Polyelectrolyte Precipitation of Wild-type and Charge-modified β-Glucurondiase from Canola, Corn, and Soy Extracts

摘要

A selective precipitation early in the process of purifying a recombinant protein from transgenic crops would be beneficial to reduce the load sent to downstream units. In recovery of relatively charged proteins from microbial extracts, precipitation with oppositely charged poly-electrolytes has proven to be an effective means of accomplishing this. Here we have investigated the potential of utilizing a polycationic precipitating agent (polyethylenimine ― PEI), to precipitate an acidic model protein (β-glucuronidase ― GUS), from the aqueous extracts of canola, corn, and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein have been investigated - the wild-type enzyme (WTGUS) and a genetically engineered version containing ten additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola would be the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (50 mg PEI/g total protein) and over 80% of the initial WTGUS activity could be recovered with an 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS, but had only a 1.3-fold enrichment factor. Corn, although requiring the most PEI to precipitate (210 mg PEI/g total protein for 100% precipitation), gave moderate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yield of recoverable activity and greater purification factors.