Planta

, Volume 229, Issue 5, pp 1109–1122

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

  • Nunzia Scotti
  • Fiammetta Alagna
  • Enrico Ferraiolo
  • Gelsomina Formisano
  • Lorenza Sannino
  • Luigi Buonaguro
  • Angelo De Stradis
  • Alessandro Vitale
  • Luigi Monti
  • Stefania Grillo
  • Franco M. Buonaguro
  • Teodoro Cardi
Original Article

DOI: 10.1007/s00425-009-0898-2

Cite this article as:
Scotti, N., Alagna, F., Ferraiolo, E. et al. Planta (2009) 229: 1109. doi:10.1007/s00425-009-0898-2

Abstract

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.

Keywords

HIV-1 Pr55gag Plant factories Plastid transformation Protein accumulation Tobacco 

Abbreviations

ELISA

Enzyme-linked immunosorbent assay

HIV-1

Human immunodeficiency virus type 1

MS

Murashige and Skoog

PBS

Phosphate-buffered saline

Supplementary material

425_2009_898_MOESM1_ESM.pdf (228 kb)
Supplementary figure S1 (PDF 228 kb)
425_2009_898_MOESM2_ESM.pdf (87 kb)
Supplementary figure S2 (PDF 86 kb)
425_2009_898_MOESM3_ESM.pdf (91 kb)
Supplementary figure S3 (PDF 91 kb)
425_2009_898_MOESM4_ESM.pdf (55 kb)
Supplementary Table S1 (PDF 55 kb)

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Nunzia Scotti
    • 1
  • Fiammetta Alagna
    • 1
  • Enrico Ferraiolo
    • 1
  • Gelsomina Formisano
    • 1
  • Lorenza Sannino
    • 1
  • Luigi Buonaguro
    • 2
  • Angelo De Stradis
    • 3
  • Alessandro Vitale
    • 4
  • Luigi Monti
    • 1
  • Stefania Grillo
    • 1
  • Franco M. Buonaguro
    • 2
  • Teodoro Cardi
    • 1
  1. 1.CNR-IGV, Institute of Plant Genetics, National Research CouncilPorticiItaly
  2. 2.Istituto Nazionale Tumori “Fond. G. Pascale”NaplesItaly
  3. 3.CNR-IVV, Institute of Plant Virology, National Research CouncilBariItaly
  4. 4.Istituto di Biologia e Biotecnologia AgrariaConsiglio Nazionale delle RicercheMilanEU

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