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Investigating the subcellular trafficking of human recombinant proteins expressed in plants
Perspectives of extension of this common project to a more ambitious proposal to be submitted to funding agencies : Long term Added value of the cooperation : The Portuguese team has been investigating the subcelular localization of several recombinant proteins produced in Medicago plants using immunolocalization with specific antibodies followed by confocal fluorescence microscopy analysis. However, the resolution of confocal microscopy and in general of all photon-based microscopy techniques is insufficient for an accurate localization of the proteins at the subcellular level. This can only be achieved by immunogold labeling and electron microscopy analysis, which will be carried out by the Spanish team. This team has been working on in situ molecular localization techniques for 15 years, applying them to the subcellular localization of proteins and nucleic acid sequences in plant material.
Summary of the proposal :
The production of recombinant proteins in plant-based systems is a promising field in plant biotechnology. Many plant platforms are currently used for the production of important molecules including pharmaceutical products; however many aspects of the intracellular trafficking of recombinant proteins are still unknown and may impair the successful production and accumulation of the final molecule. The factors that affect production include tissue and species specific features, as well as cell line dependent factors, and also intrinsic properties of the recombinant protein to be produced.
In this work, we aim to study the deposition pattern of a human recombinant protein produced in the legume model Medicago truncatula, using immunogold labeling and electron microscopy analysis. The protein – Lipocalin-type prostaglandin D synthase or L-PGDS - is a glycosylated human protein that is present in the cerebrospinal fluid and is involved in various physiological and pathological functions. We are particularly interested in comparing the subcellular fate of L-PGDS in different tissues of the plant (leaves, roots, seeds), as well as in undifferentiated cells in suspension cultures derived from the transgenic plants. This knowledge will help in choosing the most adequate plant-based platform for production, not only in terms of yield but also to determine the most cost effective extraction and purification procedures to be used. The results expected for the present project will complete the characterization of the expression of L-PGDS at both tissue and subcellular levels, significantly contributing to a better understanding of its dynamics when expressed in an heterologous system such as Medicago.
Antonio PINEDA-LUCENA, Valencia Claudio GOMES, Lisboa
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