectors after in vivo homologous recombination. Ferritin homopolymers of either H or L chain were expressed up to approximately 1.5 mg per 100 ml of infected cultures (2.0 x 10(6) cells/ml) of Spodoptera frugiperda, Sf-21, 4 days postinfection. Both recombinant H chain ferritin (rH-Ft) and recombinant L chain ferritin (rL-Ft) assembled as multi-subunit complexes with predicted electrophoretic mobility. Neither rH-Ft nor rL-Ft homopolymers had ferroxidase activity in 50 mM NaCl, as we have reported previously for native ferritin [D. DeSilva, D. M. Miller, D.W. Reif, and S.D. Aust (1992) Arch. Biochem. Biophys. 293,409-415]. When ceruloplasmin, a copper-containing protein, was used as a ferroxidase, rH-Ft loaded iron at rates comparable those obtained with native rat liver apoferritin, but rL-Ft failed to load any iron. The initial rate of Fe(II) oxidation catalyzed by ceruloplasmin was increased in the presence of rH-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 and rL-Ft homopolymer can be properly produced by the baculovirus expression system and ceruloplasmin can only load iron into H chain ferritin. The physiological significance of these results is discussed.
Guo
J H
JH
Biotechnology Center, Utah State University, Logan 84322-4705, USA.
Abedi
M
M
Aust
S D
SD
D000818:Animals; D002460:Cell Line; D002570:Ceruloplasmin / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D017924:Nucleopolyhedroviruses; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D018076:DNA, Complementary; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron; D002570:Ceruloplasmin