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Molecular characterization of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp.
A Duilio1, M L Tutino, V Matafora
1Dipartimento di Chimica Organica e Biochimica, Università di Napoli 'Federico II', Complesso universitario Monte S. Angelo, via Cinthia, 80100, Naples, Italy.
Abstract:
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.
Insights
Researchers studied a cold-adapted enzyme from Antarctic bacteria responsible for plasmid replication. A mutated version (PsyRep*) retained DNA nicking activity but lost sequence specificity, aiding functional characterization.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Psychrobacter sp. TA144, an Antarctic bacterium, possesses two cryptic plasmids, pTAUp and pTADw.
- The pTAUp plasmid relies on the PsyRep replication enzyme for rolling circle replication.
- Wild-type PsyRep production in E. coli was challenging, potentially due to auto-regulation.
Purpose of the Study:
- To characterize the functional properties of a mutated PsyRep enzyme (PsyRep*) obtained through a frameshift mutation.
- To investigate the impact of C-terminal modifications on PsyRep's DNA nicking activity and sequence specificity.
Main Methods:
- Over-production of a mutated PsyRep* protein in E. coli via a frameshift mutation in the expression vector.
- Purification of PsyRep* inclusion bodies and subsequent refolding of the cold-adapted enzyme.
- Functional characterization of the refolded PsyRep* enzyme, including assessment of DNA nicking activity and sequence specificity.
Main Results:
- A frameshift mutation led to the over-production of a truncated PsyRep* protein that precipitated as inclusion bodies.
- Purification and refolding of PsyRep* allowed for functional analysis.
- The mutated PsyRep* enzyme retained double-stranded DNA nicking activity.
- The C-terminal alteration in PsyRep* abolished its specific recognition of the pTAUp Ori+ sequence.
Conclusions:
- The study successfully produced and characterized a functional, albeit altered, replication enzyme from an Antarctic bacterium.
- The C-terminal modification of PsyRep is crucial for its sequence-specific DNA binding and nicking activity.
- This work provides insights into the mechanism of rolling circle replication and enzyme engineering for plasmid maintenance.