The four-way (Holliday) DNA junction is a key intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The final stages of recombination require resolution of the junction into nicked-duplex species by the action of a junction-resolving enzyme. The enzymes involved are nucleases that are highly selective for the structure of branched DNA. Here we present the isolation, expression and purification of a novel T7 endonuclease from the Kogelberg Biosphere Reserve (KBR), which possesses junction resolving capabilities. An initial approach was employed where the process was scaled up to 3 L with IPTG concentration of 0.1 mM at 30 °C and purified via immobilised metal affinity chromatography (IMAC). Expression titres of 20 ± 0.003 µg.L