Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins

The terminase enzyme from bacteriophage lambda is composed of two viral pro teins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging vir al DNA into the confines of an empty procapsid, We are interested in the ge netic, biochemical, and biophysical properties of DNA packaging in phage la mbda and, in particular, the nucleoprotein complexes involved in these proc esses. These studies require the routine purification of large quantities o f wild-type and mutant proteins in order to probe the molecular mechanism o f DNA packaging. Toward this end, we have constructed a hexahistidine (hexa -His)tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA s ubunits. We present a simple, one-step purification scheme for the purifica tion of large quantities of the holoenzyme and the individual subunits dire ctly from the crude cell lysate, Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a singl e step. Hexa-His terminase holoenzyme is functional in vise and possesses s teady-state and single-turnover ATPase activity that is indistinguishable f rom wild-type enzyme. The nuclease activity of the modified holoenzyme is n ear wild type, but the reaction exhibits a greater dependence on Escherichi a coil integration host factor, a result that is mirrored in vivo, These re sults suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-bind ing defect that is masked, at least in part, by integration host factor. Th e mild defect in hexa-His terminase holoenzyme is more significant in the i solated gpA-hexa-His subunit that does not appear to bind DNA Moreover, whe reas the hexa-His-tagged gpNul subunit may be reconstituted into a holoenzy me complex with wild-type catalytic activities, gpa-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reporte d here underscore that a complete biochemical characterization of the effec ts of purification tags on enzyme function must be performed prior to their use in mechanistic studies.