Development of a quality control material for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an in vivo marker of oxidative stress, and comparison of results from different laboratories

The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

Computer-aided biotechnology:from immuno-informatics to reverse vaccinology

Genome sequences from many organisms, including humans, have been completed, and high-throughput analyses have produced burgeoning volumes of 'omics' data. Bioinformatics is crucial for the management and analysis of such data and is increasingly used to accelerate progress in a wide variety of large-scale and object-specific functional analyses. Refined algorithms enable biotechnologists to follow 'computer-aided strategies' based on experiments driven by high-confidence predictions. In order to address compound problems, current efforts in immuno-informatics and reverse vaccinology are aimed at developing and tuning integrative approaches and user-friendly, automated bioinformatics environments. This will herald a move to 'computer-aided biotechnology': smart projects in which time-consuming and expensive large-scale experimental approaches are progressively replaced by prediction-driven investigations.

The finance of biotechnology:investment in new biotechnology firms by British venture capitalists-attitudes to, and evaluation of, business proposals

A notable feature of the recent commercialisation of biotechnology has been the success of 200 or so new firms, established in America since 1976, in exploiting specialised market niches. A key factor in their formation has been the ready availability of venture capital funding. These firms have been instrumental in establishing America's lead in exploiting biotechnology. It is this example which Britain has attempted to emulate as part of its strategy for developing its own biotechnology capabilities. This thesis investigated some aspects of the relationship between biotechnology and venture capital, concentrating on the determinants of the venture capitalist's investment decision. Following an extensive literature survey, two hypothetical business proposals were used to find what venture capitalists themselves consider to be the key elements of this decision. It was found that venture capitalists invest in people, not products, and businesses, not industries. It was concluded that venture capital-backed small firms should, therefore, be seen as an adjunct to the development of biotechnology in Britain, rather than as a substitute for a co-ordinated, co-operative strategy involving Government, the financial institutions, industry and academia. This is chiefly because the small size of the UK's domestic market means that many potentially important innovations in biotechnology may continue to be lost, since the short term identification of market opportunities for biotechnology products will dictate that they are insupportable in Britain alone. In addition, the data analysis highlighted some interesting methodological issues concerning the investigation of investment decision making. These related especially to shortcomings in the use of scoresheets and questionnaires in research in this area. The conclusion here was that future research should concentrate on the reasons why an individual reaches an investment decision. It is argued that only in this way can the nature of the evaluation procedures employed by venture capitalists be properly understood.

University-industry-relations - their role in technological innovation:the case of biotechnology in the UK

Biotechnology is one of a series of new `generic technologies' that have been identified by western governments as possessing stategic economic opportunities. In this thesis I examine the characteristics of the technology and the government policies that have been developed to both promote and exploit the underpinning scientific research for biotechnology. The approach I have taken involves an in-depth analysis of the role of university-industry research relations in the development of biotechnology. To this end I carried out a detailed survey of biotechnology companies in the UK on the nature of their interactions and objectives. Through individual case studies of the SERC and DTI club mechanisms in biotechnology, I provide a contemporary appraisal of the development of new mechanisms involving co-ordination and cooperation between industry, government and academia, established to couple state funded science and national economic development. The public policy implications of the club funding systems for science in the UK are examined.

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.

A European competence framework for industrial pharmacy practice in biotechnology

Project Report: The PHAR-IN ("Competences for industrial pharmacy practice in biotechnology") looked at whether there is a difference in how industrial employees and academics rank competences for practice in the biotechnological industry. A small expert panel consisting of the authors of this paper produced a biotechnology competence framework by drawing up an initial list of competences then ranking them in importance using a three-stage Delphi process. The framework was next evaluated and validated by a large expert panel of academics (n = 37) and industrial employees (n = 154). Results show that priorities for industrial employees and academics were similar. The competences for biotechnology practice that received the highest scores were mainly in: . "Research and Development", . "Upstream" and "Downstream" Processing', " . "Product development and formulation", " . "Aseptic processing", ."Analytical methodology", . "Product stability", and . "Regulation". The main area of disagreement was in the category "Ethics and drug safety" where academics ranked competences higher than did industrial employees.