• BMB Reports
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• v.28 no.3
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• pp.238-242
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• 1995

The tadpole H-ferritin produced in E. coli was purified and its molecular properties were investigated to obtain information about the contribution of the H-subunit in the reaction of iron core formation. All the expressed subunits were assembled into complete holoprotein in vitro, presumably 24-mer, and the protein was heat-stable. Electron microscopy revealed that the recombinant ferritin forms spherically and contains iron core. No difference was observed in the absorption spectrum of the expressed protein compared to that of the natural ferritin. The Ouchterlony double diffusion of the expressed protein showed that the H-chain ferritin shares an antigenic determinant with natural tadpole ferritin. Rabbit anti-horse spleen ferritin discriminated the H-ferritin from natural ferritin. The rate of ferritin formation by the recombinant H-chain apoferritin was determined to be higher than that shown by natural tadpole ferritin, which consists of H, M and L-subunits. This phenomenon may be caused by the absence of M and L-subunits in the recombinant H-chain apoferritin.

• BMB Reports
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• v.29 no.5
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• pp.411-416
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• 1996

In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.67-70
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• 1998

In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferrin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced in Eshcerichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine. Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritns were capable of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further functional analysis was also made on the individual amino acid residues of ferroxidase center.