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

作者：Nishiya Y^*; Hibi T; Oda JL

来源：Protein Expression and Purification, 2002, 25(3): 426-429.

DOI：10.1016/S1046-5928(02)00022-0

• 6-histidine tags
• processing
• proteinase K
• Ni ion-chelating
• automation

A simple purification method of the Bacillus uricase (Uao) was newly developed, The gone coding for Uao with it C-terminal 6-histidine tag (Uao-HT) was constructed and overexpressed. Using the non-specific proteases. Such its 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 it single band oil SDS-PAGE and was fully active. This method is extremely useful for high-throughput purification of mutants because of compatibility with automation.

• 出版日期2002-8