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2.7.1.163: hygromycin B 4-O-kinase

This is an abbreviated version!
For detailed information about hygromycin B 4-O-kinase, go to the full flat file.

Word Map on EC 2.7.1.163

Reaction

ATP
+
hygromycin B
=
ADP
 +
4-O-phosphohygromycin B

Synonyms

Hm phosphotransferase, HPH, Hph5, HPT, hptII, hygromycin B phosphotransferase, hygromycin B4-O-phosphohygromycin, hygromycin phosphotransferase, hygromycin-B kinase, hygromycin-phosphotransferase, HyR

ECTree

     2 Transferases
         2.7 Transferring phosphorus-containing groups
             2.7.1 Phosphotransferases with an alcohol group as acceptor
                2.7.1.163 hygromycin B 4-O-kinase

Purification

Purification on EC 2.7.1.163 - hygromycin B 4-O-kinase

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
from cell culture by centrifugation and sonication, supernatant is loaded onto a column of chelating sepharose fast flow and then proteins are subjected to gel filtration
-
Harvested cells are suspended in buffer with DNase I and phenylmethylsulfonyl fluoride. The cells are lysed by sonication. Cell debris is separated by centrifugation and the soluble fraction is applied onto an Ni2+-affinity column equilibrated with buffer. The protein is eluted with a linear gradient of imidazole using a fast protein liquid-chromatography system. The eluted protein is dialyzed and applied onto a column of Q-Sepharose FF. The Hph5 protein is eluted with a linear gradient of 0-500 mM NaCl. The purified Hph5 is concentrated to 20-30 mg/ml for crystallization and stored at 20°C. Protein purification is monitored by SDS-PAGE. The selenomethionine-substituted Hph5 is purified using an Ni2+-affinity column and a Q-Sepharose FF column; this process is identical to that uses to purify the native Hph5 and is performed using buffers containing 20 mM mercaptoethanol.
-
recombinant enzyme
-
recombinant His-tagged enzyme denatured by 8 M urea, by nickel affinity chromatography to over 95% purity showing high immunoactivity
-
soluble renatured recombinant enzyme by anion exchange chromatography to over 95% purity
-