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Protein biochips: A new and versatile platform technology for molecular medicine
Abstract
The human genome has been sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. High-throughput technologies such as DNA microarrays have been developed for the profiling of gene expression patterns in whole organisms or tissues. Protein arrays are emerging to follow DNA chips as possible screening tools. Here, we review the generation and application of microarray technology to obtain more information on the regulation of proteins, their biochemical functions and their potential interaction partners. Already, a large variety of assays based on antibody-antigen interactions exists. In addition, the medical relevance of protein arrays will be discussed.
... Assays of biochemical activities are fundamental to biological research, drug discovery, clinical diagnostics, food and environmental safety, biological warfare and other areas (1)(2)(3)(4)(5). The development and application of bioanalytical methods therefore continue to be dominant themes in analytical chemistry. ...
... (1) surface chemistries that employ polymeric materials, (2) those that use hydrogels, and (3) those that use SAMs as supports for the array. The first group includes the use of polystyrene, polycarbonate, and poly(ethylenimine) to immobilize molecules (29). ...
Biochip arrays have enabled the massively parallel analysis of genomic DNA and hold great promise for application to the analysis of proteins, carbohydrates, and small molecules. Surface chemistry plays an intrinsic role in the preparation and analysis of biochips by providing functional groups for immobilization of ligands, providing an environment that maintains activity of the immobilized molecules, controlling nonspecific interactions of analytes with the surface, and enabling detection methods. This review describes recent advances in surface chemistry that enable quantitative assays of a broad range of biochemical activities. The discussion emphasizes the use of self-assembled monolayers of alkanethiolates on gold as a structurally well-defined and synthetically flexible platform for controlling the immobilization and activity of molecules in an array. The review also surveys recent methods of performing label-free assays, and emphasizes the use of matrix-assisted laser desorption/ionization mass spectrometry to directly observe molecules attached to the self-assembled monolayers.
... Array-based technologies have been developed to assess and validate potential biomarkers 179,180 . There are two classes of protein array formats, forward phase protein microarray (FPA) and reverse phase protein microarray (RPA). ...
... Most of the arrays described are based on oligonucleotides (Wang, 2000;Barrett and Kawasaki, 2003), some on antibodies, proteins (Lee, 2001;Barry and Soloviev, 2004;Lueking et al., 2005) or enzymes (Park and Clark, 2002). A few years ago, the idea of whole-cell arrays has been advanced by Van Dyk and coworkers (2001) who described the LuxArray: a collection of 689 non-redundant functional promoter fusions to Photorhabdus luminescens luxCDABE in live E. coli strains, representing close to 30% of the predicted transcriptional units in this bacterium. ...
At the heart of every biosensor is a biological entity, the purpose of which is to react with the target analyte(s) and generate
a readily quantifiable signal. Traditional biosensors are based on the unique specificity of enzymes to their substrates,
antibodies to antigens or that of nucleic acids to their complementary sequences.
... Arrays for protein detection use the same principle of simultaneous separation as nucleic acid arrays [13]. Protein arrays can be separated into two classes, (i) those designed to identify interactions of a specific protein with other molecules or (ii) those designed to measure many protein levels in a sample. ...
The complex responses of cells to stimuli are the aggregate of alterations at the genetic, protein, metabolic and cellular levels. The immense quantities of data now available from high-throughput genomic, proteomic and metabolomic sources require specialized analytical approaches. The integration of such data for the computational elucidation and analysis of cellular pathways and networks is an area of considerable current interest. As the quantity of available data continues to increase, strategies to extract the useful information from the data will continue to provide answers to important biological questions. Chemical engineers are actively involved in this field that has taken on the global title of “Systems Biology”. These research groups have made considerable impact from both the computational and experimental standpoints. This commentary describes some of the recent contributions of chemical engineering researchers to the computational analysis of high-throughput, multi-source data as described in recent publications and presentations from the 2005 AIChE Annual Meeting.
... [12][13][14][15] By identifying distinct antibody patterns of anti-OmpC, anti-I2, anti-Saccharomyces cerevisiae antibody (ASCA), antiperinuclear antineutrophil cytoplasmic antibody (pANCA), and anti-pancreatic exocrine antibody (PAB) among others, such a technique may potentially be able to distinguish IBD from functional bowel syndromes, differentiate patients with UC and CD, and allow for the subclassification of CD. 66 The use of functional immunomics may also potentially lead to an understanding of an individuals risk, with a family history such as CD, to develop the same condition. 67 Use of the technique could also be of benefit to patients already with the disease by identifying and measuring the concentration levels of antibodies, like ASCA, and correlating the findings with the individual's disease progression and prognosis. 68 The identification of auto-antigens could also potentially provide future targets for patient treatment. ...
... Because of the critical role membrane proteins play in biological function, biosensors utilizing membrane proteins have become increasingly attractive in bioanalytical and medical applications such as drug screening, medical diagnostics and biophyscial characterization of channels [1] [2]. Characterization of membrane proteins using electrochemical impedance spectroscopy (EIS) facilitates continuous use and label-free sensing with significant advantages over affinity-based sensors [3] [4] [5] [6]. ...
Tethered bilayer lipid membranes (tBLM) offer a promising means to immobilize membrane proteins for sensor applications and study biological phenomena including membrane-nanoparticle interactions. tBLM biointerfaces are typically characterized using electrochemical impedance spectroscopy (EIS) in the 1 mHz to 1 Hz range due to interface parasitics. To enable rapid characterization of biointerfaces for high throughput applications, this paper introduces a method for high resolution EIS characterization of tBLMs at higher frequencies. The tBLM equivalent electrical model is analyzed, and the benefit of extracting the real portion of interface admittance is described. Mathematical analysis shows that the maximum frequency for measuring membrane resistance is a function of membrane characteristics and that small area membranes could enable measurement well into the kHz range, permitting observation of millisecond membrane protein activity in biosensor arrays.
... [199][200][201] Antibodies are also fundamental to the determination of biomarkers that are central to the diagnosis of cancers and other diseases. [202][203][204][205][206][207] As antibody production methods have improved, 202,208,209 it has become possible to create numerous different antibodies faster than it is possible to perform the characterization of their binding. 199 There are several methods available for antibody characterization, however most rely on labeling or on surface immobilization. ...
... The development of DNA microarray technologies in global gene expression profiling (Morley et al., 2004) could be standardized and implemented into several areas of functional genomics yet biological functions are carried out by protein rather than nucleic acids (Gygi et al., 1999). Protein microarrays are a great tool for the large-scale analysis of both functional genomics and proteomics (Predki, 2004;Lueking et al., 2005). Currently protein arrays are extensively used for various applications such as antibody profiling, biomarker identification, protein-protein interactions, and enzymatic assays (Kawahashi et al., 2003;Feilner et al., 2005;Ramachandran et al., 2008b;Anderson et al., 2011;Hu et al., 2011;Ramani et al., 2012). ...
Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.
... The processing of suspensions containing living cellular materials is an emerging field of research that is rapidly and constantly evolving. The demand for such technology is centre-stage in the nano-and micro-biotechnology industry for the production of bio-chips, biosensors, tissue engineering as well as for gene manipulation and extends into a whole host of widespread technological applications [1][2][3][4][5][6][7][8][9][10][11][12][13]. ...
This paper reports for the first time the ability to process living cellular materials by means of electrified jets at electric field strengths of up to 2 kV/mm. Bio-suspensions containing living human Jurkat cells at different concentrations were processed via this jetting approach. The jetting process was carried out at an electric field strength between 0.67 kV/mm and 2 kV/mm, corresponding to an applied voltage of 10–30 kV between two electrodes ∼15 mm apart. The Jurkat cells were jetted under sterile conditions, collected in petri dishes and incubated for 24 and 48 hours. During and after incubation, cells were assessed for survival and structural damage; cells were found to be unharmed and to retain their integrity under all electric field strengths examined. At all field strengths jetting took place in the unstable mode. Good correlation was observed between droplet distribution plots generated by way of laser spectroscopy and estimated values from measurements of droplet relics.
... Mikroarrays sind chemisch modifizierte Oberflächen, wie Glas-oder Siliziumobjektträger, auf denen viele verschiedene Substanzen, wie DNA, Proteine, Peptide oder Wirkstoffe ortsaufgelöst immobilisiert sein können. Da mehr Proben auf kleinerem Raum untergebracht werden können, ermöglichen Mikroarrays die parallele Untersuchung von mehreren 10000 Proben in einem Experiment[33,93]. ...
Die humorale Immunantwort eines Organismus auf ein Pathogen äußert sich in einer Veränderung des Antikörperrepertoires. Eine quantitative Untersuchung dieses Prozesses erfordert aufgrund der enormen Komplexität des Immunsystems, die Verwendung von Hochdurchsatztechniken, wie Peptid-Mikroarrays. Bisher gibt es nur wenige Studien, die die Verlässlichkeit von Mikroarray-Bindungsmessungen untersuchen. In dieser Arbeit werden Bewertungskriterien für die Qualität von Antikörper-Peptid-Bindungsstudien unter Verwendung der Peptid-Mikroarraytechnologie herausgearbeitet, mit dem Ziel, diese Hochdurchsatzmethode für qualitative und quantitative Antikörper-Peptid-Bindungsmessungen zu optimieren. Anhand eines Modellsystems, das aus dem monoklonalen anti-p24 (HIV-1) Antikörper CB4-1 und 26 verschiedenen Peptiden, die mit unterschiedlicher Affinität an CB4-1 binden, besteht, werden systematisch die Bindungsdissoziationskonstanten der jeweiligen Antikörper-Peptid-Komplexe mit den durch Peptid-Mikroarray-Bindungsmessungen erhaltenen Signalintensitäten verglichen. Darüber hinaus wird in dieser Arbeit die Messung von Serumantikörperbindungsprofilen gegenüber Zufallspeptidbibliotheken als Methode der serologischen Diagnostik verwendet. Anhand dreier Beispieldatensätze wird die serologische Diagnose von Infektionskrankheiten, Autoimmunkrankheiten und von Krebs mittels Zufallspeptid-Mikroarrays demonstriert. Mithilfe von Merkmalsselektion werden Peptide selektiert, die besonders geeignet sind, um zwischen gesunden und kranken Individuen zu unterscheiden. Besondere Bedeutung wird der Untersuchung der Robustheit der Methode gegenüber schwankenden experimentellen Bedingungen beigemessen. Die vorliegende Arbeit gibt Aufschluss über vorhandene Probleme der Mikroarray-Technologie, stellt Lösungsansätze vor und arbeitet bedeutende Weiterentwicklungen auf dem Weg hin zu einer minimal-invasiven serologischen Diagnostik heraus, die kein a priori Wissen über Antigene voraussetzt.
... Though not exactly the same, DNA microarray technology became the first application of this theory and has been tremendously successful in gene expression profiling and other derivatized applications, such as ChIP-chip (DeRisi et al., 1997;Morley et al., 2004;Pease et al., 1994;Schadt et al., 2003;Schena et al., 1995). However, RNA expression levels do not always correlate with protein expression levels, and biological functions are carried out primarily by proteins rather than nucleic acids (Gygi et al., 1999;Lueking et al., 2005b). Therefore, it was the next logical step to develop a miniaturized protein-centered device, namely protein microarrays, for studies of protein functionalities in a high-throughput and highly flexible fashion. ...
The protein microarray technology provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. It is viewed as a new tool that overcomes the limitation of DNA microarrays. On the basis of its application, protein microarrays fall into two major classes: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be directly spotted on a slide to form the so-called "reverse-phase" protein microarray. In the last decade, applications of functional protein microarrays in particular have flourished in studying protein function and construction of networks and pathways. In this chapter, we will review the recent advancements in the protein microarray technology, followed by presenting a series of examples to illustrate the power and versatility of protein microarrays in both basic and clinical research. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade.
... Die Kenntnis des passenden Antigen-Antikörper-Paares ist hierbei unabdingbar und muss zum Teil sehr aufwendig evaluiert werden. [95][96][97][98] Schema II. 24 [73,74] Eines der am weitest verbreiteten Systeme stellt aber das (Strept-) Avidin-Biotin-System dar. ...
In der vorliegenden Arbeit wurden erfolgreich verschiedene effiziente Syntheserouten für perfluoralkylmodifizierte, hydroxysuccinimidaktivierte Trimethylsilylethoxycarbonyl (Teoc-)-Homologe evaluiert und die Verbindungen sehr detailliert charakterisiert. Aus den synthetisierten Teoc-Bausteinen mit unterschiedlichem Fluorgehalt, einem Carbostyrilchromophor und einem AminocarboxyPEG wurde eine Bibliothek von PEG-Linkern durch modulare kombinatorische Synthese erstellt. Die Auswahl der Bausteine und damit ihrer Eigenschaften wurde nach einem zuvor auf die Anwendungen Einzelmolekülspektroskopie und Einzelmolekülinteraktionsmessung abgestimmten Design durchgeführt. Durch die geschickte Kombination der Module und deren Reaktion in Lösung konnten die PEG-Linker effizient dargestellt werden. Alle getesteten Darstellungsmethoden bedienten sich einer ausgeklügelten Synthese- und Präcipitationsstrategie, wodurch die Isolierung der Zielverbindungen in hervorragenden Ausbeuten gelang. Trotz der Polydispersität des verwendeten PEGs und der damit verbundenen Schwierigkeiten für die Analyse gelang die eindeutige Charakterisierung der Zielverbindungen mittels Maldi-Tof.
In Zusammenarbeit mit der Arbeitsgruppe um Prof. Nienhaus wurde das Enzym RNase H nach Konjugation mit den Perfluoralkyllinkern auf perfluormodifizierten Oberflächen bioorthogonal, spezifisch und stabil immobilisiert. In einzelmolekülspektroskopischen Messungen wurden gezielt verschiedene Faltungszustände des Proteins nachgewiesen und mit hohem Erfolg die enzymatische Aktivität demonstriert.
Des Weiteren wurde das PerfluorPEG-Linkersystem zusammen mit den perfluorbeschichteten Oberflächen mittels Rasterkraftmikroskopie in einer Kollaboration mit der Arbeitsgruppe um Prof. Anselmetti in Studien zur Ermittlung der Interaktion zwischen Perfluoralkyleinheiten getestet. Es konnten nicht nur Wechselwirkungen nachgewiesen werden, die die Quantität von Einzelmolekülinteraktionen um ein Weites überschritten, sondern es gelang auch einen Zusammenhang zwischen Beladungsdichte der Oberfläche und Adhäsion zu demonstrieren.
Um die Anwendungsbreite der Linker zu erhöhen, wurden diese für die Clickkonjugationen mit azidofunktionalisierten Proteinen ausgelegt und neben den bewährten Fluor-Fluor-Interaktionen auch Biotin zur Immobilisierung verwendet. Die Untersuchung dieser Derivate für Separation und Quantifizierung von zellfrei exprimierten Proteinen wurde in einer Zusammenarbeit mit der Fa. RiNA GmbH vorgenommen.
Im weiteren Verlauf dieser Arbeit wurden Machbarkeitsstudien zur Immobilisierung von Organokatalysatoren oder Biokatalysatoren in Mikroreaktoren via Fluor-Fluor-Wechselwirkungen durchgeführt und bereits erste Hydroformylierungsreaktionen mit selbst organisierenden und zum Teil perfluormodifizierten Liganden in der Arbeitsgruppe um Prof. Breit in gutem Umsatz und Selektivität durchgeführt.
... Furthermore, RNA expression levels do not always correlate with protein expression levels, and it is almost impossible to predict biochemical properties of a protein encoded by a given gene simply based on its expression profiles. 7,8 Therefore, by focusing on studies of protein structures, thousands of different proteins (e.g., antigens, antibodies, enzymes, substrates, etc.) are immobilized in discrete spatial locations, forming a high-density protein dot matrix. Depending on their applications, protein microarrays can be classified into two types: the analytical and functional protein microarrays. ...
Functional protein microarrays are emerging as a promising new tool for large-scale and high-throughput studies. In this article, we review their applications in basic proteomics research, where various types of assays have been developed to probe binding activities to other biomolecules, such as proteins, DNA, RNA, small molecules, and glycans. We also report recent progress of using functional protein microarrays in profiling protein post-translational modifications, including phosphorylation, ubiquitylation, acetylation, and nitrosylation. Finally, we discuss potential of functional protein microarrays in biomarker identification and clinical diagnostics. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology. WIREs Syst Biol Med 2011 3 255–268 DOI: 10.1002/wsbm.118
For further resources related to this article, please visit the WIREs website
... Moreover, RNA expression levels are not always correlated well to protein expression levels, and it turned out to be almost impossible to predict the functional characteristics of a polypeptide encoded by a given gene simply based on its expression profiles (Gygi et al. 1999). Therefore, the focus on studies on protein structures, functions, and protein-protein interactions should facilitate a more thorough characterization of the physiological function of a given gene (Lueking et al. 2005;Chen and Snyder 2010;Lynch et al. 2004). ...
Personalized medicine, i.e., the use of information about a person’s genes, proteins, metabolites, and environment to prevent, diagnose, and treat disease, has been much talked about in recent years. So some observers are wondering what the excitement is all about cumulating in the following statement: “Personalized health care is nothing new. Doctors have always tried to fit the therapy to the patient’s need if possible.” But what has happened more recently is that one has now begun to go a level deeper, i.e., to explore the biology of the disease and its treatment at the molecular level. However, molecular medicine does not per se define personalized medicine, but the molecular tools are important as they should enable greater relevance in the information provided by corresponding diagnostic tests (see below) (Edwards et al. 2008; Weedon et al. 2006; Romeo et al. 2007; Hegel et al. 1999; Wildin et al. 2001; Grant et al. 2006; Rothman and Greenland 2005; Raeder et al. 2006; Hegele et al. 2000; Capell and Collins 2006; Delepine et al. 2000; Janssens et al. 2006; Xiayan and Legido-Quigley 2008; Figeys and Pinto 2001; Müller 2002, 2010; Pearson et al. 2007; Janssens et al. 2008; Risch and Merikangas 1996; Janssens and van Duijn 2008; McCarthy 2003; McCarthy et al. 2003; Stumvoll et al. 2005; Lyssenko et al. 2005; Florez et al. 2003).
... Compared with DNA-microarrays, developing protein microarrays requires a lot more steps in its creation and faces many challenges (Zhu and Snyder 2003;Liotta et al. 2003;Lueking et al. 2005;Hall et al. 2007;Cretich et al. 2013;Gahoi et al. 2015). Technical challenge are to find a surface and a method of attachment that allows the proteins to maintain their secondary or tertiary structure, and to produce an array with a long shelf life so that the proteins on the chip do not denature over a short time. ...
Biological assay has been based on analysis of all individuals collected from sample populations. Bulked sample analysis (BSA), which works with selected and pooled individuals, has been extensively used in gene mapping through bulked segregant analysis with biparental populations, mapping by sequencing with major gene mutants and pooled GWAS using extreme variants. Compared to conventional entire population analysis, BSA significantly reduces the scale and cost while simplifies the procedure. The bulks can be built by selection of extremes or representative samples from any populations and all types of segregants and variants that represent wide ranges of phenotypic variation for the target trait. Methods and procedures for sampling, bulking and multiplexing are described. The samples can be analyzed by using individual markers, microarrays and high-throughput sequencing at all levels of DNA, RNA and protein. The power of BSA is affected by population size, selection of extreme individuals, sequencing strategies, genetic architecture of the trait and marker density. BSA will facilitate plant breeding through development of diagnostic and constitutive markers, agronomic genomics, marker-assisted selection and selective phenotyping. Applications of BSA in genetics, genomics and crop improvement are discussed with their future perspectives. This article is protected by copyright. All rights reserved.
... Bioassays are essential tools in basic life science research, drug discovery, human and veterinary diagnostics, food and environmental safety, and bioterrorism prevention (1)(2)(3)(4)(5)(6)(7). Although such assays are already a mainstay in the diagnostics arena, enhancements in sample throughput, analysis time, and levels of detection will clearly advance early disease diagnosis, improve patient prognosis, reduce outbreaks, and decrease hospitalization. ...
Bioassays are indispensable tools in areas ranging from fundamental life science research to clinical practice. Improving assay speed and levels of detection will have a profound impact in all of these areas. We recently developed a rapid, sensitive format for immunosorbent assays that expedites antigen mass transport by rotating the capture substrate. This review outlines the theoretical foundation of rotationally induced hydrodynamics and its application in heterogeneous assays. We describe a general solution that solves the rates of immunoreactions on rotating capture substrates, taking into account both diffusion and the rate of reaction between antibody and antigen. The general solution applies to a wide range of rotation rates, including mass transport-limited to reaction rate-limited assays, and is validated experimentally. We discuss several applications that demonstrate how immunoassays can be tailored to increase speed as well as lower the limit of detection of viral particles, pathogens, toxins, and proteins.
... "Tissue on chip" based approaches allow miniatur-scaled experiments [36,37]. Only a small amount of expensive biomaterials or GFs is necessary e.g. to test the effects of GFs or other mediators on tenogenesis by precursor cells or tenocytes. ...
... [1][2][3] The higher physical and chemical stability relative to biomacromolecules make MIPs potentially very suitable as recognition elements for chemical sensors, biosensors, or biochips. 4 Recently, a strong trend goes toward microbiochips-arrays of micrometer dots of biological molecules, 5,6 which have a great potential for applications in the bioanalytical field, drug development, environmental, food analysis, etc. 7,8 To obtain MIP microarrays, we have previously demonstrated the deposition of microdroplets of MIP solution using microcantilevers or a nanofountain pen 9,10 and detection of specific binding of fluorescent target analytes. While this proved the feasibility of the patterning approach, this system is limited to detection of fluorescent targets. ...
We report on sensitive and specific detection and quantification of a template in a molecularly
imprinted polymer MIP using Raman microspectroscopy. The b -blocking drug S-propranolol and
its enantiomer, R-propranolol, were used as target molecules since the selectivity of this MIP is well
established and serves as an appropriate validation standard. Specific peaks originating in the
template were identified in the Raman spectrum, allowing quantification of bound target molecule.
We demonstrate that label-free monitoring can be achieved from volumes as small as 1 m m 3 of
MIP, based on a single identifying peak
Protein-based microarrays is a novel, rapidly evolving proteomic technology with great potential for analysis of complex biological
samples. The technology will provide miniaturized set-ups enabling us to perform multiplexed profiling of minute amounts of
biological samples in a highly specific, selective, and sensitive manner. In this review, we describe the potential and specific
use of protein microarray technology, including both functional protein microarrays and affinity protein microarrays, for
the detection and identification of bacteria, bacterial proteins as well as bacterial diseases. To date, the first generations
of a variety of set-ups, ranging from small-scale focused biosensors to large-scale semi-dense array layouts for multiplex
profiling have been designed. This work has clearly outlined the potential of the technology for a broad range of applications,
such as serotyping of bacteria, detection of bacteria and/or toxins, and detection of tentative diagnostic biomarkers. The
use of the protein microarray technology for detection and identification of bacterial and protein analytes is likely to increase
significantly in the coming years.
Array-based technologies, providing “-omic” level understanding of tumors at the DNA, RNA, and protein levels, have led to
the uncovering of new disease susceptibility genes, therapeutic targets, expression profiles of genes or proteins related
to disease outcomes as well as markers of therapeutic sensitivity and resistance. Analysis of signaling network activation
at the protein level is of critical importance because nearly all current molecular-targeted therapeutics directed at modulating
protein kinase activity, hence, the proteins themselves are the drug targets. Newer array-based and multiplexed approaches
that can measure signaling network activation in very small tissue samples of the patient, and can perform broad-scale pathway
mapping, will be the best to deliver effectively the needed predictive, prognostic, and therapy-guiding information to the
bedside. The power of protein microarrays lies in their ability to provide a “map” of known cellular signaling proteins that
generally reflect the state of information flow through protein networks in individual specimens. Combined with continued
efforts to identify and monitor protein markers indicative of therapeutic response or resistance, protein array-based technologies
are uniquely poised to provide direct functional information for individual patient tumors in time frames which was never
before possible and could have a tremendous positive impact on therapeutic decision-making and ultimately on disease outcome.
KeywordsTissue microarray-Protein microarrays-Forward-phase protein microarray-Reverse-phase protein microarray-Personalized medicine-Cancer-Proteomics-Targeted therapeutics-Therapeutic resistance
Introduction HSP90 is known as a stabilizer of the proteome and is required for many newly synthesized proteins and introducing damaged proteins back into the refolding chaperone cycle. Due to its key position and interaction with several hundreds of proteins in a cell, it is a target for noxious cells and a responsive sensitive biomarker for cellular stress. Cellular stress is when unfolded proteins are formed by, e.g. mutational events, changes in the osmolality, or redox status. One challenge is to monitor and discriminate the cellular answers to relevant and reliable signals. The aim of this narrative review is to provide an overview of currently available microarray applications using HSP90 as the target.
Cell function is dependent upon the co-ordinated and dynamic formation of complex
interaction networks between molecules of diverse biochemical properties. These networks,
or interactomes, are comprised of macromolecular biopolymers; proteins, DNA, RNA and
polysaccharides, in addition to non-polymer compounds such as small molecular
metabolites. This myriad of interactions is highly regulated and any perturbation or
alteration has potential to result in disease.
Profiling protein-protein interactions has been the major focus of interactomics in the past
few years (Charbonnier et al. 2008) largely due to the advances in technological platforms
that have the capacity to probe globally. Early efforts have included two-hybrid screens to
identify binary binding interactions; more recent studies have used a range of mass
spectrometry based methods to identify protein complexes that are a better reflection of
multi-interactive nature of such complexes. Protein/small molecule interactions are equally
important in modulating the function of their target proteins but few studies have analyzed
these interactions on a large scale. The field is indeed still in its infancy due to difficulties in
identifying metabolites but has recently benefitted from technological advances in mass
spectrometry, data analysis software and metabolites database development for the
measurement and identification of metabolites. The next step is to integrate metabolomic
profiling to functional characterization of metabolic pathways by identifying systematically
metabolite-protein interactions.
Research efforts have in general been more focused on lipid-mediated interactions and this
chapter reviews the global methods as well as their applications used to map lipid-protein
interactomes based on mass spectrometry or arrays. The potential of these studies to deepen
our understanding on the biological function of metabolites as protein effectors is also
discussed.
The authors show that 100 nm unilamellar thiol-tagged vesicles bind discretely and specifically to Au nanodots formed on a Si surface. An array of such dots, consisting of 20 nm Au–Si three-dimensional islands, is formed by self-assembly on terraces of small-angle-miscut Si(111) after Au deposition. Consequently, both the formation of the nanopattern and the subsequent attachment of the vesicles are self-organized and occur without the need for any “top-down” lithographic processes. This approach has the potential to provide the basis of a low-cost, high-density nanoarray for use in proteomics and drug discovery.
We report a new strategy for immunochemical screening of small organic molecules based on the use of a hapten microarray. Using DNA-directed immobilization strategies, we have been able to convert a DNA chip into a hapten microarray by taking advantage of all the benefits of the structural and electrostatic homogeneous properties of DNA. The hapten microarray uses hapten-oligonucleotide probes instead of proteins, avoiding the limitations of preparing stochiometrically-defined protein-oligonucleotide bioconjugates.As proof of concept, we show here the development of a microarray for analysis of anabolic androgenic steroids. The microchip is able to detect several illegal substances with sufficient detectability to be used as a screening method, according to the regulations of the World Anti-Doping Agency for sport and the European Commision for food safety.The results that we show corroborate the universal possibilities of the DNA chip, and, in this case, they open the way to develop hapten microarrays for the immunochemical analysis of small organic molecules.
Proteinbiochips sind integrale Bestandteile einer wachsenden Zahl medizinischer und bioanalytischer Anwendungen, weshalb das Interesse an Methoden zur Proteinimmobilisierung für die Herstellung solcher Chips in den letzten Jahren stark gestiegen ist. Dieser Aufsatz gibt einen Überblick über chemische Verfahren zur kovalenten und nichtkovalenten Anbindung von Proteinen auf Oberflächen, wobei ein spezielles Augenmerk auf chemische Selektivität und die Erhaltung der Proteinfunktion bei der Immobilisierung gelegt wird. Ferner werden Strategien zur Herstellung strukturierter Proteinoberflächen umrissen. Abschließend wird ein Ausblick auf mögliche Entwicklungsrichtungen im Bereich der Proteinbiochipforschung und -anwendung gegeben.
Molecularly imprinted polymers (MIPs) are tailor-made receptors that possess the most important feature of biological antibodies and receptors - specific molecular recognition. They can thus be used in applications where selective binding events are of importance, such as chemical sensors, biosensors and biochips. For the development of microsensors, sensor arrays and microchips based on molecularly imprinted polymers, micro and nanofabrication methods are of great importance since they allow the patterning and structuring of MIPs on transducer surfaces. It has been shown that because of their stability, MIPs can be easily integrated in a number of standard microfabrication processes. Thereby, the possibility of photopolymerizing MIPs is a particular advantage. In addition to specific molecular recognition properties, nanostructured MIPs and MIP nanocomposites allow for additional interesting properties in such sensing materials, for example, amplification of electromagnetic waves by metal nanoparticles, magnetic susceptibility, structural colors in photonic crystals, or others. These materials will therefore find applications in particular for chemical and biochemical detection, monitoring and screening.
With the completion of the Aspergillus fumigatus genome it is now possible to study protein regulation on a global scale. One of the most suitable protein separation techniques is based on 2D-gel electrophoresis, which allows the separation of proteins based on their charge and size in a gel matrix. In addition, gel-free proteomics techniques based on liquid-chromatography coupled with mass spectrometry have gained importance. With the application of proteomic tools a comprehensive overview about the proteins of A. fumigatus present or induced during environmental changes and stress conditions can be obtained. For A. fumigatus, several proteomic studies have already been published including the response of the fungus to oxidative stress that induced the up-regulation of many proteins including catalases and thioredoxin peroxidase. Since many of the identified proteins/genes were apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified, designated Afyap1 and further characterized. In addition, some of the gene products expressed under stress conditions are also known fungal antigens, such as the thioredoxin peroxidase AspF3. Thus, besides pathogenicity studies, proteomics also delivers the tools to screen for new antigens which could improve the diagnosis of diseases caused by A. fumigatus.
Microscope projection photolithography is combined with nanomolding and molecular imprinting for the fast microfabrication of molecularly imprinted polymer (MIP) arrays in the form of micrometric islands of nanofilaments. Dot diameters from 70-90 μm are easily obtained using a 10× objective and a photomask carrying the desired pattern. The dots are composed of parallel nanofilaments of a high aspect ratio, 150 nm in diameter and several micrometers in length, which are obtained through a nanomolding procedure on porous alumina. The arrays are molecularly imprinted with the small molecule fluorescein or with the protein myoglobin. The fluorescein MIP arrays are able to specifically recognize their target, as demonstrated by fluorescence microscopy. A four-fold increase in binding capacity and imprinting factor (IF = 13) is obtained compared to non-nanostructured porous dots. Imprinting of the nanofilament arrays with the protein myoglobin as the template is also possible and allows for a high imprinting factor of 4.3. Such nanostructured microarrays of synthetic receptors obtained by projection photolithography have great potential in biosensor and biochip development.
Obesity has emerged as one of the major global epidemics of the 21st century and is now reaching alarming proportions. Obese subjects have an increased morbidity and mortality, decreased quality of life and a major risk of developing pathologies such as diabetes mellitus, insulin resistance and cardiovascular disease. Obesity is a complex disease characterised by an increase in body fat mass resulting from an imbalance between energy intake and expenditure. Signal integration between adipose tissue, other peripheral organs and the CNS seems to regulate energy homeostasis. Proteomics may be useful in unravelling the pathogenesis of obesity, since a combination of genetic predisposition and environmental factors account for its development. Most of the proteomic studies performed to date have focused on protein profiling of adipose tissue in different models of experimental obesity and the study of the adipocyte differentiation process. Another issue that has recently attracted attention is the characterisation of the adipocyte secretome, which may be important in signalling to other organs and in regulating energy balance. Target identification of potential therapies has also been investigated by proteomics. This review focuses on the contributions of proteomics to understanding the molecular mechanisms of obesity and their potential therapies.
Copyright © 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
Unlabelled:
We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest.
Biological significance:
DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.
Submerged laser ablation (SLAB) was analyzed to prepare solid-supported multicomponent patterned self-assembled monolayers (SAMs). The SLAB process prevents contamination arising from metal particles formed during laser ablation. Monolayers, which was made from a wide spectrum of the molecules are processed by the SLAB. The laser beam determines the template-stripped gold (TSG) by passing through the reactant solution and the SAM formed by the thiol. The primary thiol is released from the surface in the direction of the laser beam movement. The thiol in the supernatant (reactant) assembles on the bare gold from the solution due to Brownian diffusion. Scanning electron microscopy (SEM) was used to produce different SEM contrast-based on variations in end group, chain length, and conformational order. Results show that the SLAB process enables the preparation of surface-supported patterned monolayers with a wide variety of molecules.
This chapter provides a brief and general overview of some of the strategies available to modify different surfaces at the nanoscale with biological materials. It briefly discusses a few examples of applications in more specialized areas. Experimental techniques used to characterize biofunctional surfaces are similar to the ones used to study monolayers of other organic materials: scanning probe microscopies (AFM, STM), electron microsopies, fluorescence, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), or electrochemistry, to name some of them. The chapter focuses on the different approaches currently used to deposit functional biomembranes on different surfaces. It describes protocols on modifications that can be either homogeneously covering the surface or localized in defined regions. Protocols developed to pattern monolayers can also be applied to localize the surface modifications used to incorporate biological materials onto surfaces.
This review aims to introduce and cover some of the requirements of today's analytical chemists and biochemists together with a number of techniques that are being applied to satisfy these demands. The wide variety of analytes for which reliable detection methods are required are discussed initially. This is followed by a basic introduction to a variety of detection methods such as electrochemical approaches, fluorescence-based techniques, and the quartz crystal microbalance. This is followed by a section covering techniques that are designed for analyses using very small samples such as lab-on-a-chip type measurements and microfluidic devices. The next section contains more detailed information on analyses for specific analytes such as DNA. The use of array technology to perform many simultaneous measurements and the application of this to the growing fields of genomics and proteomics is also presented. Conclusions and a future outlook for these systems are finally discussed.
Keywords:
analytical techniques;
lab-on-a-chip;
biosensors;
DNA array;
electrochemistry;
fluorescence;
quartz crystal microbalance (QCM)
Polymers are extensively used in many biomedical applications such as body implants, biosensors, ophthalmology, bioseparation, etc. These applications often demand the polymer to possess certain surface properties so that it does not cause any harmful effect when it comes in contact with biological system or blood. Such surface properties can be imparted to a polymer by plasma treatment. The atmospheric pressure plasma is considered to be more advantageous over low pressure plasma because of its ease of operation and low cost. This article gives an overview of different atmospheric plasma sources and elaborates on the atmospheric plasma treatments of polymers to modify their surfaces for various biomedical applications.
Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm(3) of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories.
A guidebook for DNA Microarray Data Analysis
This guidebook is written in collaboration between several Finnish researchers from different universities and research institutions. The first edition of the DNA microarray data analysis guidebook was written by M. Minna Laine, Tomi Pasanen, Janna Saarela, Ilana Saarikko, Teemu Toivanen, Martti Tolvanen, Jarno Tuimala, Mauno Vihinen, and Garry Wong.
For the second edition, Iiris Hovatta, Katja Kimppa, Antti Lehmussola, Juha Saharinen, and Pekka Tiikkainen joined the writer team.
The purpose of this book is to serve as course and teaching material to introduce basic concepts of microarray data analysis. We hope that especially researchers starting their data analysis can benefit from the book.
This is the second, revised and slightly expanded edition of the DNA microarray data analysis guidebook. As a change to the previous edition, some relatively quickly changing material such as software tutorials have been exclusively published here, on the book's web site. This allows us better keep the material up to date. Please follow links on the Contents part above to access the extra material (all as PDF files).
Copyright
CSC - the Finnish IT center for science, is a non-profit organization for high-performance computing and networking in Finland. CSC is owned by the Ministry of Education. CSC provides cross-disciplinary expertise, computational resources and fast network connections for computational science and engineering.
All rights reserved by CSC - Scientific Computing Ltd., Finland. The PDF version of this book or parts of it can be used for academic non-profit purposes, provided that this copyright notice is included. This publication may not be sold or included as part of other publications without permission of CSC.
Available online: http://www.csc.fi/csc/julkaisut/oppaat/1999-2008/arraybook_overview
High-throughput screening (HITS) is a method of scientific experimentation widely used in drug discovery and relevant to the fields of biology and chemistry. Microfluidic systems manipulate or process tiny volumes of fluids in channel with dimensions of tens to hundreds of micrometers and are well suited for HTS due to their small size, which allows massively parallel experimentation. The unique feature of fluids within microfluidic networks can give an insight on new way to resolve the current challenges of HITS and to revolutionize all stages in drug discovery. Herein, recent progress in both microarraying strategies based on microfluidics and novel microfluidic devices with high throughput rates will be discussed.
Proteins are key actors in the life of the cell, involved in many
physiological and pathological processes. Since variations in the
expression of messenger RNA are not systematically correlated with
variations in the protein levels, the latter better reflect the way a
cell functions. Protein microarrays thus supply complementary
information to DNA chips. They are used in particular to analyse protein
expression profiles, to detect proteins within complex biological media,
and to study protein-protein interactions, which give information about
the functions of those proteins [3-9]. They have the same
advantages as DNA microarrays for high-throughput analysis,
miniaturisation, and the possibility of automation. Section 18.1 gives a
brief overview of proteins. Following this, Sect. 18.2 describes how
protein microarrays can be made on flat supports, explaining how
proteins can be produced and immobilised on a solid support, and
discussing the different kinds of substrate and detection method.
Section 18.3 discusses the particular format of protein microarrays in
suspension. The diversity of protein microarrays and their applications
are then reported in Sect. 18.4, with applications to therapeutics
(protein-drug interactions) and diagnostics. The prospects for
future developments of protein microarrays are then outlined in the
conclusion. The bibliography provides an extensive list of reviews and
detailed references for those readers who wish to go further in this
area. Indeed, the aim of the present chapter is not to give an
exhaustive or detailed analysis of the state of the art, but rather to
provide the reader with the basic elements needed to understand how
proteins are designed and used.
This paper elucidates challenges in integrating different classes of proteins into a microsystem and presents an electrochemical array strategy for heterogeneous protein-based biosensors. The overlapping requirements and limitations imposed by biointerface formation, electrochemical characterization, and microsystem fabrication are identified. A planar electrode array is presented that synergistically resolves these requirements using thin film Au and Ag/AgCl electrodes on a dielectric substrate. Using molecular self-assembly, electrodes were modified by nano-structures of two diverse proteins, alkali ion-channel protein and alcohol dehydrogenase enzyme. Electrochemical impedance spectroscopy and cyclic voltammetry measurements were performed to characterize sensor response to alkali ion and alcohol, respectively. This work demonstrates the viability of the electrochemical microsystem platform for heterogeneous protein-based biosensor interfaces.
The co-emergence of microarray technologies with systems oriented approaches to discovery is testament to the technological and conceptual advancements of recent years. By providing a platform for massively parallelized reductionism, microarrays are enabling us to examine the functional features of diverse classes of bio-system components in a contextually meaningful manner. Yet, to provide economic impact, future development of these technologies demands intimate alignment with the goal of producing safer and more efficacious drugs.:
Nearly defect-free arrays of several 104 gold dots of 12±3nm in diameter inside 10nm deep cavities were fabricated for immobilization of proteins. Extreme ultraviolet interference lithography (EUV-IL) at the XIL-beamline of the Swiss Light Source was used to produce 140nm period arrays of 50nm holes in a 40nm thick PMMA layer on oxidized silicon wafers. The size of the openings was reduced by glancing angle deposition (GLAD) of metals such as chromium and silver. Reactive ion etching of the underlying substrate, followed by deposition of a few nanometers of gold, lift-off and thermal annealing resulted in perfectly ordered arrays of small gold nanoparticles with well-defined size distribution. The combination of passivation of the silica surface with polyethylene glycol (PEG) derivatives and functionalization of gold with thiols enables the preparation of large area arrays of well separated functional protein molecules.
In this research we used diverse nanofabrication techniques in order to direct the assembly on micro- and
nanostructured surfaces of purified units from the photosynthetic unit of purple bacteria. This allowed us to explore
the unique energy transfer properties of light harvesting complexes by producing biomolecular photonic wires. We
developed an approach based on the combination of site-directed mutagenesis, nanoimprint lithography and
multivalent host-guest interactions for the realization of engineered ordered functional arrays of purified components
of the photosynthetic system, the membrane-bound LH2 complex. In addition to micrometer-scale patterned
structures, we demonstrated the use of nanometer-scale hard NIL stamps to generate functional protein arrays
approaching molecular dimensions. We also report the first observation of long-range transport of excitation energy
within a bio-mimetic molecular light-guide constructed from LH2 antenna complexes organized vectorially into
functional nanoarrays. Fluorescence microscopy of the emission of light after local excitation with a diffractionlimited
light beam reveals long-range transport of excitation energy over micrometer distances, which is much larger
than required in the parent bacterial system.
Other biological systems used were visible fluorescent proteins and α-synuclein, an intrinsically unfolded protein
associated with Parkinson’s disease. We report for the first time the directed assembly and characterization of FRET
pairs on micrometer dimension patterned surfaces. In order to characterize the biological assemblies on the surfaces
AFM imaging in combination with optical imaging (spectral fluorescence microscopy and lifetime measurements)
were performed in liquid conditions.
As the field of proteomics expands, the need for functional annotation of the proteome increases to allow the rational choice of particular targets from amongst a bewildering and increasing set of candidates. Similarly, the description of the interactome will continue to generate a plethora of candidate interactions that will need to be validated in functional assays. Although knockout mice and RNAi knockdowns have proven to be invaluable as primary tools in functional genomics, it should be remembered that these techniques apply at the gene and transcript level respectively and are, therefore, not always suitable for true functional proteomics investigations. Protein levels do not always correlate with RNA levels, particularly where active protein levels are regulated by protein stability or post-translational modifications. Moreover, off-target regulation of genes by RNAi/siRNA can complicate the validation of targets using this approach. In recent years, a myriad of potential protein targets including complexes has been emerging from high-throughput proteomics. Thus, the dilemma facing many investigators is having too many potential targets and too few means of validating them. Therefore, there is an unmet need for the development of technologies which allow the targeted disruption of particular proteins. Intracellular antibodies (intrabodies) and peptide aptamers are beginning to be applied in this area. A new class of peptide, known as Phylomers(®), also has significant potential in the area of disruption of protein-protein interactions. Phylomers, which are derived from protein subdomains, are small enough to synthesise and are of a suitable size for delivery to tissues and even into cells, making them an ideal candidates for a next-generation tool for functional validation of the proteome.
In this work, hybrid bilayer lipid membrane (hBLM) was assembled on a mechanically polished metallurgical titanium plate. Hydrophobic molecular anchors needed for phospholipid overlayers were obtained by silanization of the Ti surface with octadecyltrichlorosilane (OTS). The formation of hBLM was accomplished by fusion of the multilamellar vesicles containing of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol) at molar % ratio 60/40. The fusion process was monitored in real-time by the dynamic fast Fourier transformation (FFT) electrochemical impedance spectroscopy. We found that the repetitive regeneration of hBLM can be performed up to 4 times with no major loss of the electrochemical properties. Also, we showed that the electric barrier function of hBLMs is disrupted by phospholipase A2 (PLA2) - an enzyme, which hydrolyzes the fatty acid bonds at sn2 position in membrane phospholipids. Such effect may be used to design biosensors sensitive to both concentration and activity of the membrane damaging proteins, and possibly other agents.
Holographic molecularly imprinted polymer films for the use in chemical sensors are obtained in one step through photopolymerization with interfering laser beams. This results in hierarchical structuring at four length scales: micrometer-scale patterning of millimeter- to centimeter- size polymer objects with holographic optical properties, exhibiting nanometer-scale porosity and specific molecular recognition properties at the molecular scale through self-assembly. Specific binding of the target analyte testosterone is measured by diffraction analysis.
In this postgenomic age, cancer will be understood in intricate detail beyond the genomic level. New technologies are emerging that allow global proteomic level characterization, profiling and understanding. One of the most exciting emerging technologies of cancer proteomics is protein microarray. In this review, the different types of protein microarrays are discussed, including the methods, challenges and techniques of each type. Subsequently, the application of these specific methods to cancer diagnosis, prognosis and therapy will be overviewed, providing a general review of current methods, and proposing how protein arrays will help shape the future of oncoproteomics.
A protein with high affinity (K 12 nM) for the immunomodulatory compound A77 1726 has been isolated from mouse spleen and identified as the mitochondrial
enzyme dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a pI 9.6-9.8 and a subunit M of 43,000. Peptides derived from the mouse protein displayed high microsequence similarity to human and rat dihydroorotate
dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate dehydrogenase catalyzes
the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative effects of A77 1726 are mediated by enzyme inhibition and can be overcome by addition of exogenous uridine.
The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition
of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an
in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to counteract inhibition of the
murine acute graft versus host response.
We have recently shown that the platelet integrin αIIbβ3 is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein
kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin αIIbβ3 is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster
ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited
by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent
activation of platelet integrin αIIbβ3. Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase
2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant
PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus,
our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK
pathway, leading to integrin activation.
The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.
One hundred fifty fresh bladder tumors were analyzed blindly by two-dimensional PAGE in combination with proteome identification techniques (microsequencing and mass spectrometry) and immunofluorescence of cryostat sections. Of these, six showed protein expression patterns corresponding to squamous cell carcinomas (SCCs). All tumors were already invasive at the time of presentation, and in most cases, the histopathological grade could not be determined with certainty. The more differentiated of the tumors included SCC 589-1, a lesion showing extensive keratinization, and 536-1, a pure SCC that resembled normal skin growing invasively into the muscle. Both tumors expressed keratins 5, 6, 10, 14, 16, 17, and 20, as well as the differentiation-associated proteins psoriasin, psoriasis-associated fatty acid-binding protein (PA-FABP), and galectin 7. SCC 589-1, however, exhibited higher levels of keratin 10, PA-FABP, and galectin 7 and, in addition, expressed keratins 13, 15, and 19, which were not detected in the pure SCC. Involucrin, glutathione S-transferase pi, stratifin (14-3-3 sigma), and the SCC antigen 1, on the other hand, were less abundant in SCC 589-1. In comparison, less-differentiated tumors did not express keratin 10 and were characterized by a decreased expression of keratin 14, psoriasin, PA-FABP, galectin 7, and stratifin (14-3-3 sigma). Indeed, two of these lesions (553-1 and 651-1) could be readily lined up in order of decreasing degree of differentiation based on the expression of these markers. The degree of differentiation of the other two SCCs could not be assessed with certainty because they may represent special cases (SCC 646-1, solid tumor; SCC 485-1, special differentiation pattern). All six SCCs externalized psoriasin to the urine, supporting the contention that this protein, alone or in combination with other polypeptides, may represent a useful marker for the early detection of these lesions.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation
or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible
expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded
onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6 sequence of expressed proteins (RGS·His antibody, Qiagen) detected 20% of the library as putative expression clones. Two
example genes, GAPDH and HSP90α, were identified on high-density filters using DNA probes and antibodies against their proteins.
Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive from of vaccination, with DNA encoding a minigene for residues 139-151 of myelin proteolipid protein (PLP139-151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-gamma, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-gamma were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139-151 reduced the capacity of a T cell line reactive to PLP139-151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.
The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.
We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.
We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.
With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.
To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.
We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.
Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.
We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.
The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequence-verified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.
Naive non-obese diabetic (NOD/LtJ) mice spontaneously produce natural IgG autoantibodies against self-antigens associated with the experimental autoimmune diseases to which they are susceptible: insulin-dependant diabetes mellitus, systemic lupus erythematosus and experimental autoimmune encephalomyelitis. We discovered recently that NOD/LtJ mice also spontaneously produce IgG antibodies to the acetylcholine receptor (AchR), an antigen that can induce experimental autoimmune myasthenia gravis (EAMG) in susceptible rodents. However, there are no reports indicating that NOD/LtJ mice are susceptible to EAMG. To test whether the presence of spontaneous IgG autoantibodies can predict susceptibility to an autoimmune disease, we challenged NOD/LtJ mice using a standard protocol to induce EAMG. We now report that NOD/LtJ mice developed EAMG, although to a somewhat lesser degree than did C57BL/6 mice, a strain regarded as highly susceptible to the disease. Both strains produced comparable levels of immune antibodies to AchR of the complement-fixing isotypes IgG2a and IgG2b; however, NOD/LtJ mice produced significantly more IgG1. An antigen-specific T cell proliferative response to AchR of the same magnitude was detected in both strains, together with the secretion of similar amounts of IFN-gamma. Thus, NOD/LtJ mice are susceptible to EAMG and disease induction is accompanied by immune responses comparable to those seen in the susceptible strain C57BL/6. These results support the association between specific, natural IgG autoantibodies and susceptibility to the induction of a particular autoimmune disease.
To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.
We report details on the development of a self-assembled, double-stranded DNA (dsDNA) microarray fabrication strategy suitable for protein:dsDNA screening using the atomic force microscope (AFM). Using disulfide-modified dsDNA (26-mer) synthesized to contain the recognition sequence for ECoR1, we have created micron-sized mixed monolayer surfaces where both the spatial orientation and packing density of the immobilized oligonucleotides, two critical parameters for screening protein:dsDNA interactions, are controlled. Before exposure to ECoR1, the topography of microarrays that were composed of 26-mers containing the recognition sequence for EcoR1 was 8.8 nm +/- 1.5 nm (n = 5), a value consistent with that predicted by X-ray diffraction studies. After enzyme digestion, the topography of the microarray decreased to 4.3 nm +/- 0.8 nm (n = 14), a value consistent with predictions based on the position of the recognition sequence within the oligonucleotides. In contrast, the topography of microarrays that were composed of 26-mers that did not contain the recognition sequence for ECoR1 remained essentially the same before (8.9 nm +/- 1.5 nm (n = 5)) and after (8.3 nm +/- 1.4 nm (n = 5)) exposure to ECoR1. Furthermore, because the dsDNA were synthesized to include a fluorescein moiety above the recognition sequence, the loss of fluorescence after exposure to ECoR1 was also used to detect enzymatic cleavage. We believe that this technology holds promise as a tool for the rapid and facile screening of multiple protein interactions using massively parallel dsDNA microarrays.
The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production.
A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties. J. Cell. Biochem. 80:187–191, 2000.
The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel® matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore® analysis. Kd values of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the Kd value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT-4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.
The focus in the field of protein arrays has shifted from technology development to applying the technology in biological research. This review will highlight several recently published examples of biologically relevant experiments using both planar and bead-based arrays. Examples of the use of antibody arrays, antigen arrays and protein activity arrays will be discussed.Section Editors:Wolfgang Fischer—University of Oxford, Oxford, UK; Rob Hooft—Serono Pharmaceutical Research Institute, Geneva, Switzerland; Michael Walker—Bristol-Myers Squibb, Wallingford, USAOur ability to identify proteins and protein interactions operative under pathological conditions has grown significantly over the past few years. No longer do scientists examine proteins one by one but, rather, they rely on arrays to provide a qualitative and quantitative snapshot of a larger portion of the proteome in a single glance. Several protein array techniques are currently available using different solid supports, capturing agents and read-outs. In this review, Witte and Nock provide a birds-eye view of the available technologies and, more importantly, some interesting examples of their application. As such it can be appreciated that no single technique is suitable for all applications. The authors also point out that, with the increase in utilization of protein arrays, new tools are needed to analyze these data.
The gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is expressed at high levels in almost all tissues. However,
the molecular mechanism which sustains high-level expression of this house-keeping enzyme is still unknown. Here we show that
transcrip-tional activity is reduced by deletion of the nucleotides from −181 to −144 (relative to the transcriptional start
site) in the promoter of human GAPDH gene, both in CHO (derived from Chinese hamster ovary) and HepG2 (derived from human
hepatoma) cells. Gel retardation assays revealed that at least two nuclear factors, termed GAPBF1 and GAPBF2, bind to this
region. Mutations in the GAPBF1 binding site (−178 to −169) or the GAPBF2 binding site (−168 to −163) reduced this promoter
activity in vivo, showing that these two sites contribute to the activity of the GAPDH gene promoter. Since mutations in the region from −162
to −146 also reduced the promoter activity, this region seemed to function as an added cis-element, although we failed to find a factor that interacted specifically with this region in vitro. Accordingly, we propose that there are multiple cis-elements in the region from −181 to −144, each of which contributes to the promoter activity of GAPDH gene; the GAPBF1 binding
site has the unique feature of having a stretch of repeated A nucleotides.
All integrin alpha subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989-995, of the platelet integrin subunit glycoprotein GpIIb (alphaIIb), palmitoyl-KVGFFKR (Ppep; 10 microM), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A2 (TXA2) synthesis (EC50 = 45 +/- 5 microM), secretion of alpha-granules detected as enhanced surface expression of P-selectin (EC50 = 52 +/- 8 microM), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC50 = 3.7 +/- 1 microM). The TXA2 receptor antagonist, SQ29548, PGE1, and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptide-mediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.
A fluorescence-based immunosensor has been developed for simultaneous analysis of multiple samples. A patterned array of recognition elements immobilized on the surface of a planar waveguide is used to "capture" analyte present in samples; bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This immunosensor was used to detect physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D-dimer, a marker of sepsis and thrombotic disorders, in spiked clinical samples.
Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.
A microchip-based enzyme assay for protein kinase A is described. The microchips were prepared by standard photolithographic techniques. The assay reagents were placed in wells on the microchips, and electroosmosis was used to transport aliquots of these reagents into the network of etched channels, where the enzymatic reaction takes place. Protein kinase A catalyzes the transfer of a phosphate group from ATP to the serine residue of the heptapeptide LeuArgArgAlaSerLeuGly (Kemptide). The outcome of the enzymatic reaction was assessed by performing an on-chip electrophoretic separation of the fluorescently labeled peptide substrate and product. All liquid-handling steps were performed by controlling the electroosmotically driven flow from reagent and buffer wells using electrical current. On-chip dilutions of the peptide substrate, ATP and H-89, a known protein kinase A inhibitor, were performed and the kinetic constants (K(m), K(i)) of these compounds were determined. This prototype assay demonstrates the usefulness of the microchips for performing enzymatic assays for which fluorogenic substrates cannot easily be designed.
Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.
Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size, however, is almost always limited by the initial transformation steps following its assembly and ligation, as other all screening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening and selection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems. We review here the principle and applications of ribosome display for generating high-affinity antibodies from complex libraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA-ribosome-protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, very stringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of the initial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By this method, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosome display are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of the protein species investigated and the objectives of the selection or evolution experiment.
Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-microm gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.
Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM.
The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers.
Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.
Driven by chemistry but increasingly guided by pharmacology and the clinical sciences, drug research has contributed more
to the progress of medicine during the past century than any other scientific factor. The advent of molecular biology and,
in particular, of genomic sciences is having a deep impact on drug discovery. Recombinant proteins and monoclonal antibodies
have greatly enriched our therapeutic armamentarium. Genome sciences, combined with bioinformatic tools, allow us to dissect
the genetic basis of multifactorial diseases and to determine the most suitable points of attack for future medicines, thereby
increasing the number of treatment options. The dramatic increase in the complexity of drug research is enforcing changes
in the institutional basis of this interdisciplinary endeavor. The biotech industry is establishing itself as the discovery
arm of the pharmaceutical industry. In bridging the gap between academia and large pharmaceutical companies, the biotech firms
have been effective instruments of technology transfer.
We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.
Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX. Aptamers have been evolved to bind proteins which are associated with a number of disease states. Using this method, many powerful antagonists of such proteins have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, sugar modifications of nucleoside triphosphates are necessary to render the resulting aptamers resistant to nucleases found in serum. Changing the 2'OH groups of ribose to 2'F or 2'NH2 groups yields aptamers which are long lived in blood. The relatively low molecular weight of aptamers (8000-12000) leads to rapid clearance from the blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. When modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. A new approach to diagnostics is also described. Aptamer arrays on solid surfaces will become available rapidly because the SELEX protocol has been successfully automated. The use of photo-cross-linkable aptamers will allow the covalent attachment of aptamers to their cognate proteins, with very low backgrounds from other proteins in body fluids. Finally, protein staining with any reagent which distinguishes functional groups of amino acids from those of nucleic acids (and the solid support) will give a direct readout of proteins on the solid support.
Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.
Recombinant antibodies are becoming increasingly important in the field of proteomics. Recent advances include the development of large phage-antibody libraries that contain high-affinity binders to almost any target protein, and new methods for high-throughput selection of antibody-antigen interactions. Coupled with a range of new screening technologies that use high-density antibody arrays to identify differentially expressed proteins, these antibody libraries can be applied to whole proteome analysis.
Publisher Summary Complete genome sequences are available for three model organisms— Escherichia coli , Saccharomyces cerevisiae , and Caenorhabditis elegans —and for several pathogenic microorganisms such as Helicobacter pylori . Complete genome sequences are expected to become available soon for other model organisms and for humans. This information is expected to revolutionize the way biological questions can be addressed. Molecular mechanisms should now be approachable on a more global scale in the context of (nearly) complete sets of genes, rather than by analyzing genes individually. However, most open reading frames (ORFs) predicted from sequencing projects have remained completely uncharacterized at the functional level. The emerging field of functional genomics addresses this limitation by developing methods to characterize the function of large numbers of predicted ORFs simultaneously.
The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.
We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.
The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.
DNA vaccination is an effective means of protecting experimental animals against infectious pathogens and cancer and has more recently been used to prevent autoimmune disease. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T-cell-mediated destruction of the insulin-secreting beta cells in the pancreas. The NOD mouse is an animal model of IDDM in which several autoantigens, including insulin, have been identified. In this study we demonstrate that vaccination of NOD mice with DNA encoding an immunodominant peptide of insulin (residues 9-23 of the B chain) protects the animals from developing diabetes. Animals injected intramuscularly with a bacterial plasmid encoding the insulin B chain peptide show significantly lower disease incidence and delayed onset of disease when compared to controls. Protection appears to be mediated by insulin B (9-23)-specific down-regulation of IFN-gamma. Our results confirm that DNA vaccination has a protective effect on autoimmunity, the understanding of which will reveal new insights into the immune system and open doors for novel therapies.
Using a combination of local gene delivery and tolerizing DNA vaccination, we demonstrate that codelivery of the interleukin-4 (IL-4) gene and a DNA vaccine encoding the self-peptide proteolipid protein 139-151 (PLP139-151) provides protective immunity against experimental autoimmune encephalomyelitis (EAE). We provide evidence for a mechanism whereby IL-4 expressed from the naked DNA is secreted and acts locally on autoreactive T cells via activation of STAT6 to shift their cytokine profile to T helper 2. We also show that DNA vaccines can be used to reverse established EAE by covaccination with the genes for myelin oligodendrocyte glycoprotein and IL-4. This treatment strategy combines the antigen-specific effects of DNA vaccination and the beneficial effects of local gene delivery.
We developed versatile low-cost arrays of sol-gel-encapsulated enzymes (referred to as solzymes) suitable for repeated assays of bioactivity or enzyme inhibition. Sol-gel microstructures containing active enzymes were stabilized on glass at moderate pH and room temperature without harsh calcination. A multi-well bilayer of polydimethylsiloxane was used to support the solzyme array and contain the reaction medium. Each of the 147 microwells has a working volume of 5 muL and contains 50 mug of immobilized enzyme. The solzyme arrays maintained high activity through repeated applications and exhibited superior thermostability compared to soluble enzymes. Among the enzymes used were lipases, glucose oxidase, and horseradish peroxidase. Twenty different lipases and proteases were also used to prepare a hydrolase array, for which bromthymol blue served as a generic indicator of activity. The relative activities of the encapsulated hydrolases correlated closely with those of the soluble hydrolases, illustrating that sol-gel encapsulation preserved the hierarchy of enzyme activity. The development of solzyme arrays paves the way to higher throughput screening of diverse proteins and enzymes, including those that are available only in trace amounts.
Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels-Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH(2) on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.
Methods have been developed aimed at applying at high-throughput technology for expression of cloned cDNAs in yeast. Yeast is a eukaryotic host, which produces soluble recombinant proteins and is capable of introducing post-translational modifications of protein. It is, thus, an appropriate expression system both for the routine expression of various cDNAs or protein domains and for the expression of proteins, which are not correctly expressed in Escherichia coli. Here, we describe a standard system in Saccharomyces cerevisiae, based on a vector for intracellular protein expression, where the gene products are fused to specific peptide sequences (tags). These epitope tags, the N-terminal His(6) tag and the C-terminal StrepII tag, allow subsequent immunological identification and purification of the gene products by a two-step affinity chromatography. This method of dual-tagged recombinant protein purification eliminates contamination by degraded protein products. A miniaturization of the procedures for cloning, expression, and detection was performed to allow all steps to be carried out in 96-well microtiter plates. The system is, thus, suitable for automation. We were able to analyze the simultaneous protein expression of a large number of cDNA clones due to the highly parallel approach of protein production and purification. The microtiter plate technology format was extended to quantitative analysis. An ELISA-based assay was developed that detects StrepII-tagged proteins. The application of this high-throughput expression system for protein production will be a useful tool for functional and structural analyses of novel genes, identified by the Human Genome Project and other large-scale sequencing projects.
The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide proteins for the 'protein structure factory', a structural genomics initiative, we are working on the high-throughput expression of human proteins. Therefore, cDNAs are cloned for intracellular expression. The resulting fusion proteins carry affinity tags (6*HIS and StrepII, respectively) at the N- and C-terminus for the immunological detection and chromatographic purification of full-length proteins. Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. The screening procedure is based on a culture volume of 2 ml in a 24-well format. Lysis of the cells occurs via a chemical lysis without mechanical disruption. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l(-1) culture volume or higher.
Thanks to the results of the multiple completed and ongoing genome sequencing projects and to the newly available recombination-based cloning techniques, it is now possible to build gene repositories with no precedent in their composition, formatting, and potential. This new type of gene repository is necessary to address the challenges imposed by the post-genomic era, i.e., experimentation on a genome-wide scale. We are building the FLEXGene (Full Length EXpression-ready) repository. This unique resource will contain clones representing the complete ORFeome of different organisms, including Homo sapiens as well as several pathogens and model organisms. It will consist of a comprehensive, characterized (sequence-verified), and arrayed gene repository. This resource will allow full exploitation of the genomic information by enabling genome-wide scale experimentation at the level of functional/phenotypic assays as well as at the level of protein expression, purification, and analysis. Here we describe the rationale and construction of this resource and focus on the data obtained from the Saccharomyces cerevisiae project.
A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K(D) = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.