Molecular characterization of a recombinant replication protein (Rep) fromthe Antarctic bacterium Psychrobacter sp TA144

The Antarctic Gram-negative bacterium Psychrobacter sp. TA144 contains two small cryptic plasmids, called pTAUp and pTADw. pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway. Several attempts to produce the wil d-type biologically active PsyRep in Escherichia coil failed, possibly due to auto-regulation of the protein population. However, the serendipitous oc currence of a Frameshift mutation during the preparation of an expression v ector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble f orm. The purification of PsyRep* inclusion bodies and the successful refold ing of the cold adapted enzyme allowed us to carry out its functional chara cterization. The mutated protein still displays a double stranded DNA nicki ng activity, while the change at the C-terminus impairs the enzyme specific ity for the pTAUp cognate Ori(+) sequence. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights re served.