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Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing
Author(s) -
Ostrem James A.,
Van Oosbree Thomas R.,
Marquez Rodolfo,
Barstow Leon
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150111113
Subject(s) - isoelectric focusing , chromatography , chemistry , aspergillus oryzae , ammonium sulfate precipitation , isoelectric point , electrophoresis , protein purification , nuclease , ammonium sulfate , denaturation (fissile materials) , enzyme , biochemistry , size exclusion chromatography , nuclear chemistry
Abstract We describe the purification of a single‐strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3 TM ) designed by Milan Bier et al. for preparative‐scale insoelectric focusing. Protein separation takes place entirely in solution, with shear‐stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE‐Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially‐purified protein (250 mg, 6700 U/mg) was focused in pH 3‐4 carrier ampholytes, 67% of the activity was recovered in pooled peak fractions with a specific activity of 54 000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four‐fold increase in specific activity (from 4000 U/mg protein to 15 000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.