Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

The influence of culture and market orientation on services brands:insights from Irish banking and retail firms

Purpose: This paper aims to examine the influence of the culture of the service firm on its interpretation of the role of the brand and on the development and implementation of its brand values. Design/methodology/approach: A grounded theory approach was used. Interviews were conducted with 20 managers within two leading banking firms in Ireland and two leading grocery retailers in Ireland. Findings: The development of the brand, and its role within the firm, is closely related to the firm's culture. The research shows obstacles and opportunities created by the cultural context of firms wishing to disseminate and embed a set of brand values. The paper presents an "involvement model" of brand values implementation and outlines changes required to implement brand values. Research limitations/implications: The study was bound by access to firms, and managers' availability. The authors sought an insight into the relationship between each firm's culture and its brands. They advocate quantitative research to further investigate the findings within these service sectors and to test proposed antecedents (transformational leadership, employee involvement) and outcomes (employee-based brand equity and consumer-based brand equity) of values adoption. Practical implications: The paper identifies aspects of retail and banking cultures which support or detract from brand development. In particular, it presents the learnings from successful brand values implementation in a clan culture, aspects of which are applicable across other cultures. Originality/value: The paper provides valuable insights into the role of the brand within the service firm and the positive and negative influence of context on brand values and their development and implementation. © Emerald Group Publishing Limited.

The influence of socio-cultural environments on the performance of nascent entrepreneurs:community culture, motivation, self-efficacy and start-up success

The importance of informal institutions and in particular culture for entrepreneurship is a subject of ongoing interest. Past research has mostly concentrated on cross-national comparisons, cultural values and the direct effects of culture on entrepreneurial behaviour, but in the main found inconsistent results. We add a fresh perspective to this research stream by turning attention to community-level culture and cultural norms. We hypothesize indirect effects of cultural norms on venture emergence: Community-level cultural norms (performance-based culture and socially supportive institutional norms) impact important supply-side variables (entrepreneurial self-efficacy and entrepreneurial motivation) which in turn influence nascent entrepreneurs' success in creating operational ventures (venture emergence). We test our predictions on a unique longitudinal dataset, tracking nascent entrepreneurs' venture creation efforts over a five-year time span, and find evidence supporting them. Our research contributes to a more fine-grained understanding of how culture, in particular perceptions of community cultural norms, influences venture emergence. Based on these findings, we discuss how venture creation efforts can be supported. Our research highlights the embeddedness of entrepreneurial behaviour and its immediate antecedent beliefs in the local, community context. © 2012 Copyright Taylor and Francis Group, LLC.

Performance-based vs socially supportive culture:a cross-national study of descriptive norms and entrepreneurship

This paper is a cross-national study testing a framework relating cultural descriptive norms to entrepreneurship in a sample of 40 nations. Based on data from the Global Leadership and Organizational Behavior Effectiveness project, we identify two higher-order dimensions of culture – socially supportive culture (SSC) and performance-based culture (PBC) – and relate them to entrepreneurship rates and associated supply-side and demand-side variables available from the Global Entrepreneurship Monitor. Findings provide strong support for a social capital/SSC and supply-side variable explanation of entrepreneurship rate. PBC predicts demand-side variables, such as opportunity existence and the quality of formal institutions to support entrepreneurship.

Culture and the psychological impacts of natural disasters: Implications for disaster management and disaster mental health

In recent decades, natural disasters have caused extensive losses and damages to human psychological wellbeing, economy, and society. It has been argued that cultural factors such as social values, traditions, and attachment to a location influence communities facing and responding to natural disasters. However, the issue of culture in disaster mental health seems to have received limited attention in policy and practice. This review highlights the importance of cultural background in the assessment of vulnerability to the psychological impacts of disasters, disaster preparedness, and provision of disaster mental health services. In particular, this paper suggests the importance of cultural competence in the planning and delivery of effective disaster mental health services. In order to address the varying circumstances of people with different cultural backgrounds, disaster mental health services must be developed in a culturally sensitive manner. Development of culturally competent disaster mental health services requires significant changes in policy making, administration, and direct service provision

Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate

Background aims: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. Methods: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. Results: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. Conclusions: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

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.

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

A comparative analysis of cultural value orientations of Indians and migrant Indians in the USA

Understanding the cultural value systems of nations is a key factor in anticipating the behaviour of business managers and employees in a specific business environment. Many research studies have acknowledged the impact of culture on communication across nations and its impact on business operations, however no study has attempted to measure and quantify the cultural orientations of people originating from one nation, but working in two different national settings. This study adopted Kluckhohn and Strodtbeck's framework to examine cultural dimensions of a total of 580 Indian respondents comprising two groups: 429 Indian natives living and working in India and 151 Indian migrants living and working in the USA. It initially compares the cultural orientations of the total population of each of the two groups and then examines cultural differences in the same based on demographic characteristics consisting of occupation, gender, age, and level of education. The study found significant cultural value differences between the two groups on both levels of analysis. The theoretical and practical implications of these findings are discussed in detail.

The Team Climate Inventory (TCI): a psychometric test on a Taiwanese sample of work groups

Understanding the feasibility of applying the Team Climate Inventory (TCI) in non-Western cultures is essential for researchers attempting to understand the influence of culture on workers' perceived climate. This study describes the application of the TCI in such a setting using data from 203 administrators employed in a Taiwanese medical center. Reliability and factor analyses were performed to establish the feasibility and psychometric properties of the TCI Taiwan version. Reliabilities of both the four- and five-factor solutions exceeded .80. Factor analyses indicated a satisfactory four-factor structure, despite some variations in comparison with the U.K. version. The TCI Taiwan version is feasible and has acceptable psychometric properties. Further research is warranted regarding the degree to which disparities result from cultural differences and the specific nature of organizational systems in Chinese communities.

Liberalization and the public sector:the pre-eminent role of governments in the ‘sale’ of higher education abroad

Much recent scholarship concerning liberalization has emphasized the role of regulatees, rather than governments, in promoting liberalization. This article examines such scholarship in the light of an important development in the British and French public sectors—the creation of new agencies (the Education Counselling Service and EduFrance) to ‘sell’ British and French higher education to potential international students. The new agencies attempted to induce two things: competition amongst higher education institutions for the recruitment of international students from developed and emerging economy countries, and the commodification of these students. This article shows that, contrary to existing theories of liberalization, governments were pre-eminent in pushing forward this liberalization, while higher education institutions attempted to hold it back.

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage. We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O2 in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP. In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.