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 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.