title: Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization.
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)(8)GFP. The culture of the transgenic cells in a 5-L bioreactor showed that the production of (GA)(8)GFP was cell growth-associated. The extracellular yield of (GA)(8)GFP was 116.8 mg/L after 14 days of culture and accounted for 87% of the total fusion protein expressed. (GA)(8)GFP 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)(8)GFP 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.
authors.affiliation: Department of Chemistry and Biochemistry, Ohio University, 350 W. State St., Athens, Ohio 45701, USA. xuj@ohio.edu
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Xu
authors.forename: Elena
authors.initials: E
authors.lastname: Shpak
authors.forename: Tingyue
authors.initials: T
authors.lastname: Gu
authors.forename: Murray
authors.initials: M
authors.lastname: Moo-Young
authors.forename: Marcia
authors.initials: M
authors.lastname: Kieliszewski
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006023:Glycoproteins / Q000819:agonists / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006170:Gum Arabic / Q000378:metabolism*; D010940:Plant Proteins / Q000819:agonists / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C005488:extensin protein, plant; D006170:Gum Arabic