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Genetic Engineering Strategies for Purification of Recombinant Proteins From Canola by Anion Exchange Chromatography: An Example of Beta-Glucuronidase

Genetic Engineering Strategies for Purification of Recombinant Proteins From Canola by Anion Exchange Chromatography: An Example of Beta-Glucuronidase

C Zhang, R T Love, J M Jilka, C E Glatz

Biotechnol Prog. Jan-Feb 2001;17(1):161-7.

PMID: 11170494

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9001450-A	Fast β-Glucuronidase, Recombinant		9001-45-0	Price

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