海洋科学技術センター試験研究報告　第47号　JAMSTECR, 47（March 2003） 1 Cloning and overexpression of the rpoZ gene encoding RNA polymerase ω subunit from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12 Hiroaki KAWANO ＊１，２ Yasuo SUZAKI ＊１ Junko FUKUCHI ＊２ Kaoru NAKASONE ＊３ Fumiyoshi ABE ＊２ Chiaki KATO ＊４ Yasuhiko YOSHIDA ＊１ Ron USAMI ＊１ Koki HORIKOSHI ＊１，２ We have cloned the rpoZ gene, encoding RNA polymerase ω protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285 bp in length, was found to encode a protein consisting of 93 amino acid residues with a molecular mass of 10,227 Da. Significant homol- ogy was evident comparing the rpoZ protein of S. violacea with that of Escherichia coli K-12 (71% identity), Vibrio cholerae (70% identity) and Haemophilus influenzae (61% identity). Phylogenetic analysis of RpoZ proteins of several bacteria suggested that S. violacea is independent from other bacteria. We constructed expression plasmid to overpro- duce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombi- nant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni 2+ affinity column. The result of SDS-PAGE during purification suggests that a chimeric RNA polymerase (α 2 ββ'σ 70 ω) was assembled in E. coli. Keywords : Shewanella violacea, RNA polymerase ω protein, rpoZ gene, expression plasmid ＊１ Department of Applied Chemistry, Faculty of Engineering, Toyo University ＊２ The DEEP STAR Group, Japan Marine Science and Technology Center ＊３ Department of Chemistry and Environmental Technology, School of Engineering, Kinki University ＊４ Marine Ecosystems Research Department, Japan Marine Science and Technology Center