Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12.
We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.
Kawano
Hiroaki
H
The Deepstar Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan. kawanoh@jamstec.go.jp
Suzaki
Yasuo
Y
Fukuchi
Junko
J
Nakasone
Kaoru
K
Abe
Fumiyoshi
F
Kato
Chiaki
C
Yoshida
Yasuhiko
Y
Usami
Ron
R
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020592:Shewanella / Q000235:genetics* / Q000378:metabolism
C111232:RNA polymerase omega subunit; D012321:DNA-Directed RNA Polymerases