Issue 4 Summer 2002 - Applied Biosystems

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10 technical communications The InteraX System Monitoring Protein-Protein Interactions in Different Compartments of the Mammalian Cell A better understanding of the specific interactions of proteins with each other, under physiologically relevant conditions, is extremely helpful in elucidating new drug targets and the discovery of new cell signalling pathways. This in turn may reveal new action points for new drugs correcting molecular disfunctions. The InteraX system allows the direct detection of in vivo proteinprotein interactions in a wide range of cell types and species. This technology employs the intracistronic complementation or alpha-complementation of two mutant forms (∆α and ∆ω) of ß-galactosidase. These two specific mutant forms have low affinity for each other and do not form an active enzyme complex. They can be expressed as fusions with the target proteins under investigation. Upon interaction of the two target proteins with each other, the two mutant forms of ß-galactosidase restore an active ß-galactosidase complex. Consequently, the ß-galactosidase activity measured after ß-galactosidase complementation is a direct function of the interaction of the two proteins under investigation (See figure 1). Figure 1. Probing Protein-Protein Interaction ß-Galactosidase Mutant Complementation A and B do not interact A A add substrate No Light A and B do interact B A B B A B add substrate The InteraX system is capable of detecting protein-protein interactions in different cell lines of different organisms. The system can be applied to mammalian cell lines with intact regulatory machinery, which not only allows a functional readout for orphan and known G-Protein coupled receptors (GPCRs), but extend to a more general use of the technology as a tool to map signalling pathways. Blau et al1 used the above principle of intracistronic complementation underlying the InteraX system to monitor EGF receptor dimerisation in live cells. Their experimental set-up is illustrated in figure 2. Blau et al expressed chimeric EGF - ß-gal receptors in cell culture. Treatment with EGF and EGF-like compounds resulted in ligand specific, reversible dose responses. The dimerisation of the chimeric EGF-receptor in the cell membrane was inhibited by antibodies blocking the ligand binding. In addition the kinetics of EGF receptor dimerisation was investigated. Low levels of receptor chimeras can be detected avoiding over-expression of protein. Figure 2. Functional Receptor Binding Assay: Monitoring EGFR Dimerization FRAP rapamycin rapamycin rapa apa yc FRAP FKBP12 FKBP12 Cytosolic interaction Signal / Noise 40 30 20 10 0 Parental ∆ω alone ∆α/∆ω clone A ∆α/∆ω clone B Media For the cytosolic protein-protein interaction of ∆αFRAP and ∆ωFKBP12 (See figure 3) high S/N ratios have been achieved. This documents not only the quantitative nature of the technology, but also the ability to use it to develop functional readouts for protein-protein interactions in different cell compartments. Figure 3. Protein Induced Interaction Measured Using Gal-Screen ® Assay. ß-Gal complementation is demonstrated upon induction with rapamycin (10 ng/mL) in two clones of transfected cells (10,000 cells/well) co-expressing the fusion proteins ∆αFRAP and ∆ωFKBP12. The InteraX system can be applied as well to develop assays amenable for high-throughput screening. Rees et al2 developed a homogeneous 384-well assay protocol based on the Your feedback on Biosystems Solutions R ecently, with Biosystems Solutions, we included some market research questions on the content of the magazine and how you like to be kept informed with up-to-date information from Applied Biosystems. Encouragingly most of you found the articles we chose to include, informative, well balanced and of general interest. We would welcome your proposals and suggestions for future articles on the advancement of life science research throughout Europe. If you want to comment on the content of previous issues of Biosystems Solutions or request to see specific topics technical communications ß-galactosidase complementation technology and studied pharmacological characteristics of a chimeric EGF-receptor towards EGF antagonists. The DMSO tolerance of this assay was observed as up to 2%. In a 1280 compound screen, retest hit rates of 0.4% were observed, which is far better than that achieved with reporter gene assays, wherein hit rates of greater than 5% were obtained. Also, an average mean Z-factor of 0.55 was obtained throughout the screen. Therefore, one of the benefits of the technology was determined to be the low false-positive hit rate compared to functional antagonist assays and the fact that the readout is detection platform independent. In summary, the InteraX system employing ß-galactosidase complementation represents a functional readout for protein-protein interactions in a variety of different cells and in different compartments of a cell under physiological conditions. It is a promising new system for assay development and high-throughput screening with platform independent detection. References 1. Bruce T. Blakely, Fabio M.V. Rossi, Bonnie Tillotson, Michelle Palmer, Angeles Estelles, and Helen Blau; Epidermal Growth Factor Receptor Dimerization monitored in live cells; Nature Biotechnology, Volume 18, Number 2, p.218-222, February 2000 2. Debbie L.Graham, Nicola Bevan, Peter N. Lowe, Michelle Palmer, and Stephen Rees; Application of ß-Galactosidase Enzyme Complementation Technology as a High- Throughput Screening Format for Antagonists of the Epidermal Growth Factor Receptor; Journal of Biomolecular Screening, Volume 6, Number 6, p.401- 411, 2001 For more information on: InteraX system datasheet and reprints enter: No. 406 included in future issues then please contact us by emailing abdirect@eur.appliedbiosystems.com using ‘BS comment’ as your email subject. Finally, to continue to keep you informed it is important that our records of your contact details are as up-to-date as possible. So if you have recently changed your address, or are about to, please complete the postage free reply card in this magazine and send it back to us. Or alternatively email us at the above address. 11
12 technical communications Choosing the Right Target! T he first challenge for today’s pharmaceutical companies is not so much hitting the target, but choosing the right target in the first place. Over the last hundred years drugs have been designed against 400-500 disease targets. The post genome era presents the industry an interesting dilemma – with the completion of the human genome the number of ‘druggable’ targets is expected to increase dramatically with estimated numbers between 3,000 to 10,000. So how can a company choose which targets to aim at! Technologies and platforms from Applied Biosystems are being used by pharmaceutical companies across the globe to more fully understand the molecular cause of disease. By studying disease mechanisms researchers are able to identify important genes and proteins, understanding how they influence and control biological processes. At Applied Biosystems we transformed gene discovery research with Automated DNA Analyzers that decipher entire genomes in months instead of years. These systems of choice for identifying disease-related mutations and correlating genetic markers with disease, are also the primary technology for revealing genes with altered expression levels in disease. Our Gene Expression Analysis Systems help to assign function to potential target genes and also provide novel assays for lead discovery and biomarkers for clinical trials. Proteomics offers distinct opportunities for target discovery and validation, novel assays for lead discovery, and research to discover biomarkers for clinical trials. Applied Biosystems is advancing the science of proteomics with Automated Protein Sequencers, Time-of-Flight (TOF) Mass Spectrometers, and differential protein expression analysis using ICAT reagents and software. Applied Biosystems Proteomics Research Center and Applied Biosystems/MDS SCIEX, work with key leaders in the field to speed the development of emerging technologies and novel R & D applications. Even with the high quality data generated with these technologies it is still necessary to make sense of this information before deciding which targets to take forward into the drug development process. Applied Biosystems has developed and refined informatics systems that allow the automation and integration of genomic and proteomic systems allowing researchers to make informed choices on the targets for tomorrow’s drugs. See articles on pages 20, 29 & 40 For more information on: Systems for DNA Analysis and Gene Expression enter: Solutions for Proteomics and LC/MS enter: Informatics Solutions enter: No. 407 No. 408 No. 409 technical communications Revolution in 5' Nuclease Assay TaqMan ® MGB Probes Deliver Simple and Robust SNP Genotyping! D etection of single nucleotide polymorphisms (SNPs) is now central to modern molecular genetics. Large-scale population scoring of known SNPs requires a technology with minimal steps and an ability to automate the assay process. Applied Biosystems vision: to create a single-step SNP assay making it easy to unambiguously assign SNP genotypes in a flexible and scaleable format. Here’s how we’ve delivered on that vision… Figure 1. SNP scoring (also called allelic discrimination) assay using 5' nuclease chemistry and TaqMan MGB probes. Tm=Tm of perfectly matched probe -Tm of mismatched probe. Discrimination of the two SNP alleles is achieved by using an annealing/extension temperature within Tm window. A substantial increase in VIC ® fluorescence only indicates homozygosity for Allele 1, while a substantial increase in FAM ® fluorescence only indicates homozgosity for Allele 2. Both fluorescent signals increase substantially when sample is heterozygous. NFQ = Non fluorescent quencher Three factors contribute to allelic discrimination based on a single mismatch (Figure 1). 1 A mismatched probe has a lower Tm than a perfectly matched probe. Shorter probes display greater mismatch discrimination because the single mismatch has a higher disruptive effect on the hybridisation kinetics of the shorter probe. 2 The assay is performed with both probes present in the reaction tube. The mismatched probes are virtually prevented from binding to the target due to the stable binding of the perfectly matched probes. 3 For efficient probe cleavage, the 5' end of the probe must start to be displaced. Once a probe starts to be displaced, complete dissociation occurs faster with a mismatch than with an exact match. Thus, the presence of a mismatch promotes dissociation rather than cleavage of the probe. The minor groove binder (MGB) contributes a major enhancement to the 5' Nuclease Assay. The addition of an MGB molecule to an oligonucleotide has been shown to stabilise nucleic acid duplexes, causing a dramatic increase in the T m of the oligo. Employing the MGB attachment in a TaqMan probe facilitates the use of shorter probes, thus resulting in improved mismatch discrimination for SNP assays and increased design flexibility for both allelic discrimination assays and gene expression assays. With a more robust assay, a new generation of products are being created using 5' Nuclease assay with TaqMan MGB probes. Building on the efforts of both Celera Genomics and public sequencing programs, Applied Biosystems is generating validated SNP assays that can easily be applied for genetic studies. Using our bioinformatics pipeline for ‘genome-aided’ assay design and our industrial scale production genotyping lab, as many as 200,000 validated, ready-for-use Human SNP Assays-on-Demand products and nearly 30,000 Human gene expression assays will be fully released by Summer 2002. These Assays-on-Demand products together with the Assays-by-Design SM service represent Applied Biosystems Genomic Assays product line (Figure 2), a set of unique enabling tools that provide the most rapid and productive path to disease-gene discovery. Figure 2. Genomic Assays from Applied Biosystems. Genomic assays are based upon 5' Nuclease assay using TaqMan MGB probes. Assays-on-Demand products are ready-to-use Human SNP and Gene expression assays. Assays-by-Design Service provides assays for customer-specified SNPs or genes of interest for any species. For more information on: Assays-on-Demand Products enter: No. 410 Assays-by-Design Service enter: No. 411 13
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