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Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco
Department of Plant Cell Biology, Centre for Organismal Studies, University of Heidelberg, Heidelberg D-69120, Germany
Institute of Agricultural Biology and Biotechnology, National Research Council (CNR), via Bassini 15, Milano 20133, Italy
Laboratory of Cell and Molecular Biology, University of Neuchatel, Rue Emile-Argand 11, Neuchâtel CH-2000, Switzerland
* Authors to whom correspondence should be addressed.
Received: 7 April 2013; in revised form: 28 May 2013 / Accepted: 17 June 2013 / Published: 26 June 2013
Abstract: Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.
Keywords: HIV p24; transgenic plants; membrane proteins; endomembrane system; protein targeting; thrombin cleavage
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Virgili-López, G.; Langhans, M.; Bubeck, J.; Pedrazzini, E.; Gouzerh, G.; Neuhaus, J.-M.; Robinson, D.G.; Vitale, A. Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco. Int. J. Mol. Sci. 2013, 14, 13241-13265.
Virgili-López G, Langhans M, Bubeck J, Pedrazzini E, Gouzerh G, Neuhaus J-M, Robinson DG, Vitale A. Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco. International Journal of Molecular Sciences. 2013; 14(7):13241-13265.
Virgili-López, Goretti; Langhans, Markus; Bubeck, Julia; Pedrazzini, Emanuela; Gouzerh, Guillaume; Neuhaus, Jean-Marc; Robinson, David G.; Vitale, Alessandro. 2013. "Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco." Int. J. Mol. Sci. 14, no. 7: 13241-13265.