A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease

A simple purification method of the Bacillus uricase (Uao) was newly developed. The gene coding for Uao with a C-terminal 6-histidine tag (Uao-HT) was constructed and overexpressed. Using the non-specific proteases, such as proteinase K, the tag was easily removed because Uao-HT includes its C-terminal region to be specifically cleaved by them. Such treatment of Uao-HT with the proteases did not affect its enzymatic properties and enabled us to purify it from the crude extract with a single-step protocol; the cell lysate containing Uao-HT was mixed with the Ni ion-chelating magnetic beads and then the adsorbed enzyme was eluted with the proteinase K-containing buffer after untagged proteins were washed out. The isolated enzyme yielded a single band on SDS-PAGE and was fully active. This method is extremely useful for high-throughput purification of mutants because of compatibility with automation.