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

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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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References

  1. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD: Molecular Biology of the Cell, 3rd ed. Garland Publishing, New York/London (1994).

    Google Scholar 

  2. An G, Mitra A, Choi HK, Costa MA, An K, Thornburg RW, Ryan CA: Functional analysis of the 3′ control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1: 115–122 (1989).

    Google Scholar 

  3. Armstrong CL, Green CE, Phillips RL: Development and availability of germplasm with high Type II culture formation response. Maize Genet Coop Newsl 65: 92–93 (1991).

    Google Scholar 

  4. Austin S, Bingham ET, Koegel RG, Mathews DE, Shahan MN, Straub J, Burgess RR: An overview of a feasibility study for the production of industrial enzymes in trangenic alfalfa. Ann NY Acad Sci 721: 234–244 (1994).

    Google Scholar 

  5. Bassham DC, Gal S, de Silva Conceicao A, Raikhel NV: An Arabidopsis syntaxin homologue isolated by functional complementation of a yeast pep12 mutant. Proc Nat Acad Sci USA. 92: 7262–7266 (1995).

    Google Scholar 

  6. Benfey PN, Chua N-H: Regulated genes in transgenic plants. Science 244: 174–181 (1989).

    Google Scholar 

  7. Bradford M: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  8. Callis J, Fromm M, Walbot V: Introns increase gene expression in cultured maize cells. Genes Devel 1: 1183–1200 (1987).

    Google Scholar 

  9. Chay CH, Buehler EG, Thorn JM, Whelan TM, Bedinger PA: Purification of maize pollen exines and analysis of associated proteins. Plant Physiol 100: 756–761 (1992).

    Google Scholar 

  10. Christensen AM, Sharrock RA, Quail PH: Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18: 675–689 (1992).

    Google Scholar 

  11. Cornejo M, Luth D, Blankenship K, Anderson O, Blechl A: Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23: 567–581 (1993).

    Google Scholar 

  12. DeLange RJ, Huang TS: Egg white avidin. III. Sequence of the 75-residue middle cyanogen bromide peptide. Complete amino acid sequence of the protein subunit. J Biol Chem 246: 698–709 (1971).

    Google Scholar 

  13. Durance TD: Residual avidin activity in cooked egg white assayed with improved sensitivity. J Food Sci 56: 707 (1991).

    Google Scholar 

  14. Fisk HJ, Dandekar AM: The introduction and expression of transgenes in plants. Scient Hort 55: 5–36 (1993).

    Google Scholar 

  15. Fritz SE, Hood KR, Hood EE: Localization of soluble and insoluble fractions of hydroxyproline-rich glycoproteins during maize kernel development. J Cell Sci 98: 545–550 (1991).

    Google Scholar 

  16. Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucl Acids Res 15: 3257–3273 (1987).

    Google Scholar 

  17. Gope ML, Keinanen RA, Kristo PA, Conneely OM, Beattie WG, Zarucki-Schulz T, O'Malley BW, Kulomaa MS: Molecular cloning of the chicken avidin cDNA. Nucl Acids Res 15: 3595–3606 (1987).

    Google Scholar 

  18. Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brian JV, Chambers SA, Adams Jr. WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG: Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603–618 (1990).

    Google Scholar 

  19. Green NM: Spectrophotometric determination of avidin and biotin. Meth Enzymol 118A: 418–422 (1970).

    Google Scholar 

  20. Gribskov M, Devereux J, Burgess RR: The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucl Acids Res 12: 539–549 (1984).

    Google Scholar 

  21. Hallauer AR, Russell WA, Lamkey KR: Corn breeding. In: Sprague, GF, Dudley JW (eds) Corn and corn Improvement, 3rd ed., pp. 463–564. American Society of Agronomy, Madison, WI (1988).

    Google Scholar 

  22. Hancock K, Tsang VCW: India ink staining of protein on nitrocellulose paper. Anal Biochem 133: 157–162 (1983).

    Google Scholar 

  23. Heney G, Orr GA: The purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-sepharose 4B. Anal Biochem 114: 92–96 (1981).

    Google Scholar 

  24. Hiller Y, Gershoni JM, Bayer EA, Wilchek M: Biotin binding to avidin. Biochem J 248: 167–171 (1987).

    Google Scholar 

  25. Holzman D: Agracetus grows monoclonals in soybeans and corn plants. Genet Eng News 14(16): 1, 34 (1994).

    Google Scholar 

  26. Jhingan AK: A novel technology for DNA isolation. Meth Mol Cell Biol 3: 15–22 (1992).

    Google Scholar 

  27. Jones RL, Robinson DG: Protein secretion in plants. New Phytol 111: 567–597 (1989).

    Google Scholar 

  28. Kay R, Chan A, Daly M, McPherson J: Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302 (1987).

    Google Scholar 

  29. Keinanen RA, Laukkanen M-L, Kulomaa MS: Molecular cloning of three structurally related genes for chicken avidin. J Steroid Biochem 30: 17–21 (1988).

    Google Scholar 

  30. Keinanen RA, Wallen MJ, Kristo PA, Laukkanen MO, Toimela TA, Helenius MA, Kulomaa MS: Molecular cloning and nucleotide sequence of chicken avidin-related genes 1–5. Eur J Biochem 220: 615–621 (1994).

    Google Scholar 

  31. Krebbers E, Bosch D, Vandekerckhove J: Prospects and progress in the production of foreign proteins and peptides in plants. In: Shewry PR, Gutteridge S (eds) Plant Protein Engineering, pp. 315–325. Cambridge University Press (1993).

  32. Laemmli UK: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680–685 (1970).

    Google Scholar 

  33. Lowe K, Bowen B, Hoerster G, Ross M, Bond D, Pierce D, Gordon-Kamm B: Germline transformation of maize following manipulation of chimeric shoot meristems. Bio/technology 13: 677–682 (1995).

    Google Scholar 

  34. Matsudaira P: Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035–10038 (1987).

    Google Scholar 

  35. Nagy F, Odell JT, Morelli G, Chua NH: Properties of expression of the 35S promoter from CaMV in transgenic tobacco plants. In: Zaitlin M, Day P, Hollaender A (eds) Biotechnology in Plant Science: Relevance to Agriculture in the Eighties, pp. 227–235. Academic Press, Orlando, FL (1985).

    Google Scholar 

  36. Pen J, Verwoerd TC, van Paridon PA, Beudeker RF, van den Elzen PJM, Geerse K, van der Klis JD, Versteegh HAJ, van Ooyen AJJ, Hoekema A: Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization. Bio/technology 11: 811–814 (1993).

    Google Scholar 

  37. Pen J, Sijmons PC, Van Ooijen AJJ, Hoekema A: Protein production in transgenic crops: analysis of plant molecular farming. In: Industrial Crops Production, pp. 241–250. Elsevier, Amsterdam (1993).

    Google Scholar 

  38. Register JC III, Peterson DJ, Bell PJ, Bullock WP, Evans IJ, Frame B, Greenland AJ, Higgs NS, Jepson I, Jiao S: Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol Biol 25: 951–961 (1994).

    Google Scholar 

  39. Rogers JC: Two barley alpha-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260: 3731–3738 (1985).

    Google Scholar 

  40. Rogers SO, Bendich AJ: Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5: 69–73 (1985).

    Google Scholar 

  41. Sharp AH, Campbell KP: Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies: evidence for a 32 000 Da subunit. J Biol Chem 264: 2816–2825 (1989).

    Google Scholar 

  42. Staehelin LA, Moore I: The plant golgi apparatus: Structure, functional organization and trafficking mechanisms. Annu Rev Plant Physiol Plant Mol Biol 46: 261–288 (1995).

    Google Scholar 

  43. Tomes DT, Ross MC, Songstad DD: Direct DNA transfer into intact plant cells via microprojectile bombardment. In: Gamborg OL, Phillips GC (eds) Plant Cell Tissue and Organ Culture: Fundamental Methods, pp. 197–213. Springer-Verlag, Berlin/Heidelberg (1995).

    Google Scholar 

  44. White J, Chang S-YP, Bibb MJ, Bibb JM: A cassette containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucl Acids Res 18: 1062 (1992).

    Google Scholar 

  45. Whitelam GC, Cockbrurn B, Gandeacha AR, Owen MRL: Heterologous protein production in transgenic plants. Biotechnol Genet Eng Rev 11: 1–29 (1993).

    Google Scholar 

  46. Witcher DR, DeWaard M, Liu H, Pragnell M, Campbell KP: Association of native Ca2+ channel B subunits with the α1 subunit interaction domain. J Biol Chem 270: 18088–18093 (1995).

    Google Scholar 

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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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  14. Zivko Nikolov
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  15. Cynthia Wooge
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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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