Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis

Animating the Ethical Demand:Exploring user dispositions in industry innovation cases through animation-based sketching

This paper addresses the challenge of attaining ethical user stances during the design process of products and services and proposes animation-based sketching as a design method, which supports elaborating and examining different ethical stances towards the user. The discussion is qualified by an empirical study of Responsible Research and Innovation (RRI) in a Triple Helix constellation. Using a three-week long innovation workshop, UCrAc, involving 16 Danish companies and organisations and 142 students as empirical data, we discuss how animation-based sketching can explore not yet existing user dispositions, as well as create an incentive for ethical conduct in development and innovation processes. The ethical fulcrum evolves around Løgstrup's Ethical Demand and his notion of spontaneous life manifestations. From this, three ethical stances are developed; apathy, sympathy and empathy. By exploring both apathetic and sympathetic views, the ethical reflections are more nuanced as a result of actually seeing the user experience simulated through different user dispositions. Exploring the three ethical stances by visualising real use cases with the technologies simulated as already being implemented makes the life manifestations of the users in context visible. We present and discuss how animation-based sketching can support the elaboration and examination of different ethical stances towards the user in the product and service development process. Finally we present a framework for creating narrative representations of emerging technology use cases, which invite to reflection upon the ethics of the user experience.</jats:p