Extraction of Recombinant Dog Gastric Lipase from Transgenic Corn Seed

Abstract

Several approaches were examined for extracting the relatively hydrophobic protein recombinant dog gastric lipase (rDGL) expressed in the endosperm of transgenic corn seed. The first approach used minimal processing of the seed before extraction (i.e. simple grinding of whole seed) followed by selective extraction to eliminate 72% of contaminant proteins without compromising rDGL recovery from the meal of whole grain. The second approach added defatting of the whole grain meal to reduce the amount of detergent in the subsequent step for extracting rDGL. The third approach incorporated dry-milling of the corn to recover an endosperm rich fraction, followed by extraction of this fraction. The dry milling strategy was most effective, resulting in recovery of 35 U rDGL/g of corn seed (50 U/g of endosperm) with a specific activity of 9 U/mg compared to 22 U and 3 U/mg for the first strategy and 36 U and 3.7 U/mg for the second. The reductions in host protein contamination and lower detergent levels of the endosperm route should simplify downstream purification steps. Several approaches were examined for extracting the relatively hydrophobic protein recombinant dog gastric lipase (rDGL) expressed in the endosperm of transgenic corn seed. The first approach used minimal processing of the seed before extraction (i.e. simple grinding of whole seed) followed by selective extraction to eliminate 72% of contaminant proteins without compromising rDGL recovery from the meal of whole grain. The second approach added defatting of the whole grain meal to reduce the amount of detergent in the subsequent step for extracting rDGL. The third approach incorporated dry-milling of the corn to recover an endosperm rich fraction, followed by extraction of this fraction. The dry milling strategy was most effective, resulting in recovery of 35 U rDGL/g of corn seed (50 U/g of endosperm) with a specific activity of 9 U/mg compared to 22 U and 3 U/mg for the first strategy and 36 U and 3.7 U/mg for the second. The reductions in host protein contamination and lower detergent levels of the endosperm route should simplify downstream purification steps.

Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen‐related proteins

“…in the production of lipases. 15 Well-established dry-and wet-milling processes have been utilized by milling industries to fractionate corn components for value-added products, but they have not been extensively studied to fractionate corn as a means to enrich recombinant proteins. Only a few studies on the dry-milling process for that purpose have been published 7,13 and no extensive study on the fractionation efficiency of the wet-milling process for the recovery of recombinant proteins could be identified.…”

Wet‐milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine

“…Most grain fractionation processes for recovery of recombinant proteins produced in maize have been based mainly on hand separation or drymilling procedures. 12,15,20,26,27,34 On the other hand, an early preliminary study on fractionation of transgenic maize producing b-glucuronidase (GUS) by wet-milling showed detrimental effects on recombinant GUS activity. 27 In order to study the effects of the steeping composition on LT-B recovery from wet-milled fractions, we carried out the milling process using traditional (SO 2 þ lactic acid, TS) and water steeping (WS).…”

“…25 Standard dry-milling procedures were also used to study the benefit of fractionation in purification of a recombinant dog lipase targeted to be expressed in the endosperm of transgenic maize. 20 In this study, it was reported that recombinant lipase can be extracted from both endosperm and germ fractions, but utilization of endosperm fraction can eliminate the need for germ defatting and its effects on recombinant protein purification.…”

“…The two most widely used tissues for recombinant protein expression are the germ 12,13,18 and the endosperm. 19,20 The smaller amount of germ per kernel (11%) on mass basis compared to endosperm (82.9%) could be a potential advantage for achieving greater recombinant protein concentration. The endosperm, however, is a natural storage tissue for proteins (primarily water-insoluble zeins) and starch represents up to 83% of the total kernel mass, which also makes it an attractive target for foreign protein accumulation.…”

“…24 Although efforts have been focused on optimizing wet-milling conditions for maize fractionation, much less has been done to study the effects of wet-milling on recombinant protein recovery from transgenic maize. Fractionation studies for recovery of recombinant proteins have been carried out for germ-targeted 12,[25][26][27] and endosperm-targeted proteins 20,25,28 mainly using dry-milling or other non-wet-milling procedures.…”