Cloning, nucleotide sequence and expression of the cytochrome c-552 gene from Hydrogenobacter thermophilus.
A cytochrome c-552 gene from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein. A 780-bp PstI-SphI fragment of the cloned DNA was sequenced and found to contain the entire structural gene coding for cytochrome c-552 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. Cytochrome c-552 is synthesized in vivo as a precursor having an N-terminal signal sequence consisting of 18 amino acid residues. The cloned cytochrome c-552 gene without its own signal sequence was introduced into the pKK223-3 vector and expressed in E. coli upon induction with isopropyl beta-D-thiogalactoside. An expressed cytochrome c-552 protein had a methionine residue at the N-terminus since an initiation signal was introduced before the first amino acid residue of the mature cytochrome c-552. The heme c was attached to apo-type cytochrome c-552 in the cytoplasm of E. coli and the holoprotein had spectral properties, similar to the authentic cytochrome c-552 from H. thermophilus.
Sanbongi
Y
Y
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Yang
J H
JH
Igarashi
Y
Y
Kodama
T
T
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D014158:Transcription, Genetic
D003574:Cytochrome c Group; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; C083951:cytochrome c553; C022302:cytochrome C-552