Process engineering strategy for recombinant protein recovery from canola by cation exchange chromatography

The suitability of canola as a recombinant protein production host was eval uated in terms of the potential for simple chromatographic recovery by ion exchange. To investigate the influence of the charge of a recombinant prote in on recovery from canola, a series of mutants of T4 lysozyme of varying c harge were used to model the situation of transgenic expression by being sp iked into nontransgenic canola protein extracts. This mixture was then frac tionated by cation exchange chromatography. Two different means of charge m odification were compared, namely, point mutations and fusions. Point mutat ions proved the better means for adding positive charges. A linear relation ship between the protein charge and the eluent conductivity, which could be used to guide the genetic engineering for protein recovery from canola, wa s found. It showed that an increase of fl charge on T4 lysozyme increased t he required conductivity (molarity) of the eluent by 0.068 mS/cm (27.8 mM N aCl). For this specific case, T4 lysozyme with a nominal charge of +7 moves the point of elution into a valley between two major native canola protein peaks, where substantial one-step enrichment can be obtained. Equivalent c harge changes provided by polyarginine fusions gave very wide elution patte rns that were ascribed to either proteolytic degradation within the polyarg inine fusion or interaction of the polyarginine with polyanions present in canola. While the above results came after a dialysis step to adjust the ca nola extract, elimination of the dialysis step did not significantly influe nce the purification behavior of the cation-exchange column. However, a mor e severe resin regeneration scheme was needed to maintain the column's perf ormance.