Pillay, PriyenMoralo, MaaboMtimka, SibongileShai, TaolaBotha, KirstyKwezi, LusisizweTsekoa, Tsepo L.2025-03-102025-03-102025-03Pillay, P., Moralo, M., Mtimka, S. et al. 2025, 'Identification and purification of a novel bacteriophage T7 endonuclease from the Kogelberg Biosphere Reserve (KBR) biodiversity hotspot', Biotechnology Reports, vol. 45, art. e00877, pp. 1-9, doi : 10.1016/j.btre.2025.e00877.2215-017X (online)10.1016/j.btre.2025.e00877http://hdl.handle.net/2263/101400DATA AVAILABILITY : Data will be made available on request.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.en© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/).T7 endonuclease IHolliday junction resolvase/nucleaseDeoxyribonucleic acid (DNA)DNA-protein interactionNucleasesJunction-resolving enzymeGenome editing detectionCrispr/Cas9Kogelberg Biosphere Reserve (KBR)SDG-15: Life on landArticle