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Commercial production of aprotinin in transgenic maize seeds

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Abstract

The development of genetic transformation technology for plants has stimulated an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutical value. In this regard, we report the generation and characterization of transgenic maize lines producing recombinant aprotinin. The transgenic aprotinin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced into immature maize embryos via particle bombardment. Aprotinin gene expression 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 genetic 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 T2 seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental transgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T2 lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of the purified aprotinin from seeds revealed that the recombinant aprotinin had the same molecular weight, N-terminal amino acid sequence, isoelectric point, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seeds has biochemical and functional properties identical to its native counterpart provides a proof-of-concept example for producing new generation products for the pharmaceutical industry.

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Authors and Affiliations

  1. Pioneer Hi-Bred International Inc., 7300 NW 62nd Avenue, Johnston, IA , 50131-1004, USA

    Gan-Yuan Zhong, David Peterson, James C. Register III, Chun-Ping Li, Lisa Marshall, Ellen Kulisek, David Ritland & Terry Meyer

  2. ProdiGene, Inc., 1500 Research Parkway, Suite 220, College Station, TX , 77845, USA

    Donna E. Delaney, Michele Bailey, Elizabeth E. Hood & John A. Howard

  3. Eli Lilly & Company, Lilly Corporate Center, Indianapolis, IN , 46285, USA

    Derrick R. Witcher

  4. PE-Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA, 94404, USA

    Diane Bond

Authors
  1. Gan-Yuan Zhong
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  2. David Peterson
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  3. Donna E. Delaney
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  4. Michele Bailey
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  5. Derrick R. Witcher
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  6. James C. Register III
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  7. Diane Bond
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  8. Chun-Ping Li
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  9. Lisa Marshall
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  10. Ellen Kulisek
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  11. David Ritland
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  12. Terry Meyer
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  13. Elizabeth E. Hood
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  14. John A. Howard
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Zhong, GY., Peterson, D., Delaney, D.E. et al. Commercial production of aprotinin in transgenic maize seeds. Molecular Breeding 5, 345–356 (1999). https://doi.org/10.1023/A:1009677809492

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