Cloning, nucleotide sequence and expression of the cytochrome c‐552 gene from Hydrogenobacter thermophilus

Abstract

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 β‐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.