title: Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: an example of beta-glucuronidase.
abstract: The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.
authors.affiliation: Department of Chemical Engineering, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: C
authors.initials: C
authors.lastname: Zhang
authors.forename: R T
authors.initials: RT
authors.lastname: Love
authors.forename: J M
authors.initials: JM
authors.lastname: Jilka
authors.forename: C E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D000837:Anion Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D005818:Genetic Engineering; D005966:Glucuronidase / Q000302:isolation & purification*; D010940:Plant Proteins / Q000302:isolation & purification*; D030821:Plants, Genetically Modified / Q000201:enzymology; D000074262:Rapeseed Oil; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D000837:Anion Exchange Resins; D005229:Fatty Acids, Monounsaturated; D010940:Plant Proteins; D000074262:Rapeseed Oil; D011994:Recombinant Proteins; D005966:Glucuronidase