Yeast ferrochelatase: expression in a baculovirus system and purification of the expression protein.
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.
Eldridge
M G
MG
Department of Microbiology, University of Georgia, Athens 30602.
Dailey
H A
HA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics; D005294:Ferrochelatase / Q000096:biosynthesis* / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D009036:Moths; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D008565:Membrane Proteins; D011994:Recombinant Proteins; D005294:Ferrochelatase