Poster: Protein targeting & vesicular trafficking

Abs # 649: Membrane Anchors for Vacuolar Targeting: Application in Plant Bioreactors

Presenter:	Tai Chi, Seto , seto520@yahoo.com
Authors	Tai Chi, Seto  (A)   Ka Leung Jacky, Fung  (A)   Sai-ming Samuel, Sun  (A)   Lorne,  Clarke A. (B)   Allison,  Kermode R. (C)   Liwen, Jiang  (A)
Affiliations:	(A): Department of Biology, the Chinese University of Hong Kong (B): Department of Medical Genetics, University of British Columbia (C): Department of Biological Sciences, Simon Fraser University

Using transgenic plants as bioreactors is an attractive and competitive approach for the economic production of recombinant proteins. However, recombinant proteins expressed in transgenic plants may be subjected to undesired post-transnational modifications and degradation in various compartments. Here we test the hypothesis that unique transmembrane domain (TMD) and cytoplasmic tail (CT) sequences can be used as anchors for delivering recombinant proteins via distinct vesicular transport pathways to specific vacuolar compartments for their stable accumulation in transgenic plants [1]. Towards this goal, yellow fluorescent protein (YFP) and a human lysosomal enzyme α-L-iduronidase (IDUA) were fused with various TMD/CT sequences and expressed in transgenic tobacco plants. Western blot analysis and confocal immunofluorescence studies demonstrated that YFP fusions were expressed in both leaves and seeds. To facilitate detections and purifications of IDUA fusions, anti-IDUA antibodies were generated using synthetic peptides that detected both IDUA peptides and recombinant IDUA. Western blot analysis demonstrated that IDUA fusions were expressed in both leaves and seeds of transgenic tobacco. Detection of IDUA fusions in transgenic tobacco seeds by confocal immunofluorescence was not conclusive. Further studies focus on subcellular localization of YFP and IDUA fusions and their transport pathways. Additionally, recombinant IDUA fusions will be purified for further analysis. Supported by RGC and AoE to L. Jiang. [1] L. Jiang and S. S. M. Sun (2002) Membrane anchors for vacuolar targeting: application in plant bioreactors. Trends in Biotechnology 20: 99-102.