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

Xu, Jianfeng; Shpak, Elena; Gu, Tingyue; Moo‐Young, Murray; Kieliszewski, Marcia J.

doi:10.1002/bit.20441

Cited by 27 publications

(24 citation statements)

References 57 publications

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“…The secretion of recombinant protein greatly enhanced by addition of a Hyp glycomodule is consistent with recent work where we expressed EGFP and IFNa2 as HRGP chimeras Shpak et al 2001;1999;Tan et al 2003;Xu et al 2005;2007;Zhao et al 2002). We suggest that Hyp glycosylation, both arabinosylation and arabinogalactosylation, facilitate protein secretion to the extracellular matrix, a role proposed earlier for protein glycosylation in general (Eylar 1966).…”

Section: Discussionsupporting

confidence: 88%

Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life

Okada

et al. 2010

Transgenic Res

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Therapeutic proteins with molecular weights lower than 40 kDa often have short serum half-lives due to their susceptibility to serum proteases and rapid renal clearance. Chemical derivatization, such as PEGylation, or expression as serum albumin fusions increases molecular mass and overcome these problems but at the expense of decreased bioactivity. Here we applied a new method that yields biologically potent recombinant human growth hormone (rhGH) with increased serum half-life when expressed as an arabinogalactan-protein (AGP) in tobacco BY-2 cells. Thus, rhGH was expressed with 10 repeats of the AGP glycomodule Ser-Hyp (SO) at the C-terminus (rhGH-(SO)(10)). We also expressed rhGH as an AGP-enhanced green fluorescent protein (EGFP) fusion, designated rhGH-(SO)(10)-EGFP, to assess the cellular distribution of the glycoprotein, which was mainly extracellular. Recombinant hGH-(SO)(10) bound the hGH receptor with an affinity similar to that of a rhGH standard, stimulated the same intracellular signaling pathway as hGH, but possessed an in vivo serum half-life more than sixfold that of the hGH control. Furthermore, rhGH-(SO)(10) gave a 500 fold greater secreted yield than the non-glycosylated control rhGH that was also targeted for secretion. Detailed analysis of the rhGH-(SO)(10) glycans indicated a conserved structure with relatively little microheterogeneity and an average size of 25 monosaccharide residues. These results were consistent with earlier work expressing interferon alpha 2b as an AGP chimera and further demonstrate the feasibility of this approach to the production of long-acting, biologically potent therapeutic proteins by plant cells.

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Section: Discussionsupporting

confidence: 88%

Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life

Okada

et al. 2010

Transgenic Res

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“…Primary Structure of Interferon Hyp-polysaccharide-1-Sizefractionated base hydrolysates of Interferon␣2-(Ser-Hyp) 20 yielded a single peak containing sugar and Hyp residues, described earlier (26). Two subfractions were chosen for structural analyses; one contained the major Hyp-AG species, designated Interferon Hyp-polysaccharide-1, with 22 glycosyl residues estimated by the Hyp to monosaccharide molar ratios, and a second fraction contained a smaller, less abundant Hyp-arabinogalactan of 20 glycosyl residues, designated interferon Hyp-polysaccharide-2.…”

Section: Resultsmentioning

confidence: 75%

“…The Structures of Interferon Hyp-polysaccharide-1, interferon Hyp-polysaccharide-2, and Ala-Hyp-polysaccharide-2 were determined based on earlier composition analyses and on the chemical shifts (15,26) observed in one-dimensional 1 H NMR spectra and two-dimensional COSY, TOCSY, HSQC, and HMBC NMR spectra as follows:…”

Section: Resultsmentioning

confidence: 99%

Plant O-Hydroxyproline Arabinogalactans Are Composed of Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Várnai

Lamport

et al. 2010

Journal of Biological Chemistry

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105

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Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon ␣2b-(Ser-Hyp) 20 , and a 14-residue Hyp-AG isolated from (Ala-Hyp) 51 -green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 133 ␤-linked trigalactosyl blocks linked by a ␤-136 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an136)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the ␤-136 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.Hydroxyproline-rich glycoproteins of the cell surface comprise groups of related structural proteins, including the extensins that form cell wall scaffolding networks essential for cytokinesis (1) and the classical arabinogalactan proteins (2) that are largely at the membrane wall interface (3) and have diverse functions (4). O-Hyp 6 glycosylation characterizes the hydroxyproline-rich glycoproteins and is of much interest as it defines molecular properties and, hence, biological function. Arabinogalactan proteins are highly glycosylated mainly with O-Hyp-arabinogalactan polysaccharides (5, 6). Extensins are less highly glycosylated mainly with small O-Hyp arabinooligosaccharides (7,8), whereas the related proline-rich proteins are minimally glycosylated also with arabinooligosaccharides (9). Peptide sequence directs O-Hyp glycosylation by the addition of small oligosaccharides to contiguous Hyp residues and larger acidic arabinogalactan polysaccharides to clustered noncontiguous Hyp (10, 11). For example, clustered Ala-Hyp and Ser-Hyp are typical AGP glycosylation motifs (12), whereas the Hyp residues in repetitive blocks of Ser-Hyp 2 orSer-Hyp 4 are arabinosylated. The "hyperglycosylation" of closely related AGPs complicates their purification, a problem that can be overcome by expressing single individual AGPs as GFP fusion glycoproteins, the hydrophobic GFP tag enabling chromatographic purification (13,14). This approach also allows purification of neoAGPs containing single repeating AGP glycosylation motifs, for example (Ala-Hyp) n or (Ser-Hyp) n , for base-catalyzed peptide bond hydrol...

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“…Cell suspension culture has been proposed as a biotechnological alternative to develop biological compounds production systems (Zhao et al 2005). Because, Arabinogalactan-proteins are predominantly secreted into cell wall space, these macromolecules can be found in the suspension cell medium (Hernández-Sánchez et al 2009;Capataz-Tafur et al 2010) and furthermore, be responsible for the emulsifying activity observed in this type of glycoproteins (Xu et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Establishment and characterization of Prosopis laevigata (Humb. & Bonpl. ex Willd) M.C. Johnst. cell suspension culture: a biotechnology approach for mesquite gum production

et al. 2011

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This study presents a protocol for the establishment of Prosopis laevigata cell suspension culture as a strategy to obtain an in vitro mesquite gum productive cell line. The callus used for this purpose was obtained with hypocotyls from 15-day-old plantlets, placed on Murashige-Skoog medium with two different plant growth regulators (PGRs), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T; 5.0 lM) and kinetin (KIN; 5.0 lM). With this PGRs treatment, after four subcultures (30 days each) an exuded gum-like substance was observed on the callus surface. The growth kinetics of the cell suspension culture showed a specific cell growth rate (l) of 0.14 d -1 and doubling time (t d ) of 6.6 days, respectively. The gum-like substance from callus culture and the broth from cell suspension culture were subjected to chemical analysis and compared with the mesquite gum exuded from wild trees. Both, gum-like substance from callus culture and the broth from cell suspension culture showed the presence of Arabinogalactanproteins, and their polysaccharide fraction presented the same monosaccharides as those isolated from mesquite gum. In addition, the emulsifying properties of gum-like substance from callus culture and the broth from cell suspension culture were compared to those of mesquite gum and all three samples exhibited similar emulsifying capacity and emulsification stability.

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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

Cited by 27 publications

References 57 publications

Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life

Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life

Plant O-Hydroxyproline Arabinogalactans Are Composed of Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Establishment and characterization of Prosopis laevigata (Humb. & Bonpl. ex Willd) M.C. Johnst. cell suspension culture: a biotechnology approach for mesquite gum production

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