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Mechanisms devoted to the secretion of proteins via extracellular vesicles (EVs) have been found in mammals, yeasts, and plants. Since they transport a number of leader-less proteins to the plasma membrane or the extracellular space, EVs are considered part of Unconventional protein secretion (UPS) routes. UPS involving EVs are a relatively new field in plants. Aside from their role in plant physiology and immunity, plant extracts containing EVs have also been shown to be beneficial for human health. Therefore, exploring the use of plant EVs in biomedicine and their potential as drug delivery tools is an exciting avenue. Here we give a summary of the state of knowledge on plant EVs, their crosstalk with mammalian systems and potential research routes that could lead to practical applications in therapeutic drug delivery.

A major obstacle for studying membrane proteins by biophysical techniques is the difficulty to produce sufficient amount of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially a eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacteria and insect cells, solubilization screening, large-scale production as well as commonly used affinity chromatography purification methods. We discuss general optimisation conditions such as promoters, tags and describe current techniques that can be used in any laboratory without specialised expensive equipment. Especially for insect cells, GFP-fusions are particularly useful for localisation and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.