Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Visualisation of quality information for geospatial and remote sensing data:providing the GIS community with the decision support tools for geospatial dataset quality evaluation

The evaluation of geospatial data quality and trustworthiness presents a major challenge to geospatial data users when making a dataset selection decision. The research presented here therefore focused on defining and developing a GEO label – a decision support mechanism to assist data users in efficient and effective geospatial dataset selection on the basis of quality, trustworthiness and fitness for use. This thesis thus presents six phases of research and development conducted to: (a) identify the informational aspects upon which users rely when assessing geospatial dataset quality and trustworthiness; (2) elicit initial user views on the GEO label role in supporting dataset comparison and selection; (3) evaluate prototype label visualisations; (4) develop a Web service to support GEO label generation; (5) develop a prototype GEO label-based dataset discovery and intercomparison decision support tool; and (6) evaluate the prototype tool in a controlled human-subject study. The results of the studies revealed, and subsequently confirmed, eight geospatial data informational aspects that were considered important by users when evaluating geospatial dataset quality and trustworthiness, namely: producer information, producer comments, lineage information, compliance with standards, quantitative quality information, user feedback, expert reviews, and citations information. Following an iterative user-centred design (UCD) approach, it was established that the GEO label should visually summarise availability and allow interrogation of these key informational aspects. A Web service was developed to support generation of dynamic GEO label representations and integrated into a number of real-world GIS applications. The service was also utilised in the development of the GEO LINC tool – a GEO label-based dataset discovery and intercomparison decision support tool. The results of the final evaluation study indicated that (a) the GEO label effectively communicates the availability of dataset quality and trustworthiness information and (b) GEO LINC successfully facilitates ‘at a glance’ dataset intercomparison and fitness for purpose-based dataset selection.

A geometrical model for surface roughness prediction when face milling Al 7075-T7351 with square insert tools

Surface quality is important in engineering and a vital aspect of it is surface roughness, since it plays an important role in wear resistance, ductility, tensile, and fatigue strength for machined parts. This paper reports on a research study on the development of a geometrical model for surface roughness prediction when face milling with square inserts. The model is based on a geometrical analysis of the recreation of the tool trail left on the machined surface. The model has been validated with experimental data obtained for high speed milling of aluminum alloy (Al 7075-T7351) when using a wide range of cutting speed, feed per tooth, axial depth of cut and different values of tool nose radius (0.8. mm and 2.5. mm), using the Taguchi method as the design of experiments. The experimental roughness was obtained by measuring the surface roughness of the milled surfaces with a non-contact profilometer. The developed model can be used for any combination of material workpiece and tool, when tool flank wear is not considered and is suitable for using any tool diameter with any number of teeth and tool nose radius. The results show that the developed model achieved an excellent performance with almost 98% accuracy in terms of predicting the surface roughness when compared to the experimental data. © 2014 The Society of Manufacturing Engineers.

Role of the educator in social software initiatives in Further and Higher Education:a conceptualisation and research agenda

Higher and further education institutions are increasingly using social software tools to support teaching and learning. A growing body of research investigates the diversity of tools and their range of contributions. However, little research has focused on investigating the role of the educator in the context of a social software initiative, even though the educator is critical for the introduction and successful use of social software in a course environment. Hence, we argue that research on social software should place greater emphasis on the educators, as their roles and activities (such as selecting the tools, developing the tasks and facilitating the student interactions on these tools) are instrumental to most aspects of a social software initiative. To this end, we have developed an agenda for future research on the role of the educator. Drawing on role theory, both as the basis for a systematic conceptualization of the educator role and as a guiding framework, we have developed a series of concrete research questions that address core issues associated with the educator roles in a social software context and provide recommendations for further investigations. By developing a research agenda we hope to stimulate research that creates a better understanding of the educator’s situation and develops guidelines to help educators carry out their social software initiatives. Considering the significant role an educator plays in the initiation and conduct of a social software initiative, our research agenda ultimately seeks to contribute to the adoption and efficient use of social software in the educational domain.

Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and nongovernmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathway-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

Integration of a graph-based model indexer in commercial modelling tools

Softeam has over 20 years of experience providing UML-based modelling solutions, such as its Modelio modelling tool, and its Constellation enterprise model management and collaboration environment. Due to the increasing number and size of the models used by Softeam’s clients, Softeam joined the MONDO FP7 EU research project, which worked on solutions for these scalability challenges and produced the Hawk model indexer among other results. This paper presents the technical details and several case studies on the integration of Hawk into Softeam’s toolset. The first case study measured the performance of Hawk’s Modelio support using varying amounts of memory for the Neo4j backend. In another case study, Hawk was integrated into Constellation to provide scalable global querying of model repositories. Finally, the combination of Hawk and the Epsilon Generation Language was compared against Modelio for document generation: for the largest model, Hawk was two orders of magnitude faster.

Acumen : an open-source testbed for cyber-physical systems research

Developing Cyber-Physical Systems requires methods and tools to support simulation and verification of hybrid (both continuous and discrete) models. The Acumen modeling and simulation language is an open source testbed for exploring the design space of what rigorousbut- practical next-generation tools can deliver to developers of Cyber- Physical Systems. Like verification tools, a design goal for Acumen is to provide rigorous results. Like simulation tools, it aims to be intuitive, practical, and scalable. However, it is far from evident whether these two goals can be achieved simultaneously. This paper explains the primary design goals for Acumen, the core challenges that must be addressed in order to achieve these goals, the “agile research method” taken by the project, the steps taken to realize these goals, the key lessons learned, and the emerging language design.