Biotechnology and Bioprocess Engineering

, Volume 3, Issue 2, pp 67–70 | Cite as

Influence of site-directed mutagenesis on protein assembly and solubility of tadpole H-chain ferritin

  • Kyung-Suk Kim


In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferritin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced inEscherichia 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 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 ferritins 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.

Key words

mutagenesis ferritin assembly solubility 


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  1. [1]
    Theil, E. C. (1987) Ferritin; structure, gene, regulation and cellular function in animals, plants and microganisms.Ann. Rev. Biochem. 56, 289–315.CrossRefGoogle Scholar
  2. [2]
    Harrison, P. M., S. C. Andrews, P. J. Artymiuk, G. C. Ford, D. M. Lawson, J. M. A. Smith, A. Treffry, and J. L. White (1990) Ferritin, In:Iron transfer and storage (Ponka, P., H. M. Schulman and R. C. Woodworth, eds.) p. 81–101 CRC press.Google Scholar
  3. [3]
    Harrison, P. M. and P. Arosio (1996) The ferritins: molecular properties, ion storage function and cellular regulation.Biochim. Biophys. Acta 1275, 161–203.CrossRefGoogle Scholar
  4. [4]
    Sun, S., P. Arosio, S. Levi, and N. D. Chasteen, (1993) Ferroxidase kinetics of human liver apoferritin, recombinant H-chain apoferritic and site-directed mutants.Biochemistry 32, 9362–9369.CrossRefGoogle Scholar
  5. [5]
    Waldo, G. S., J. Ling, J. Sanders-Loehr, and E. C. Theil (1993) Formation of an Fe(III)-tyrosinate complex during biomineralization of H-subunit ferritin.Science 259, 796–798.CrossRefGoogle Scholar
  6. [6]
    Lawson, D. M., A. Treffry, P. J. Artymiuk, P. M. Harrison, S. J. Yewdall, A. Luzzago, G. Cesareni, S. Levi and P. Arosio (1989) Identification of the ferroxidase centre in ferritin.FEBS Lett. 254, 207–210.CrossRefGoogle Scholar
  7. [7]
    Levi, S., P. Santambrogio, A. Cozzi, E. Rovida, B. Corsi, E. Tamborini, S. Spada, A. Albertini, and P. Arosio (1994) The role of the L-chain in ferritin iron incorporation.J. Mol. Biol. 238, 649–654.CrossRefGoogle Scholar
  8. [8]
    Levi, S., A. Luzzago, G. Cesareni, A. Cozzi, F. Franceschinelli, A. Albertini and P. Arosio (1988) Mechanism of ferritin iron uptake: Activity of the H-chain and deletion mapping of the ferro-oxidase site.J. Biol. Chem. 263, 18086–18092.Google Scholar
  9. [9]
    Kim, Y.-T. and K.-S. Kim (1994) Synthesis of active tadpole H-chain ferritin inEscherichia coli.Mol. Cells 4, 125–129.Google Scholar
  10. [10]
    Kramer, B., W. Kramer and H. J. Fritz (1984) Different base-base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system ofE. coli.Cell 38, 879.CrossRefGoogle Scholar
  11. [11]
    Sanger, F. (1981) Determination of nucleotide sequences in DNA.Science 214, 5463–5467.CrossRefGoogle Scholar
  12. [12]
    Laemmli, U. K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4.Nature 227, 680–685.CrossRefGoogle Scholar
  13. [13]
    Hames, B. D. and D. Rickwood (1983) In:Gel electrophoresis of proteins. IRL press. Oxford.Google Scholar
  14. [14]
    Chang, S.-R., Y.-T. Kim, and K.-S. Kim (1995) Purification and characterization of recombinant tadpole H-chain ferritin inEscherichia coli.J. Biochem. Mol. Biol. 3, 238–242.Google Scholar
  15. [15]
    Spreugart, M. L., H. P. Fatscher, and E. Fuchs (1990) The initiation of translation inE. coli: apparent base pairing between the 16 srRNA and downstream sequences of the mRNA.Nucleic Acids Res. 18, 1719–1723.CrossRefGoogle Scholar
  16. [16]
    De Boer, H. A., L. J. Comstock and M. Vesser (1983) Thetac promoter: A functional hybrid derived from thetrp andlac promoters.Proc. Natl. Acad. Sci. USA 80, 21–25.CrossRefGoogle Scholar
  17. [17]
    Keshavarz-Moore, E., R. Olbrich, M. Hoare and P. Dunnill (1991) Application of biochemical engineering principles to develop a recovery process for protein inclusion bodies. p. 307–314. In: Prokop, A. and R. K. Bajpai (ed.)Recombinant DNA technology I. Annals of NY academy of Sciences. NY.Google Scholar
  18. [18]
    Blackwell, J. R. and R. Horgan (1991) A novel strategy for production of a highly expressed recombinant protein in an active form.FEBS Lett. 295, 10–12.CrossRefGoogle Scholar
  19. [19]
    Darby, N. J. and T. E. Creghton (1990) Folding protein.Nature 334, 715–716.CrossRefGoogle Scholar
  20. [20]
    Georgopoulos, C. (1992) The emergence of the chaperone machines.TIBS 17, 295–299.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 1998

Authors and Affiliations

  1. 1.Faculty of Biological Sciences and Institute for Molecular Biology and GeneticsChonbuk National UniversityChonjuKorea

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