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

Abstract

Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed-based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen-related proteins; thereby, reducing the amount and type of corn-derived impurities in subsequent protein extraction and purification steps. The two proteins were a full-length human recombinant collagen type I alpha 1(rCIalpha1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44-kDa rCIalpha1 fragment. For each, approximately 60% of the rCIalpha1s in the seed was recovered in the dry-milled germ-rich fractions making up ca. 25% of the total kernel mass. For wet milling, approximately 60% of each was recovered in three fractions accounting for 20-25% of the total kernel mass. The rCIalpha1s in the dry-milled germ-rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCIalpha1s were enriched 4-10 times in selected wet-milled fractions. The recovered starch from wet milling was almost free of rCIalpha1. Therefore, it was possible to generate rCIalpha1-enriched fractions by both dry and wet milling along with rCIalpha1-free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on-farm thus minimizing the risk of inadvertent release of viable transgenic seeds.