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 approximately 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.
Eidsness
M K
MK
Department of Chemistry, University of Georgia, Athens 30602.
O'Dell
S E
SE
Kurtz
D M
DM
Robson
R L
RL
Scott
R A
RA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003013:Clostridium / Q000235:genetics; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D015202:Protein Engineering; D012416:Rubredoxins / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D012416:Rubredoxins; C077196:rubredoxin protein, Clostridium pasteurianum