PMID:8056770
| Citation |
Miyamoto, K, Kanaya, S, Morikawa, K and Inokuchi, H (1994) Overproduction, purification, and characterization of ferrochelatase from Escherichia coli. J. Biochem. 115:545-51 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Blotting, Western; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Ferrochelatase/biosynthesis; Ferrochelatase/chemistry; Ferrochelatase/genetics; Ferrochelatase/isolation & purification; Hydrogen-Ion Concentration; Isoelectric Focusing; Molecular Sequence Data; Molecular Weight; Plasmids/genetics; Protein Structure, Secondary; Protoporphyrins/metabolism; Substrate Specificity; Temperature |
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