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.
Nishiya
Yoshiaki
Y
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan. yoshiaki_nishiya@bio.toyobo.co.jp
Hibi
Takao
T
Oda
Jun
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D018189:Immunomagnetic Separation / Q000379:methods*; D008969:Molecular Sequence Data; D009532:Nickel; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D009532:Nickel; D014503:Urate Oxidase; D010450:Endopeptidases