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.

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

The dynamics of organizational change : an analysis of power, professions and new managerialism in the case of CoastElectric

It is widely accepted that the Thatcher years and their immediate aftermath were associated with substantive social and organizational change. The privatisation programme, 'the rolling back of the State', prosecuted by the successive Conservative Governments from 1979-1997 was a central pillar of Governmental policy. This thesis seeks to engage with privatization through the of CoastElectric, a newly privatised Regional Electricity Company. This thesis contributes to the extant understanding of the dynamics of organizational change in four major ways. Firstly, the study into CoastElectric addresses the senior management decision making within the organization: in particular, it will attempt to make sense of 'why' particular decisions were made. The theoretical backdrop to this concern will draw on the concepts of normalization, cultural capital and corporate fashion. The argument presented in this thesis is that the decision-making broadly corresponded with that which could be considered to be at the vanguard of mangerialist thought. However, a detailed analysis suggested that at different junctures in CoastElectric's history there were differences in the approach to decision making that warranted further analysis. The most notable finding was that the relative levels of new managerialist cultural capital possessed by the decision-making elite had an important bearing upon whether the decision was formulated either endogenously or exogenously, with the assistance of cultural intermediaries such as management consultants. The thesis demonstrates the importance of the broader discourse of new managerialism in terms of shaping what is considered to be a 'commonsensical, rational' strategy. The second concern of this thesis is that of the process of organizational change. The study of CoastElectric attempts to provide a rich account of the dynamics of organizational change. This is realized through, first, examining the pre-existing context of the organization; second, through analyzing the power politics of change interventions. The master concepts utilised in this endeavour are that of: dividing practices, the establishment of violent hierarchies between competing discourses; symbolic violence; critical turning points; recursiveness; creative destruction; legitimation strategies and the reconstitution of subjects in the workplace.

Stability studies on pencillin derivatives

Current analytical assay methods for ampicillin sodium and cloxacillin sodium are discussed and compared, High Performance Liquid Chromatography (H.P.L.C.) being chosen as the most accurate, specific and precise. New H.P.L.C. methods for the analysis of benzathine cloxacillin; benzathine penicillin V; procaine penicillin injection B.P.; benethamine penicillin injection; fortified B.P.C.; benzathine penicillin injection; benzathine penicillin injection, fortified B.P.C.; benzathine penicillin suspnsion; ampicillin syrups and penicillin syrups are described. Mechanical or chemical damage to column packings is often associated with H.P.L.C. analysis. One type, that of channel formation, is investigated. The high linear velocity of solvent and solvent pulsing during the pumping cycle were found to be the cause of this damage. The applicability of nonisotherrnal kinetic experiments to penicillin V preparations, including formulated paediatric syrups, is evaluated. A new type of nonisotherrnal analysis, based on slope estimation and using a 64K Random Access Memory (R.A.M.) microcomputer is described. The name of the program written for this analysis is NONISO. The distribution of active penicillin in granules for reconstitution into ampicillin and penicillin V syrups, and its effect on the stability of the reconstituted products, are investigated. Changing the diluent used to reconstitue the syrups was found to affect the stability of the product. Dissolution and stability of benzathine cloxacillin at pH2, pH6 and pH9 is described, with proposed dissolution mechanisms and kinetic analysis to support these mechanisms. Benzathine and cloxacillin were found to react in solution at pH9, producing an insoluble amide.

The social and entrepreneurial orientation of small businesses:mutually exclusive or reinforcing? (INTERACTIVE PAPER)

Principal Topic - As argued by Acs and Phillips (2002) it is not only “the creation of wealth (entrepreneurship)” but also “the reconstitution of wealth (philanthropy)”, which has been essential for the inherent dynamism of the market economy (Ibid., p.201). However, we understand little about the entrepreneurship – philanthropy link in institutional contexts that differ from that of leading developed market economies. Accordingly our research agenda is to investigate the entrepreneurship-philanthropy nexus in a very different context of Lithuania, a country which shed a command economy system twenty years ago. In particular, we are interested to see if the cluster of attitudes and strategies of firms conducive to entrepreneurship, i.e. their entrepreneurial orientation (Covin & Slevin, 1989), is consistent or contradictory with philanthropy? In other words, is philanthropy strongly associated with some core components of entrepreneurship, or is it an entrepreneurial anomaly, relying on a minority of economic actors that provide important links with wider, non-economic communities. Method - The study draws on 270 randomly sampled, phone interviews with owners and ownermanagers of small and medium-sized enterprises (SMEs), i.e. firms with less than 250 employees. Interviews were conducted in Lithuania during January- March, 2008. Our results are based on confirmatory factor analysis combined with regression analysis. Results and Implications - Despite the legacy of informal institutions that is conducive neither to entrepreneurship nor to civic society, we found that by now, (i) the companies that score highest on entrepreneurial orientation construct, (ii) that perform best and those (iii) that have foreign owners are also most likely to declare their commitment to philanthropy. Our findings that most entrepreneurial firms are also involved in philanthropy are consistent with the perspective on the pattern of development in an entrepreneurial economy as outlined by Acs and Phillips (2002).

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.