Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1992 Feb;1(2):271–277. doi: 10.1002/pro.5560010209

Yeast ferrochelatase: expression in a baculovirus system and purification of the expression protein.

M G Eldridge 1, H A Dailey 1
PMCID: PMC2142192  PMID: 1304908

Abstract

The terminal step of the heme biosynthetic pathway is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). In eukaryotes this enzyme is bound to the inner mitochondrial membrane with its active site facing the matrix side of the membrane. Previously this laboratory has characterized this enzyme via kinetic and protein chemical modification techniques, and with the recent cloning of the enzyme from yeast, mouse, and human sources it now becomes possible to approach structure-function questions by using site-directed mutagenesis. Of primary significance to this is the development of an efficient expression vector. This is of particular significance for ferrochelatase, as it is a low-abundance protein whose DNA coding sequence has a very low codon bias. In the current work we describe the production of yeast ferrochelatase in a baculovirus system. This system is shown to be an excellent one in which to produce large quantities of active ferrochelatase. The expressed enzyme is membrane associated and is not released into the growth medium either during or after virus development and cell lysis. The expressed protein can be purified in a procedure that requires only 1 day and makes use of a Pharmacia Hi Trap blue affinity column. The measured Km's for the substrates mesoporphyrin and iron are the same as those reported previously for the yeast enzyme. To our knowledge this is the first example of a mitochondrial membrane protein that has been expressed in a baculovirus system.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beames B., Summers M. D. Location and nucleotide sequence of the 25K protein missing from baculovirus few polyhedra (FP) mutants. Virology. 1989 Feb;168(2):344–353. doi: 10.1016/0042-6822(89)90275-4. [DOI] [PubMed] [Google Scholar]
  2. Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
  3. Dailey H. A., Fleming J. E., Harbin B. M. Purification and characterization of mammalian and chicken ferrochelatase. Methods Enzymol. 1986;123:401–408. doi: 10.1016/s0076-6879(86)23049-9. [DOI] [PubMed] [Google Scholar]
  4. Dailey H. A. Purification and characterization of membrane-bound ferrochelatase from Rhodopseudomonas sphaeroides. J Biol Chem. 1982 Dec 25;257(24):14714–14718. [PubMed] [Google Scholar]
  5. Guarino L. A., Smith M. W. Nucleotide sequence and characterization of the 39K gene region of Autographa californica nuclear polyhedrosis virus. Virology. 1990 Nov;179(1):1–8. doi: 10.1016/0042-6822(90)90266-t. [DOI] [PubMed] [Google Scholar]
  6. Harbin B. M., Dailey H. A. Orientation of ferrochelatase in bovine liver mitochondria. Biochemistry. 1985 Jan 15;24(2):366–370. doi: 10.1021/bi00323a019. [DOI] [PubMed] [Google Scholar]
  7. Jones O. T. Ferrochelatase of spinach chloroplasts. Biochem J. 1968 Mar;107(1):113–119. doi: 10.1042/bj1070113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Karr S. R., Dailey H. A. The synthesis of murine ferrochelatase in vitro and in vivo. Biochem J. 1988 Sep 15;254(3):799–803. doi: 10.1042/bj2540799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Labbe-Bois R. The ferrochelatase from Saccharomyces cerevisiae. Sequence, disruption, and expression of its structural gene HEM15. J Biol Chem. 1990 May 5;265(13):7278–7283. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Maeda S. Expression of foreign genes in insects using baculovirus vectors. Annu Rev Entomol. 1989;34:351–372. doi: 10.1146/annurev.en.34.010189.002031. [DOI] [PubMed] [Google Scholar]
  12. Miller L. K. Baculoviruses as gene expression vectors. Annu Rev Microbiol. 1988;42:177–199. doi: 10.1146/annurev.mi.42.100188.001141. [DOI] [PubMed] [Google Scholar]
  13. Nakahashi Y., Taketani S., Okuda M., Inoue K., Tokunaga R. Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase. Biochem Biophys Res Commun. 1990 Dec 14;173(2):748–755. doi: 10.1016/s0006-291x(05)80099-3. [DOI] [PubMed] [Google Scholar]
  14. Ooi B. G., Rankin C., Miller L. K. Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene. J Mol Biol. 1989 Dec 20;210(4):721–736. doi: 10.1016/0022-2836(89)90105-8. [DOI] [PubMed] [Google Scholar]
  15. Taketani S., Nakahashi Y., Osumi T., Tokunaga R. Molecular cloning, sequencing, and expression of mouse ferrochelatase. J Biol Chem. 1990 Nov 15;265(32):19377–19380. [PubMed] [Google Scholar]
  16. Vaughn J. L., Goodwin R. H., Tompkins G. J., McCawley P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro. 1977 Apr;13(4):213–217. doi: 10.1007/BF02615077. [DOI] [PubMed] [Google Scholar]
  17. Wilson M. E., Mainprize T. H., Friesen P. D., Miller L. K. Location, transcription, and sequence of a baculovirus gene encoding a small arginine-rich polypeptide. J Virol. 1987 Mar;61(3):661–666. doi: 10.1128/jvi.61.3.661-666.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES