Abstract
Fusion protein strategies are useful tools to enhance expression and to support the development of purification technologies. The capacity of fusion protein strategies to enhance expression was explored in tobacco leaves and seeds. C-terminal fusion of elastin-like polypeptides (ELP) to influenza hemagglutinin under the control of either the constitutive CaMV 35S or the seed-specific USP promoter resulted in increased accumulation in both leaves and seeds compared to the unfused hemagglutinin. The addition of a hydrophobin to the C-terminal end of hemagglutinin did not significantly increase the expression level. We show here that, depending on the target protein, both hydrophobin fusion and ELPylation combined with endoplasmic reticulum (ER) targeting induced protein bodies in leaves as well as in seeds. The N-glycosylation pattern indicated that KDEL sequence-mediated retention of leaf-derived hemagglutinins and hemagglutinin-hydrophobin fusions were not completely retained in the ER. In contrast, hemagglutinin-ELP from leaves contained only the oligomannose form, suggesting complete ER retention. In seeds, ER retention seems to be nearly complete for all three constructs. An easy and scalable purification method for ELPylated proteins using membrane-based inverse transition cycling could be applied to both leaf- and seed-expressed hemagglutinins.
Highlights
The efficient production of recombinant eukaryotic proteins in native conformations is a major goal for the biotechnological production of therapeutic proteins
Its widespread use is curtailed by problems related to expression levels, antigen purity and proper post-translational modification which are important for induction of robust immune responses
We show here that C-terminal fusion of elastin-like polypeptide (ELP) to HA resulted in increased accumulation of hemagglutinin subtype 5 (H5)-ELP in both leaves and seeds under control of either the constitutive CaMV 35S or the seed-specific USP promoter in comparison to the untagged H5 and H5-HFBI (Figure 2, 3)
Summary
The efficient production of recombinant eukaryotic proteins in native conformations is a major goal for the biotechnological production of therapeutic proteins. Three protein fusion systems, ZERA fusions, hydrophobin (HFBI) fusions and fusions with elastin-like polypeptide (ELP), that allow high intracellular accumulation of recombinant proteins in separate and newly formed storage organelles were described. These strategies facilitate the development of specific purification processes [9,10,11,12,13,14]. The insoluble ELP fusion proteins are re-solubilized in a cool and low-ionic buffer This purification method was described in 1999 and called centrifugation-based ITC (cITC) [16]. Hemagglutinin (HA) and neuraminidase from the avian flu virus have been expressed as ELPylated proteins in plants and purified by this simple and scalable method from leaves [24,25,26]
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