Genetic engineering strategies for purification of recombinant proteins from Canola by anion exchange chromatography: An example of beta-glucuronidase

The elution behavior of native canola proteins from different anion-exchang e resins was determined. The elution profiles showed the potential for simp lified recovery of acidic recombinant proteins from canola. When Q-sepharos e fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native protein s. The feasibility of exploiting this advantage was examined for recovery o f the acidic protein beta -glucuronidase (GUS/ GUSD0 from the Escherichia c oli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fus ed 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 ad ded to canola extracts before chromatography. The equivalence of this spiki ng experiment to that of extracting a recombinant protein from transgenic c anola was determined in a control experiment using transgenic canola expres sing the wild-type enzyme. Behavior in the transgenic and spiked experiment s was equivalent. GUSD0 eluted at the earliest optimal elution point; the a ddition of polyaspartate tails resulted in longer retention times and bette r selective recovery. If one assumes binding through a single fusion (the p rotein 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 flexibili ty in process scheduling.