Overproduction, purification, and characterization of ferrochelatase from Escherichia coli.
To establish a system for overproduction of the ferrochelatase [EC 4.99.1.1] from Escherichia coli, a plasmid designated pFC3 was constructed. The 35-kDa protein was accumulated in E. coli DH5 alpha cells that harbored pFC3 to a level equal to approximately 9% of the total protein (roughly 50 mg/liter) upon thermal induction. This 35-kDa protein was identified as the ferrochelatase of E. coli by Western blotting and amino-terminal amino acid sequence analysis. The protein with ferrochelatase activity was purified from the cells by three simple steps with a yield of 17%. The optimum pH of the purified enzyme was around 8.0. The molecular weight of the enzyme was estimated to be 35-kDa from column chromatography on Sephacryl S-300, a value consistent with that estimated from SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a monomer. The isoelectric point of the enzyme was approximately 4.7. Determination of the far-ultraviolet circular dichroism spectrum allowed us to calculate the alpha-helix and beta-sheet contents of the enzyme as 10 +/- 0.2 and 39 +/- 0.2%, respectively. High-level production of the ferrochelatase from E. coli will greatly facilitate detailed structural analysis of this protein.
Miyamoto
K
K
Department of Biophysics, Faculty of Science, Kyoto University.
Kanaya
S
S
Morikawa
K
K
Inokuchi
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005294:Ferrochelatase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011524:Protoporphyrins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D011524:Protoporphyrins; C028025:protoporphyrin IX; D005294:Ferrochelatase