Dry‐Milling and Fractionation of Transgenic Maize Seed Tissues with Green Fluorescent Protein as a Tissue Marker

Abstract

The efficiency of fractionating cereal grains (e.g., dry corn milling) can be evaluated and monitored by quantifying the proportions of seed tissues in each of the recovered fractions. The quantities of individual tissues are typically estimated using indirect methods such as quantifying fiber or ash to indicate pericarp and tip cap contents, and oil to indicate germ content. More direct and reliable methods are possible with tissue‐specific markers. We used two transgenic maize lines, one containing the fluorescent protein green fluorescent protein (GFP) variant S65T expressed in endosperm, and the other containing GFP expressed in germ to determine the fate of each tissue in the dry‐milling fractionation process. The two lines were dry‐milled to produce three fractions (bran‐, endosperm‐, and germ‐rich fractions) and GFP fluorescence was quantified in each fraction to estimate the tissue composition. Using a simplified laboratory dry‐milling procedure and our GFP‐containing grain, we determined that the endosperm‐rich fraction contained 4% germ tissue, the germ‐rich fraction contained 28% germ, 20% endosperm, and 52% nonendosperm and nonembryo tissues, and the bran‐rich fraction contained 44% endosperm, 13% germ, and 43% nonendosperm and nonembryo tissues. GFP‐containing grain can be used to optimize existing fractionation methods and to develop improved processing strategies.

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

“…Because the majority of oil accumulates in the corn germ, the oil content and recovery in each fraction were commonly used to estimate the purity of the germ and endosperm fractions. Targeted expression of green fluorescent protein (GFP) was recently used as marker 16 to study fractionation efficiency, but GFP would need to be coexpressed with the desired recombinant protein to improve on the oil method to estimate fractionation efficiency for a specific grain. The typical oil content in an industrial dry-milled germ-rich fraction for producing foodgrade corn grits and flour is about 23% db (dry basis), 17 which corresponds to about 70% purity based on the assumption that pure germ contains 33% oil.…”

“…60% germ yield. 16 The oil contents of the endosperm-rich fractions are typically less than 1% from an effective industrial dry-milling process.…”

Utilizing Protein-Lean Coproducts from Corn Containing Recombinant Pharmaceutical Proteins for Ethanol Production

“…In kernels containing r-protein expressed in germ, about 70% of the protein, can be recovered in 20-25% of the mass (9)(10)(11). The starch-rich endosperm could be directly used as a fermentation feedstock to produce biofuel and/or industrial chemicals.…”

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

“…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.…”

“…27 Dry-milling and hand fractionation of transgenic maize expressing the green fluorescent protein under control of an embryo-or endosperm-specific promoter were used to study the efficiency of the fractionation procedures. 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.…”