Abstract
Systematic analysis of protein and enzyme function typically requires scale-up of protein expression and purification prior to assay development; this can often be limiting. Miniaturization of assays provides an alternative approach, but simple, generic methods are in short supply. Here we show how custom microarrays can be adapted to this purpose. We discuss the different routes to array fabrication and describe in detail one facile approach in which the purification and immobilization procedures are combined into a single step, significantly simplifying the array fabrication process. We illustrate this approach by reference to the creation of arrays of human protein kinases and of human cytochrome P450s. We discuss methods for both ligand-binding and turnover-based assays, as well as data analysis on such arrays.
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References
Hunter S, Apweiler R et al. (2009) InterPro: the integrative protein signature database (2009). Nucleic Acids Res. 37: D224–228
Wise EY, Yew WS, Babbitt PC, Gerlt JA, Rayment I (2002) Homologous (b/a)8-Barrel Enzymes That Catalyze Unrelated Reactions: Orotidine 5’-Monophosphate Decarboxylase and 3-Keto-L-Gulonate 6-Phosphate Decar-boxylase. Biochemistry 41: 3861–3869
Schmidt, DMZ, Mundorff EC, Dojka M, Bermudez E et al (2003) Evolutionary potential of (b/a)8-Barrels: Functional promiscuity produced by single substitutions in the enolase superfamily. Biochemistry 42: 8387–8393
The genome international sequencing consortium (2001) Initial sequencing and analysis of the human genome. Nature 409: 860–921
MacBeath, G (2002) Protein microarrays and proteomics. Nature Genetics 32: 526–532
Wolf-Yadlin A, Sevecka M, MacBeath G (2009) Dissecting protein function and signaling using protein microarrays. Current Op Chem Biol 13: 398–405
Boutell JM, Hart DJ, Godber BLJ, Kozlowski RZ, Blackburn JM. (2004) Analysis of the effect of clinically-relevant mutations on p53 function using protein microarray technology. Proteomics 4: 1950–1958
Kodadek T (2001) Protein microarrays: prospects and problems. Chem Biol 8:105–115
Predki P (2004) Functional protein microarrays: Ripe for discovery. Curr Opin Chem Biol 8: 8–13
Zhu H, Klemic JF, Chang S, Bertone P et al. (2000) Analysis of yeast protein kinases using protein chips. Nat Genet 26: 283–289
Zhu H, Bilgin M, Bangham R, Hall D et al. (2001) Global analysis of protein activities using proteome chips. Science 293 : 2101–2105
Michaud GA, Salcius M, Zhou F, Bangham R. et al. (2003) Analyzing antibody specificity with whole proteome microarrays. Nature Biotechnology 21: 1509–12
Fang Y, Lahiri J, Picard L (2003) G-Protein-coupled receptor microarrays for drug discovery. Drug Discovery Today 8: 755–761
MacBeath G, Schreiber SL (2000) Printing proteins as microarrays for high-throughput function determination. Science 289: 1760–1763
Angenendt P, Glokler J, Sobek J, Lehrach H, Cahill DJ (2003) Next generation of protein microarray support materials: Evaluation for protein and antibody microarray applications. J Chromatogr A 1009: 97–104
Koopmann J-O, Blackburn JM (2003) High Affinity Capture Surface for MALDI compatible Protein Microarrays. Rapid Communication in Mass Spectrometry 17: 1–8
He M, Taussig M (2001) Single step generation of protein arrays from DNA by cell-free expression and in situ immobilization (PISA method). Nucleic Acids Res 29: E73.
Ramachandran N, Hainsworth E et al. (2004) Self-Assembling Protein Microarrays. Science 305: 86–90
Zhao Y, Chapman DAG, Jones IM (2003) Improving baculovirus recombination. Nucleic Acids Res.31: e6
Sambrook J, MacCallum P, Russell D (2001) Molecular Cloning, A Laboratory Manual, Third ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Athappilly FK, Hendrickson WA (1995) Structure of the biotinyl domain of acetyl-coenzymeA carboxylase determined by MAD phasing. Structure 3: 1407–19
Chapman-Smith A, Cronan JE (1999) The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity. Trends Biochem Sci 24: 359–363
Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714–2723
Yang Y-S, Watson WJ, Tucker PW, Capra JD (1993) Construction of recombinant DNA by exonuclease recession. Nucleic Acids Res 21: 1889–1893
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254
Terwilliger TC, Stuart D, Yokoyama S (2009) Lessons from Structural Genomics. Ann Rev Biophys 38: 371–383
Brown M (2007) Novel fluorescent kinase ligands and assays employing the same. Patent application no. WO2008071937
Hernandez-Boussard T, Whirl-Carrillo M, Hebert J.M, et al (2008) The pharmacogenetics and pharmacogenomics database: accentuating the knowledge. Nucleic Acids Research 36 (Database issue), D913–D918
Ingelman-Sundberg M (2004) Pharmacogen-etics of cytochrome P450 and its applications in drug therapy: the past, present and future. TRENDS in Pharmacological Sciences 25: 193–200
Choi-Rhee E, Cronan JE (2003) The biotin carboxylase-biotin carboxyl carrier protein complex of Escherichia coli acetyl-CoA carboxylase. J Biol Chem 278: 30806–30812
Cull MG, Schatz PJ (2000) Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol 326: 430–40
Lue RY, Chen GY, Hu Y, Zhu Q, Yao SQ (2004) Versatile protein biotinylation strategies for potential high-throughput proteomics. J Am Chem Soc 126: 1055–62
Berliner E, Mahtani HK, Karki S, Chu LF, et al. (1994) Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface. J Biol Chem 269: 8610–5
Lerner CG, Saiki AY (1996) Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells. Anal. Biochem 240: 185–96
Parrott MB, Barry MA (2001) Metabolic biotinylation of secreted and cell surface proteins from mammalian cells. Biochem Biophys Res Commun 281: 993–1000
Zheng J, Li L, et al (2005) Strong Repulsive Forces between Protein and Oligo (ethylene glycol) Self- Assembled Monolayers: A Molecular Simulation Study. Biophys J. 89: 158–166
Makris TM, von Koenig K, Schlichting I, Sligar, SG (2006) The status of high-valent metal oxo complexes in the P450 cytochromes. Journal of Inorganic Biochemistry 100: 507–518
Pylypenko O, Schlichting I (2004) Structural aspects of ligand binding to and electron transfer in bacterial and fungal P450s. Annual Reviews in Biochemistry 73: 991–1018
Sakai-Kato K, Kato M, Homma H, Toyo’oko T, Utsunomiya-Tate N (2005) Creation of a P450 array toward high-throughput analysis. Analytical Chemistry 77: 7080–7083
Lee M, Park CB, Dordick JS, Clark DS (2005) Metabolising enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses. Proceedings of the National Academy of Sciences USA 102: 983–987
Sukumaran SM, Potsaid B, Lee M, Clark DS, Dordick JS (2009) Development of a fluorescence-based, ultra high-throughput screening platform for nanoliter-scale cytochrome P450 microarrays. Journal of Biomolecular Screening 14: 668–678
Acknowledgments
The authors thank Nashied Peton, Sarah Joyce, Colin Wheeler, Jens Koopman, Nick Workman, Steve Parham, and Mike McAndrew for their help in generating the data detailed herein. We thank Procognia Ltd (UK) for provision of human kinase arrays and also thank the Centre for Proteomic & Genomic Research, Cape Town, for access to equipment. JMB thanks the National Research Foundation (NRF; South Africa) for a Research Chair; NBK thanks the NRF for a PhD studentship. The research was supported by grants from the NRF, Procognia Ltd and Genetix PLC (UK).
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Blackburn, J.M., Shoko, A., Beeton-Kempen, N. (2012). Miniaturized, Microarray-Based Assays for Chemical Proteomic Studies of Protein Function. In: Zanders, E. (eds) Chemical Genomics and Proteomics. Methods in Molecular Biology, vol 800. Humana Press. https://doi.org/10.1007/978-1-61779-349-3_10
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DOI: https://doi.org/10.1007/978-1-61779-349-3_10
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