Target Discovery and Validation Reviews and Protocols

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Protein Arrays 343 The ideal proteomics-based analytical tool would consist of a microarray containing a large number of high-affinity, high-specificity protein ligands. For humans, this outcome would mean isolating on the order of 100,000 good monoclonal antibodies or the equivalent capture proteins. In reality, the number would be much higher, because the detection of different posttranslationally modified forms of a protein is one of the principal advantages of moving from nucleic acids to protein-based analytical techniques. However, the high cost of monoclonal antibody manufacturing is a bottleneck for large-scale production of antibody microarrays. Thus, it is most likely the field will proceed in a stepwise manner, and a reasonable intermediate goal would be to construct arrays of small amount of protein-binding ligands directed against proteins of interest in a particular disease state. Reverse phase arrays and the aforementioned display techniques allow the rapid and efficient isolation of high-affinity and -specificity protein ligands. Furthermore, the uses of display techniques allow identification of unknown biomarkers. It is increasingly evident that posttranslational modifications of biomolecules could play an essential role in coding for both new and native properties. However, the selection of antigens with modifications is not accessible by peptide-on-plasmid or phage display techniques. Regardless, the establishment of a robust system that may combine the advantages of display strategies continues to be a key challenge. To achieve certain desired display advantages, several different viral systems have been used to display peptides, including lysogenic filamentous phages (6) and lytic lambda phage (50,51), T7 bacteriophage (52), and T4 bacteriophage (53). Lysogenic filamentous phages remain the most commonly used phage display system. For cases in which displayed proteins may be toxic to filamentous phage assembly or incompatible with the bacterial secretion pathway, lytic phages can be used that allow displayed sequences to minimize negative selection. However, selection of recombinant proteins displayed on T7 phage by arrays seems to be difficult because negative phage immobilized on the surface showed reactivity to patient antibodies as well (Cekaite et al., unpublished data). One way of addressing this problem is to reduce binding valency by including a binding competitor, thereby increasing the binding affinity threshold. The examples in this chapter show that protein microarray technology is still in its infancy, given the diversity of proposed approaches as well as raised problems. However, this technology is promising tool for priming of the humoral arm of the immune system. References 1. Fields, S. and Song, O. -K. (1989) A novel genetic system to detect protein Â–protein interactions. 340, 245–246. 2. Bartel, P. L., Roecklein, J. A., SenGupta, D., and Fields, S. (1996) A protein linkage map of Escherichia coli bacteriophage T7. Nat Genet. 12, 72–77.
344 Cekaite, Hovig, and Sioud 3. Uetz, P., Giot, L., Cagney, G., et al. (2000) A comprehensive analysis of proteinprotein interactions in Saccharomyces cerevisiae. Nature 403, 623–627. 4. Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98, 4569–4574. 5. Becker, F., Murthi, K., Smith, C. W., et al. (2004) A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors. Chem. Biol. 11, 211–223. 6. Smith, G. P. (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317. 7. Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom, H. R. (1994) Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455. 8. Sheets, M. D., Amersdorfer, P., Finnern, R., et al. (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. USA 95, 6157–6162. 9. Dybwad, A., Lambin, P., Sioud, M., and Zouali, M. (2003) Probing the specificity of human myeloma proteins with a random peptide phage library. Scand. J. Immunol. 57, 583–590. 10. Souriau, C. and Hudson, P. J. (2003) Recombinant antibodies for cancer diagnosis and therapy. Expert Opin. Biol. Ther. 3, 305–318. 11. Moghaddam, A., Borgen, T., Stacy, J., et al. (2003) Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J. Immunol. Methods 280, 139–155. 12. Dybwad, A., Forre, O., Kjeldsen-Kragh, J., Natvig, J. B., and Sioud, M. (1993) Identification of new B cell epitopes in the sera of rheumatoid arthritis patients using a random nanopeptide phage library. Eur. J. Immunol. 23, 3189–3193. 13. Hansen, M. H., Dybwad, A., Forre, O., and Sioud, M. (2000) Probing antinuclear antibody specificities by peptide phage display libraries. Clin. Exp. Rheumatol. 18, 465–472. 14. Hansen, M. H., Nielsen, H., and Ditzel, H. J. (2001) The tumor-infiltrating B cell response in medullary breast cancer is oligoclonal and directed against the autoantigen actin exposed on the surface of apoptotic cancer cells. Proc. Natl. Acad. Sci. USA 98, 12,659–12,664. 15. Hansen, M. H., Ostenstad, B., and Sioud, M. (2001) Identification of immunogenic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumour. Int. J. Oncol. 19, 1303–1309. 16. Hansen, M. H., Ostenstad, B., and Sioud, M. (2001) Antigen-specific IgG antibodies in stage IV long-time survival breast cancer patients. Mol. Med. 7, 230–239. 17. Hansen, M. H., Sode, L. L., Hyldig-Nielsen, J. J., and Engberg, J. (1997) Detection of PNA/DNA hybrid molecules by antibody Fab fragments isolated from a phage display library. J. Immunol. Methods 203, 199–207.
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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78 Beaty et al. using a protein ass
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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148 Sano and Taira ribozymes may cl
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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186 Houdebine and stroma. Several o
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204 Vijayaraj, Söhl, and Magin 1.
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 285 expre
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Overview of Immune System 289 solub
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Overview of Immune System 291 amino
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Page 342 and 343: Immunoproteomics 329 by “shot gun
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Page 360 and 361: Protein Arrays 347 49. Yuko Kawahas
Page 362 and 363: Index 349 Index A Acetyl-CoA carbox
Page 364 and 365: Index 351 conditional by using FRT
Page 366 and 367: Index 353 Recombinant tumor antigen
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