Expression of Gene Product 126 from Phage Gizmo and Creation of a Substrate

Endonucleases are enzymes which cleave DNA at specific nucleotide sequences. Homing Endonucleases (HEase) are a group of endonucleases that reside as open reading frames within self-splicing introns or inteins. After expression of the gene containing the HEase the HEase goes on to cleave a target site in a homolog of the hosting gene to induce homologous recombination turning the vacant homolog into a homing endonuclease gene (HEG). It is predicted that gene product (gp126) from the mycobacteriophage Gizmo encodes a HEase. To test whether the gene encodes a HEase the phage sequence of gp126 Gizmo was transferred into the his-tag containing plasmid (PET14b). The protein has been expressed and purified. To test the activity of the HEase a substrate is being created that will allow the predicted function to be directly tested in an enzyme assay