Molecular characterization of a recombinant replication protein (Rep) from the 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 wild-type biologically active PsyRep in Escherichia coli failed, possibly due to auto-regulation of the protein population. However, the serendipitous occurrence of a frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form. The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization. The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori+ sequence.
Duilio
A
A
Dipartimento di Chimica Organica e Biochimica, Università di Napoli 'Federico II', Complesso universitario Monte S. Angelo, via Cinthia, 80100, Naples, Italy.
Tutino
M L
ML
Matafora
V
V
Sannia
G
G
Marino
G
G
D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001483:Base Sequence; D003080:Cold Temperature; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D016368:Frameshift Mutation; D020563:Gammaproteobacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases