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Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification

Molecular Breeding volume 3, pages 291–306 (1997)Cite this article

Abstract

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces.

An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at −29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.

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Authors and Affiliations

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

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

  2. Pioneer Hi-Bred International, Inc., PO Box 1004, Johnston, IA, 50131-1004, USA

    Derrick R. Witcher, Sheila Maddock, Terry Meyer, Chris Baszczynski, Pam Flynn, James Register, Lisa Marshall, Diane Bond, Ellen Kulisek, David Ritland & Chun Ping Li

  3. Dept. of Food Science and Human Nutrition, Iowa State University, 2312 Food Sciences Building, Ames, IA, 50011-1060, USA

    Ann Kusnadi, Roque Evangelista & Zivko Nikolov

  4. Sigma Chemical Company, 3500 Dekalb, St. Louis, MO, 63118, USA

    Cynthia Wooge, Richard J. Mehigh, Ronald Hernan & William K. Kappel

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  1. Elizabeth E. Hood
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  3. Sheila Maddock
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  4. Terry Meyer
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Hood, E.E., Witcher, D.R., Maddock, S. et al. Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Molecular Breeding 3, 291–306 (1997). https://doi.org/10.1023/A:1009676322162

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  • DOI: https://doi.org/10.1023/A:1009676322162

  • avidin
  • heterologous gene
  • localization
  • male sterility
  • processing
  • protein production
  • protein purification
  • seed expression
  • transgenic maize