Identification and Cloning of the ClpB Gene in Psychromonas arctica by Inverse PCR and Cassette PCR Technology

摘要

The family of ClpB protein is a molecular chaperone which protects cellular proteins from being aggregated upon exposure to severe environmental stresses in association with DnaK/DanJ/GrpE in the ATP-dependent manner. In a psychro-philic bacterium which survives at a subzero temperature, any functional role of cold-active ClpB protein can be rather crucial. In order to identify a ClpB encoding gene from a cold-adapted bacterium whose genome sequence has not been fully discovered, we have employed a series of PCR technologies, including a gradient PCR with homologous primers, an inverse PCR and a cassette PCR. The full sequence of PaclpB gene was successfully identified and compared with those of other psychrophilic species. We have further cloned the gene inE.coli expression systems and were able to induce PaClpB protein expression by IPTG, which help us understand a molecular mechanism for survival against extremely cold environments.