Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin

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Abstract
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 spectroseopy 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. Hie 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.