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(rCI alpha 1) chain with telopeptides and peptide, foldon to effect triple helix formation and a 44-k-Da rCI alpha 1 fragment. For each, similar to 60% of the rCI alpha 1s in the seed was recovered in the dry-milled germ-rich fractions making up ca. 25% of the total kernel mass. For wet milling, similar to 60% of each was recovered in three fractions accounting for 20-25% of the total kernel mass. The rCI alpha 1s in the dry-milled germ-rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCI alpha 1s were enriched 4-10 times in selected wet-milled fractions. The recovered starch from wet milling was almost free of rCI alpha 1. Therefore, it was possible to generate rCI alpha 1-enriched fractions by both dry and wet milling along with rCI alpha 1-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. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1396-1401, 2009