title: Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing.
abstract: We describe the purification of a single-strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative-scale isoelectric 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.
authors.affiliation: Protein Technologies Incorporated, Tucson, AZ 85719.
authors.forename: J A
authors.initials: JA
authors.lastname: Ostrem
authors.forename: T R
authors.initials: TR
authors.lastname: Van Oosbree
authors.forename: R
authors.initials: R
authors.lastname: Marquez
authors.forename: L
authors.initials: L
authors.lastname: Barstow
mesh_terms: D000645:Ammonium Sulfate; D000665:Ampholyte Mixtures; D001236:Aspergillus oryzae / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002872:Chromosome Deletion; D002999:Clone Cells; D004271:DNA, Fungal / Q000032:analysis; D007525:Isoelectric Focusing; D010957:Plasmids; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000235:genetics / Q000302:isolation & purification*; D013379:Substrate Specificity
chemical_list: D000665:Ampholyte Mixtures; D004271:DNA, Fungal; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000645:Ammonium Sulfate