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Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants

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An Erratum to this article was published on 24 April 2007

Abstract

High-level expression of transgenes is essential for cost-effective production of valuable pharmaceutical proteins in plants. However, transgenic proteins often accumulate in plants at low levels. Low levels of protein accumulation can be caused by many factors including post-transcriptional gene silencing (PTGS) and/or rapid turnover of the transgenic proteins. We have developed an Amplicon-plus Targeting Technology (APTT), by using novel combination of known techniques that appears to overcome both of these factors. By using this technology, we have successfully expressed the highly-labile L1 protein of canine oral papillomavirus (COPV L1) by infecting transgenic tobacco plants expressing a suppressor of post-transcriptional gene silencing (PTGS) with a PVX amplicon carrying a gene encoding L1, and targeting the vaccine protein into the chloroplasts. Further, a scalable “wound-and-agrospray” inoculation method has been developed that will permit high-throughput Agrobacterium inoculation of Nicotiana tabacum, and a spray-only method (named “agrospray”) for use with N. benthamiana to allow large-scale application of this technology. The good yield and short interval from inoculation to harvest characteristic of APTT, combined with the potential for high-throughput achieved by use of the agrospray inoculation protocol, make this system a very promising technology for producing high value recombinant proteins, especially those known to be highly labile, in plants for a wide range of applications including producing vaccines against rapidly evolving pathogens and for the rapid response needed to meet bio-defense emergencies.

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Abbreviations

APTT:

Amplicon-plus Targeting Technology

CTP:

chloroplast targeting peptide

P1/HC-Pro:

a mutated version of component-proteinase from Tobacco Etch Virus

PTGS:

post transcriptional gene silencing

PVX:

potato virus X

SiRNA:

small interfering RNA

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Acknowledgements

We thank David Baulcombe for supplying us with the PVX amplicon vector pGR106, and A. tumefaciens strain GV3101 containing the helper plasmid pJIC SA_Rep, Vicki Vance for supplying us with seeds of the transgenic TEV-B line, and Nancy Magnuson for providing us with the vector pZW containing the TEV and SP sequences. We also thank A. Bennett Jenson and Shin-je Ghim for providing the B1 monoclonal antibody used in this study, and for helpful discussions regarding COPV L1. We thank Rebecca Boston and Raymond Long for useful discussions, and Jussi Joensuu and Mirkka Kotiaho for information about the chloroplast extraction buffer used in this study. We especially thank Lesley Corbin for her work with the immuno-blot analysis. We also thank Yan Liu and Tan Tuong for growing plants and collecting leaf samples for some experiments. This work was supported by a grant to A.K.W. from the United States Department of Agriculture (USDA-CSREES, Contract No. 2001-38712-10592).

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Authors and Affiliations

  1. Department of Crop Science, North Carolina State University, Raleigh, 27695, USA

    Kasi Azhakanandam, Sandra M. Weissinger, Jennifer S. Nicholson, Rongda Qu & Arthur K. Weissinger

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  1. Kasi Azhakanandam
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  2. Sandra M. Weissinger
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  3. Jennifer S. Nicholson
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  4. Rongda Qu
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  5. Arthur K. Weissinger
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Correspondence to Arthur K. Weissinger.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s11103-007-9168-5

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Azhakanandam, K., Weissinger, S.M., Nicholson, J.S. et al. Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants. Plant Mol Biol 63, 393–404 (2007). https://doi.org/10.1007/s11103-006-9096-9

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  • DOI: https://doi.org/10.1007/s11103-006-9096-9

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