Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization

Authors

  • Jianfeng Xu,

    Corresponding author
    1. Department of Chemistry and Biochemistry, Ohio University, 350 W. State St., Athens, Ohio 45701; telephone: 740-597-1225; fax: 740-597-1772
    2. Department of Chemical Engineering, Ohio University, Athens, Ohio 45701
    • Department of Chemistry and Biochemistry, Ohio University, 350 W. State St., Athens, Ohio 45701; telephone: 740-597-1225; fax: 740-597-1772
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  • Elena Shpak,

    1. Department of Chemistry and Biochemistry, Ohio University, 350 W. State St., Athens, Ohio 45701; telephone: 740-597-1225; fax: 740-597-1772
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  • Tingyue Gu,

    1. Department of Chemical Engineering, Ohio University, Athens, Ohio 45701
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  • Murray Moo-Young,

    1. Department of Chemical Engineering, Ohio University, Athens, Ohio 45701
    2. Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada, N2L 3G1
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  • Marcia Kieliszewski

    1. Department of Chemistry and Biochemistry, Ohio University, 350 W. State St., Athens, Ohio 45701; telephone: 740-597-1225; fax: 740-597-1772
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Abstract

Many plant gums, such as gum arabic, contain hydroxyproline-rich glycoproteins (HRGPs), which are also abundant components of the plant cell extracellular matrix. Here we expressed in transgenic BY2 Nicotiana tabacum (tobacco) cells, a synthetic gene encoding a novel HRGP-based gum, designated gum arabic-8 or (GA)8. (GA)8 encoded eight repeats of the consensus polypeptide sequence of gum arabic glycoprotein (GAGP): Gly-Pro-His-Ser-Pro-Pro-Pro-Pro-Leu-Ser-Pro-Ser-Pro-Thr-Pro-Thr-Pro-Pro-Leu, in which most of the Pro residues were posttranslationally modified to hydroxyproline (Hyp). (GA)8 was expressed as a green fluorescent protein (GFP) fusion protein targeted to the culture medium, (GA)8GFP. The culture of the transgenic cells in a 5-L bioreactor showed that the production of (GA)8GFP was cell growth-associated. The extracellular yield of (GA)8GFP was 116.8 mg/L after 14 days of culture and accounted for 87% of the total fusion protein expressed. (GA)8GFP was purified from the culture medium by a combination of hydrophobic interaction, gel permeation, and reversed phase chromatography. Biochemical characterization indicated that the amino acid composition of the (GA)8 module, after removal of GFP by proteolysis, was virtually identical to that of predicted by the GAGP consensus sequence and that carbohydrate, which occurred as arabinogalactan polysaccharides and small oligoarabinosides O-linked through the Hyp residues, accounted for 84% of the molecules' dry weight. Functional assays showed that (GA)8 exhibited low viscosity in aqueous solution similar to native GAGP. However, neither GFP alone nor the (GA)8 module could emulsify orange oil. However, the fusion protein (GA)8GFP possessed 1.28-fold better emulsification properties than native GAGP. This work demonstrates the feasibility and potential of a synthetic gene approach to the de novo design of novel glycoprotein-based gums and emulsifiers. © 2005 Wiley Periodicals, Inc.

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