EXPRESSION OF A SYNTHETIC GENE CODING FOR THE AMINO-ACID-SEQUENCE OF CLOSTRIDIUM-PASTEURIANUM RUBREDOXIN

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E.coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to - 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-. . . (63%), blocked (30%) and Met-Lys-. . . (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-. . . and nMet-Lys-. . ., where nMet represents an amino-blocked methionine residue.