We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway(®) recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals.

MORANDINI, Francesca;AVESANI, Linda;BORTESI, Luisa;BAZZONI, Flavia;PEZZOTTI, Mario
2011-01-01

Abstract

We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway(®) recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.
2011
molecular farming
plant made pharmaceuticals
transgenic plants
seed-specific expression
type 1 diabetes
GAD65
File in questo prodotto:
File Dimensione Formato  
2011_Plant Biotech J_Morandini.pdf

solo utenti autorizzati

Tipologia: Documento in Post-print
Licenza: Accesso ristretto
Dimensione 339.15 kB
Formato Adobe PDF
339.15 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/350989
Citazioni
  • ???jsp.display-item.citation.pmc??? 22
  • Scopus 47
  • ???jsp.display-item.citation.isi??? 43
social impact