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

Abstract

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.

β-Galactosyl Yariv Reagent Binds to the β-1,3-Galactan of Arabinogalactan Proteins

“…Although we did not examine the Yariv reactivity of such a kinked main chain, a kink spaced at seven or more residues might not disrupt the helical structure of the main chain, particularly if it is a kink of b-1,6-linked Gal, since the b-1,6-linkage is generally flexible compared with other types of linkages (Rees and Scott, 1971). On the other hand, our results do not appear consistent with structural models for AG moieties that have a short kinked main chain but are nevertheless precipitated with b-Gal-Yariv Xu et al, 2010). Additionally, in the case of mugwort pollen, O-glycans were precipitated by b-Glc-Yariv, although they have been reported to lack b-1,3-galactan (Léonard et al, 2005).…”

Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site

“…in the culture medium of suspension cells) of the recombinant glycoproteins 500-fold (up to 120 mg/L) and the glycoproteins retained nearly 100% of their biological activity (Fernandes et al, 2010;Shpak et al, 1999Shpak et al, , 2001Tan et al, 2003;Xu et al, 2005Xu et al, , 2007Xu et al, , 2010. This work was consistent with the earlier suggestion of Eylar that protein glycosylation in general facilitates protein secretion (Eylar, 1966) and raises the possibility that N-glycosylation of the same proteins will give yields similar to those in yeast (Sagt et al, 2000).…”

“…However, in reality, recombinant proteins synthesized by plant cells, despite bearing a signal peptide, are often intracellularly retained and subsequently degraded by the cellular proteases. This results in significantly reduced protein yields and makes the cost for protein purification soar (Doran, 2006;Xu et al, 2007Xu et al, , 2010.…”

Transgenic Plants as Biofactories for the Production of Biopharmaceuticals: A Case Study of Human Placental Lactogen

“…In plants, several authors have reported the suitability of engineering fusion proteins to improve expression levels (Wirth et al, 2006;Xu et al, 2010). Many fusion proteins can also serve as purification tags due to their specific binding features or easy of recovery, but those will be discussed in the next section.…”