Commercial production of aprotinin in transgenic maize seeds

The development of genetic transformation technology for plants has stimula ted an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutica l value. In this regard, we report the generation and characterization of t ransgenic maize lines producing recombinant aprotinin. The transgenic aprot inin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced i nto immature maize embryos via particle bombardment. Aprotinin gene express ion was driven by the maize ubiquitin promoter and protein accumulation was targeted to the extracellular matrix. One line that showed a high level of aprotinin expression was characterized in detail. The protein accumulates primarily in the embryo of the seed. Southern blot analysis showed that the line had at least 20 copies of the bar and aprotinin genes. Further geneti c analysis revealed that numerous plants derived from this transgenic line had a large range of levels of expression of the aprotinin gene (0-0.069%) of water-soluble protein in T-2 seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental tra nsgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T-2 lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of t he purified aprotinin from seeds revealed that the recombinant aprotinin ha d the same molecular weight, N-terminal amino acid sequence, isoelectric po int, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seed s has biochemical and functional properties identical to its native counter part provides a proof-of-concept example for producing new generation produ cts for the pharmaceutical industry.