Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12.

Abstract

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.