Jlh. Busch et al., EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF A RECONSTITUTED RECOMBINANT 7FE FERREDOXIN FROM DESULFOVIBRIO-AFRICANUS, Biochemical journal, 314, 1996, pp. 63-71
Desulfovibrio africanus ferredoxin III is a monomeric protein (molecul
ar mass of 6585 Da) that contains one [3Fe-4S](1+/0) and one [4Fe-4S](
2+/1+) cluster when isolated aerobically. The amino acid sequence cons
ists of 61 amino acids, including seven cysteine residues that are all
involved in co-ordination to the clusters. In order to isolate larger
quantities of D. africanus ferredoxin III, we have overexpressed it i
n Escherichia coli by constructing a synthetic gene based on the amino
acid sequence of the native protein. The recombinant ferredoxin was e
xpressed in E. coli as an apoprotein. We have reconstituted the holopr
otein by incubating the apoprotein with excess iron and sulphide in th
e presence of a, reducing agent. The reconstituted recombinant ferredo
xin appeared to have a lower stability than that of wildtype D. africa
nus ferredoxin III. We have shown by low-temperature magnetic circular
dichroism and EPR spectroscopy that the recombinant ferredoxin contai
ns a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) cluster similar to those fou
nd in native D. africanus ferredoxin III. These results indicate that
the two clusters have been correctly inserted into the recombinant fer
redoxin.