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

A synthetic gene based on the published amino acid sequencefor Clostridium pasteurianum rubredoxin was constructed, clonedin Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promotersystem in E.coli HMS273. UV/visible spectroseopy and metal analysesindicated that the as–isolated synthetic gene productis a mixture of holo–(i.e. iron–containing) rubredoxinand zinc–substituted rubredoxin, with the latter amountingto {small tilde} 70% of the total rubredoxin. Hie UV/visibleabsorption and resonance Raman spectra of the cloned holorubredoxinare characteristic of the native rubredoxin–type ironsite. N–terminal amino acid sequencing suggests that thegene product consists of at least three polypeptide specieswith the initial sequences (approximate relative abundances):Met–Met–Lys–... (63%), blocked (30%) and Met–Lys–...(7%). The blocked portion presumably consists of a mixture ofnMet–Met–Lys–... and nMet-Lys–..., wherenMet represents an amino–blocked methionine residue.