Barbante, Alessandra, Irons, Sarah, Hawes, C. R., Frigerio, Lorenzo, Vitale, A. (Alessandro) and Pedrazzini, Emanuela. (2008) Anchorage to the cytosolic face of the endoplasmic reticulum membrane : a new strategy to stabilize a cytosolic recombinant antigen in plants. Plant Biotechnology Journal, Volume 6 (Number 6). pp. 560-575. ISSN 1467-7644

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Abstract

The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QP Physiology S Agriculture > SB Plant culture
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Cytochromes, Viral proteins, Endoplasmic reticulum, Viral proteins -- Physiological transport, Transgenic plants, Recombinant proteins
Journal or Publication Title:	Plant Biotechnology Journal
Publisher:	Wiley-Blackwell Publishing Ltd.
ISSN:	1467-7644
Date:	August 2008
Volume:	Volume 6
Number:	Number 6
Number of Pages:	16
Page Range:	pp. 560-575
Identification Number:	10.1111/j.1467-7652.2008.00342.x
Status:	Not Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Sixth Framework Programme (European Commission) (FP6)
Grant number:	LSH-2002-1.2.5-2 (FP6)
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URI:	http://wrap.warwick.ac.uk/id/eprint/29701

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