Abstract:
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-1 culture were achieved with the amount of KBR-T7 endonuclease required per reaction ranging from as low as 10 to 100 nanograms. The solubility of the enzyme was relatively poor; however, enzyme activity was not affected. A derivative for improved solubility and efficacy was then designed from this original wild-type version, MBP-KBR-T7 and was expressed under similar conditions at 20 °C yielding 1.63 ± 0.154 mg.L-1 of formulated enzyme. This novel high value enzyme derivative is a valuable asset within the molecular reagent space as a tool for confirming both in vivo and in vitro genome editing; therefore, a means to produce it recombinantly in a scalable and technoeconomicaly viable process is highly desirable.
