BACTERIOPHAGE-T7 DNA-LIGASE - OVEREXPRESSION, PURIFICATION, CRYSTALLIZATION, AND CHARACTERIZATION

The bacteriophage T7 DNA ligase gene was amplified using polymerase ch ain reaction-based methods and cloned into a T7 promoter-based express ion vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of pro tein per liter of bacterial culture. An initial physical and biochemic al characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibi tion of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethyle ne glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 Angstrom. The unit cell dimensions are a 66.1 Ang strom, b = 87.6 Angstrom, and c = 78.6 Angstrom with one monomer in th e asymmetric unit (V-m = 2.77 Angstrom(3)/Da). This is the first membe r of the DNA ligase family of enzymes to be crystallized.