Protein cleavage strategies for an improved analysis of the membrane proteome

Background  

Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria.     

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway? recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio? fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

Bioimprinting strategies: From soft lithography to biomimetic sensors and beyond

Imprinting is a straightforward, yet a reliable technique to develop dynamic artificial recognition materials—so called as synthetic antibodies. Surface imprinting strategies such as soft lithography allow biological stereotyping of polymers and sol–gel phases to prepare extremely selective receptor layers, which can be combined with suitable transducer systems to develop high performance biomimetic sensors. This article presents an overview of the remarkable technical advancements in the field of surface bioimprinting with particular emphasis on surface imprinted bioanalyte detection systems and their applications in rapid bioanalysis and biotechnology. Herein, we discuss a variety of surface imprinting strategies including soft lithography, template immobilization, grafting, emulsion polymerization, and others along with their biomimetic sensor applications, merits and demerits. The pioneering research works on surface patterned biosensors are described with selected examples of detecting biological agents ranging from small biomolecules and proteins to living cells and microorganisms.     

Protein immobilization strategies for protein biochips

In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein immobilization, in order to fully realize the potential of protein biochips. In fact, protein immobilization is the key to the success of microarray technology. Proteins need to be immobilized onto surfaces with high density in order to allow the usage of small amount of sample solution. Nonspecific protein adsorption needs to be avoided or at least minimized in order to improve detection performances. Moreover, full retention of protein conformation and activity is a challenging task to be accomplished. Although a large number of review papers on protein biochips have been published in recent years, few have focused on protein immobilization technology. In this review, current protein immobilization strategies, including physical, covalent, and bioaffinity immobilization for the fabrication of protein biochips, are described. Particular consideration has been given to oriented immobilization, also referred to as site-specific immobilization, which is believed will improve homogeneous surface covering and accessibility of the active site.     

Applications of biomolecular interaction analysis in drug development

Biomolecular interaction analysis (BIA) is now utilised increasingly in drug development to kinetically characterise binding events of relevant components. BIA thereby covers a broad range of applications in early ADME (adsorption, distribution, metabolism and excretion), secondary screens, functional and metabolic assays and in assay development. This versatile technology allows measurements in real time without the need of labelling. A wide range of affinities can be characterised in a near-native state, with high reproducibility, high sensitivity and low sample consumption. However, limitations due to experimental design, and limitations in sample throughput, may occur. Future developments of BIA aim at parallelisation and automatisation and at coupling to platform technologies already established in the drug development process.     

Microbial sensors for small molecules: development of a mevalonate biosensor

We describe a novel biosensor strain for detection and quantification of a small molecule, mevalonate. The biosensor strain is an Escherichia coli mevalonate auxotroph that expresses the green fluorescent protein and reports on the mevalonate concentration in the growth medium through a change in growth rate. A model describing the growth rate dependence on mevalonate was developed in order to use the biosensor strain for high-throughput screening (HTS) and quantitative measurement of mevalonate in the extracellular environment. In general, this method should be applicable to the quantification of any small molecule for which an auxotroph can be developed and will be useful for HTS of evolved metabolic pathways for which there is no readily available screen or selection.     

Crystallization chaperone strategies for membrane proteins

From G protein-coupled receptors to ion channels, membrane proteins represent over half of known drug targets. Yet, structure-based drug discovery is hampered by the dearth of available three-dimensional models for this large category of proteins. Other than efforts to improve membrane protein expression and stability, current strategies to improve the ability of membrane proteins to crystallize involve examining many orthologs and DNA constructs, testing the effects of different detergents for purification and crystallization, creating a lipidic environment during crystallization, and cocrystallizing with covalent or non-covalent soluble protein chaperones with an intrinsic high propensity to crystallize. In this review, we focus on this last category, highlighting successes of crystallization chaperones in membrane protein structure determination and recent developments in crystal chaperone engineering, including molecular display to enhance chaperone crystallizability, and end with a novel generic approach in development to target any membrane protein of interest.     

The development of photometric sensors for boronic acids

Boronic acids bind certain 1,2- and 1,3-diols with high affinity through reversible formation of boronate esters. They have been utilized as the recognition moiety for artificial receptors, particularly receptors for carbohydrates that have cis-diol moieties. Therefore, sensors for boronic acids could serve as universal reporters for monitoring boronate formation. This paper reports the design and synthesis of a series of photometric chemosensors for phenylboronic acid using diethanolamine as the recognition moiety. Diethanolamine, which binds strongly to boronic acids, has been linked to three different types of optical reporters. A photoinduced electron transfer system based on the anthracene fluorophore has been used to create sensors that show up to a fivefold increase in fluorescent intensity in the presence of millimolar concentrations of phenylboronic acid. Sensor designs based on the restriction of free rotation of extended pi systems and on the perturbed electronic properties of azo dyes are also included. This work demonstrates that sensors based on several different designs can be used for the detection of boronic acids.     

Cytotoxic immunotherapy strategies for cancer: mechanisms and clinical development

Traditional therapies for cancer include surgery, chemotherapy, and radiation. Chemotherapy has widespread systemic cytotoxic effects against tumor cells but also affects normal cells. Radiation has more targeted local cytotoxicity but is limited to killing cells in the radiation field. Immunotherapy has the potential for systemic, specific killing of tumor cells. However, if the immune response is specific to a single antigen, tumor evasion can occur by down-regulation of that antigen. An immunotherapy approach that induces polyvalent immunity to autologous tumor antigens can provide a personalized vaccine with less potential for immunologic escape. A cytotoxic immunotherapy strategy creates such a tumor vaccine in situ. Immunogenic tumor cell death provides tumor antigen targets for the adaptive immune response and stimulates innate immunity. Attraction and activation of antigen presenting cells such as dendritic cells is important to process and present tumor antigens to T cells. These include cytotoxic T cells that kill tumor cells and T cells which positively and negatively regulate immunity. Tipping the balance in favor of anti-tumor immunity is an important aspect of an effective strategy. Clinically, immunotherapies may be most effective when combined with standard therapies in a complimentary way. An example is gene-mediated cytotoxic immunotherapy (GMCI) which uses an adenoviral vector, AdV-tk, to deliver a cytotoxic and immunostimulatory gene to tumor cells in vivo in combination with standard therapies creating an immunostimulatory milieu. This approach, studied extensively in animal models and early stage clinical trials, is now entering a definitive Phase 3 trial for prostate cancer.     

Ecologically-based strategies for conservation and development in the tropics

Tropical countries are endowed with a rich array of biotic and geophysical resources. Only in the past 30 years has the rate of their resource transformation been so rapid as to threaten the environment, largely because of the demands of a growing human population resulting from the demographic transition. While resource transformations have produced the goods and services necessary to meet the needs of the growing populations, they have also generated a variety of effects on the environment and the societies of these countries. The rapid pace of technological advances and social change seriously threaten the sustainability of natural assets and ecological processes in these countries, with global consequences. This threat is exacerbated (1) by inadequate capability in these tropical countries to manage degradations in natural resources and the environment arising from technological interventions affecting large-scale ecological processes; and (2) by the coincidence of down-turns in long-term cycles of unequal amplitudes, which concern elitism and administrations, dependency on fossil fuels for industrialisation and shifts in cultural periods, all of which threaten the global structure and continuity of prevailing social institutions, Both phenomena endanger the prospects for international investments in the transformation of natural resources and management of the environment for sustainable livelihoods, especially in developing countries. most tropical countries have developing economies, and have inadequate capacity to manage the impacts and trade-offs of technological insertions largely because of financial constraints, poor technical expertise and the international character of their economies. In spite of harsh socio-biological, technological and financial constraints, there is a pressing need for investing in human expertise in tropical countries because of their effects on the sustainability of global climate, resource and environmental heritage, cultural heritage and societal organisation. Assurance of this sustainability demands that investments in tropical countries must be founded on traditional knowledge, organisation and community participation; on comparative advantages in terms of resource endowments and technological skills; on strategies and actions promoting innovative futures; on the strengthening of institutional capacities for assessing impacts and trade-offs; on a universally-acceptable system for exchanging experiences about technological insertions with reference to spatial areas, levels of sophistication, assessment of impacts and standards; and on the attraction of various forms of international cooperation at professional, governmental and non-governmental levels, which has been recommended by the World Commission on Environment and Development (Bruntland Commission). This overall challenge for international development in the tropics amounts to advancing macro-ecological and-economic sciences of large-scale processes having local impacts, andvice versa, involving the dynamic interactions between culture and philosophy, politics, investment (economics), society, technology and environment at different spatial and temporal scales.     

Modulation of glutamate receptors: strategies for the development of novel antidepressants

Summary.  On a biochemical level, conventional antidepressants have been shown to modulate synaptic levels of biogenic amines (i.e., serotonin, norepinephrine, and dopamine), most often by interfering with reuptake processes or inhibiting metabolism. Strategies directed at modulating glutamatergic transmission may overcome the principal limitations (i.e., delayed onset and low efficacy) that appear to be inherent to these conventional agents. In this brief overview, I summarize two glutamate-based approaches to develop novel antidepressants. These distinct and (on a cellular level) seemingly diametric strategies may converge on intracellular pathways that are also impacted upon by chronic treatment with biogenic amine based agents. Received July 6, 2001 Accepted August 6, 2001 Published online June 17, 2002     

Genetically encoded fluorescent sensors of membrane potential

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.