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

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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.

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Abbreviations

ELISA:

Enzyme-linked immunosorbent assay

HIV-1:

Human immunodeficiency virus type 1

MS:

Murashige and Skoog

PBS:

Phosphate-buffered saline

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Acknowledgments

This work was partially funded by the grant of the Italian Ministry of Research (MIUR) DD 1105/2002. Thanks are due to Prof. P. Maliga, Rutgers University, NJ, USA, for generously providing plastid transformation vectors. We are also grateful to Dr. P. Lenzi, CNR-IGV, Portici, Italy, and to Dr. A. Pompa, CNR-IBBA, Milan, Italy, for help in ELISA and pulse-chase experiments, respectively. HIV-1 antisera (specific for p24 and p17 subunits), and recombinant proteins (p24, p17 and proteinase) were provided by the NIBSC Centralized Facility for AIDS reagent (EU Programme EVA, UK Medical Research Council).

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Authors and Affiliations

  1. CNR-IGV, Institute of Plant Genetics, National Research Council, Via Università 133, 80055, Portici, Italy

    Nunzia Scotti, Fiammetta Alagna, Enrico Ferraiolo, Gelsomina Formisano, Lorenza Sannino, Luigi Monti, Stefania Grillo & Teodoro Cardi

  2. Istituto Nazionale Tumori “Fond. G. Pascale”, Via M. Semmola 1, 80131, Naples, Italy

    Luigi Buonaguro & Franco M. Buonaguro

  3. CNR-IVV, Institute of Plant Virology, National Research Council, Via G. Amendola 165/A, 70126, Bari, Italy

    Angelo De Stradis

  4. Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133, Milan, Italy, EU

    Alessandro Vitale

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  1. Nunzia Scotti
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  2. Fiammetta Alagna
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  3. Enrico Ferraiolo
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  4. Gelsomina Formisano
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  10. Stefania Grillo
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  11. Franco M. Buonaguro
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Correspondence to Nunzia Scotti.

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IGV publication no. 330

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Scotti, N., Alagna, F., Ferraiolo, E. et al. High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts. Planta 229, 1109–1122 (2009). https://doi.org/10.1007/s00425-009-0898-2

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