High‐yields and extended serum half‐life of human interferon α2b expressed in tobacco cells as arabinogalactan‐protein fusions

Abstract

Therapeutic proteins like human interferon a2 generally possess short serum half-lives due to their small size, hence rapid renal clearance, and susceptibility to serum proteases. Chemical derivatization, such as addition of polyethylene glycol (PEG) groups overcomes both problems, but at the expense of greatly decreased bioactivity. We describe a new method that yields biologically potent interferon a2b (IFNa2) in high yields and with increased serum half-life when expressed as arabinogalactan-protein (AGP) chimeras in cultured tobacco cells. Thus IFNa2-AGPs targeted for secretion typically gave 350-1400-fold greater secreted yields than the non-glycosylated IFNa2 control. The purified AGP domain itself was not immunogenic when injected into mice and only mildly so when injected as a fusion glycoprotein. Importantly, the AGPIFNa2 chimeras showed up to a 13-fold increased in vivo serum half-life while the biological activity remained similar to native IFNa2. The use of arabinogalactan glycomodules may provide a general approach to the enhanced production of therapeutic proteins by plants.

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

“…Gene Construction and Expression in Tobacco Cells-Genes encoding Interferon ␣2-(Ser-Hyp) 20 and (Ala-Hyp) 51 -GFP were constructed and expressed as described in detail earlier (12,18). Briefly, proteins were targeted for secretion using a tobacco extensin signal sequence, and gene expression was under control of the 35 S cauliflower mosaic virus promoter.…”

“…Briefly, proteins were targeted for secretion using a tobacco extensin signal sequence, and gene expression was under control of the 35 S cauliflower mosaic virus promoter. The genes were subcloned into the plant transformation vector pBI121 and expressed in tobacco Bright Yellow-2 cells selected and maintained as described earlier (12,18).…”

“…Isolation of the Interferon ␣2-(Ser-Hyp) 20 and (Ala-Hyp) 51 -GFP Fusion Glycoproteins-Interferon ␣2-(Ser-Hyp) 20 and (Ala-Hyp) 51 -GFP were isolated from the medium of suspension-cultured tobacco Bright Yellow-2 cells by hydrophobic interaction chromatography on a phenyl-Sepharose column as described earlier (18).…”

“…Two of the HypAGs designated interferon-polysaccharide-1 (interferon Hyppolysaccharide 1) and interferon-polysaccharide-2 were isolated from a fusion glycoprotein of human interferon ␣2b fused to a (Ser-Hyp) 20 AGP glycomodule (18). The third Hyp-AG, designated Ala-Hyp polysaccharide-2, was isolated from (AlaHyp) 51 -GFP similar to Ala-Hyp-polysaccharide-1 described earlier (15).…”

Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

“…As shown in Fig. 2A, characteristic AGP-like smeared bands in a high molecular weight range were observed on respective SDS/PAGE gels stained with either Coomassie brilliant blue or 0.2% (wt/vol) β-GlcY dye (35,36). Consistent with the gel information, the presence of AGPs in the purified ivy nanoparticles was further evidenced by Western blotting analysis using two mAbs, JIM13 and JIM14, which are commonly used to specifically recognize glycan epitopes of typical AGPs (see Table S1), as shown in Fig.…”