Strategies for recombinant protein recovery from canolaby precipitation

Abstract

Transgenic plants may prove to be one of the most economical systems for the large-scale production of proteins and peptides. Our goal is to develop an approach for protein recovery from canola that will be adaptable to a wide variety of recombinant proteins. For recombinant protein recovery, the two downstream processes considered were extraction of target protein and purification of recombinant protein from host proteins and other impurities. In these experiments, T4 lysozyme has been added to the canola extracts as an example protein to simulate recovery of recombinant proteins while evaluating the merits of several candidate precipitation strategies to understand selectivity behavior and how it might be affected by the presence of canola components. The ability of precipitating agents, such as acid and the polyelectrolytes Glass H and poly(acrylic acid) (PAA), was investigated. Approximately 70% of extracted canola proteins were precipitated by acid addition to pH 5, leaving about 90% of T4 lysozyme still in solution. Targeting T4 lysozyme for the precipitate phase by addition of oppositely charged polyelectrolyte was not successful in the presence of canola components. Addition of 2.5 times the PAA dosage required to precipitate pure T4 lysozyme to the spiked canola extract brought down only 40% of the T4 lysozyme. For the comparable result with Glass H, a 9 times higher dosage was required.