The expression of a xylanase targeted to ER-protein bodies provides a simple strategy to produce active insoluble enzyme polymers in tobacco plants
Llop Tous, Immaculada (Centre de Recerca en Agrigenòmica)
Ortiz Alcaide, Miriam (Centre de Recerca en Agrigenòmica)
Torrent, Margarita (Centre de Recerca en Agrigenòmica)
Ludevid, M. Dolors (Centre de Recerca en Agrigenòmica)

Fecha:	2011
Resumen:	Background: Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings: Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance: In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.
Ayudas:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-703 Ministerio de Ciencia e Innovación CDS2007/00036 Ministerio de Ciencia e Innovación TRA2009/0124
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Publicado en:	PloS one, Vol. 6, Issue 4 (April 2011) , art. e19474, ISSN 1932-6203

DOI: 10.1371/journal.pone.0019474
PMID: 21559333

11 p, 1.0 MB

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