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Genetic Engineering Strategies for Purification of Recombinant Proteins from Canola by Anion Exchange Chromatography: An Example of β‐Glucuronidase
Author(s) -
Zhang Chenming,
Love Robert T.,
Jilka Joseph M.,
Glatz Charles E.
Publication year - 2001
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp000140c
Subject(s) - canola , elution , chromatography , chemistry , recombinant dna , ion chromatography , enzyme , biochemistry , escherichia coli , affinity chromatography , fusion protein , gene , food science
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 β‐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.