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
Original language | English |
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Article number | e99347 |
Journal | PLoS ONE |
Volume | 9 |
Issue number | 6 |
DOIs | |
Publication status | Published - 2014 |
MoE publication type | A1 Journal article-refereed |