Exceptional overproduction of a functional human membrane protein

Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. © 2007 Elsevier Inc. All rights reserved.

Improving recombinant eukaryotic membrane protein yields in Pichia pastoris:the importance of codon optimization and clone selection

In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

Interregionalism

Interregionalism is deeply rooted in the foreign policies and external relations of the EU. Interregional cooperation today not only encompasses trade and aid but also political dialogue, cultural relations and even security cooperation. Although the EU’s official ambition has been to formalize and institutionalize its interregional relations with other regional bodies or organizations (so-called ‘pure interregionalism’), in practice there are a bewildering variety of interregional or group-to-group relations on display (Hänggi 2006; Baert et al. 2014). The EU is rapidly evolving as a global actor and while doing so it has been trying to export its own civilian and normative values. Interregionalism is an important tool in this process, contributing to the EU’s policy of fostering regionalism worldwide, not only in the triad (Europe, North America and East Asia) (De Lombaerde and Schulz 2009). Through interregionalism, the EU and its regional others enhance their presence, gain recognition, tighten institutional cohesion and define identities. Interregionalism, therefore, occupies a special position in the construction of regional actorness in global affairs (Wunderlich 2012). However, the link between interregionalism and regionalism is both complex and underexplored (Baert et al. 2014; Doidge 2007). Much depends on the type of interregional relations, and the balance of other forms of cooperation, which appears to play out differently in different regions. All this leads to a number of research questions that should be addressed by the academic literature, including is there a preference for interregional relations in EU’s foreign policy? If so, for what reason(s)? What are the consequences of such a preference? What is the role of interregionalism in the broader context of EU’s external policies? How are expressions of regionalism related to expressions of interregionalism? Does the sui generis character of the EU lead to a sui generis character of EU interregionalism? This chapter provides a general overview of the evolution of the field, the key conceptual and analytical debates, as well as the main research questions that drive the research agenda. Emphasis is also placed on identifying the main gaps in the field and suggesting directions.

Plasma membrane abundance of human aquaporin 5 is dynamically regulated by multiple pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.