This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway® cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Reference
Burley, S. K. (2000) An overview of structural genomics. Nat. Struct. Biol. 7, 932–934.
Chandonia, J. M., and Brenner, S. E. (2006) The impact of structural genomics: expectations and outcomes. Science 311, 347–351.
Gordon, S., Crocker, P. R., Morris, L., Lee, S. H., Perry, V. H., and Hume, D. A. (1986) Localization and function of tissue macrophages. Ciba Found. Symp. 118, 54–67.
Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., and Ezekowitz, R. A. (1999) Phylogenetic perspectives in innate immunity. Science 284, 1313–1318.
Mammen, E. F. (2000) Disseminated intravascular coagulation (DIC). Clin. Lab. Sci. 13, 239–245.
Morley, J. E., Thomas, D. R., and Wilson, M. M. (2006) Cachexia: pathophysiology and clinical relevance. Am. J. Clin. Nutr. 83, 735–743.
Duffield, J. S. (2003) The inflammatory macrophage: a story of Jekyll and Hyde. Clin. Sci. (Lond.) 104, 27–38.
Puri, M., Robin, G., Cowieson, N., Forwood, J. K., Listwan, P., Hu, S. H., Guncar, G., Huber, T., Kellie, S., Hume, D. A., Kobe, B., and Martin, J. L. (2006) Focusing in on structural genomics: the University of Queensland structural biology pipeline. Biomol. Eng. 23, 281–289.
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The Protein Data Bank. Nucleic Acids Res. 28, 235–242.
Fleming, K., Kelley, L. A., Islam, S. A., MacCallum, R. M., Muller, A., Pazos, F., and Sternberg, M. J. (2006) The proteome: structure, function and evolution. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 441–451.
Shi, J. B. T., and Mizuguchi K. (2001) FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure- dependent gap penalties. J. Mol. Biol. 310, 243–257.
Chen, Y., Yu, P., Luo, J., and Jiang, Y. (2003) Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm. Genome 14, 859–865.
Bono, H., Kasukawa, T., Furuno, M., Hayashizaki, Y., and Okazaki, Y. (2002) FANTOM DB: database of Functional Annotation of RIKEN Mouse cDNA Clones. Nucleic Acids Res. 30, 116–118.
Yokoyama, S. (2003) Protein expression systems for structural genomics and pro-teomics. Curr. Opin. Chem. Biol. 7, 39–43.
Listwan, P., Cowieson, N., Kurz, M., Hume, D. A., Martin, J. L., and Kobe, B. (2005) Modification of recombinatorial cloning for small affinity tag fusion protein construct generation. Anal. Biochem 346, 327–329.
Studier, F. W. (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234.
Gilbert, M., and Albala, J. S. (2002) Accelerating code to function: sizing up the protein production line. Curr. Opin. Chem. Biol. 6, 102–105.
Moy, S., Dieckman, L., Schiffer, M., Maltsev, N., Yu, G. X., and Collart, F. R. (2004) Genome-scale expression of proteins from Bacillus subtilis. J. Struct. Funct. Genomics 5, 103–109.
Huang, R. Y., Boulton, S. J., Vidal, M., Almo, S. C., Bresnick, A. R., and Chance, M. R. (2003) High throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins. Biochem. Biophys. Res. Commun. 307, 928–934.
Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C. H., and Edwards, A. M. (2000) Structural proteomics: prospects for high throughput sample preparation. Prog. Biophys. Mol. Biol. 73, 339–345.
Cowieson, N. P., Wensley, B., Listwan, P., Hume, D. A., Kobe, B., and Martin, J. L. (2006) An automatable screen for the rapid identification of proteins amenable to refolding. Proteomics 6, 1750–1757.
Miles, A. J., and Wallace, B. A. (2006) Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics. Chem. Soc. Rev. 35, 39–51.
Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 227–293.
Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., and Lesley, S. A. (2003) Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome. Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037.
Majeed, S., Ofek, G., Belachew, A., Huang, C. C., Zhou, T., and Kwong, P. D. (2003) Enhancing protein crystallization through precipitant synergy. Structure 11, 1061–1070.
Senger, A. B., and Mueser, T. C. (2005) A method for the rapid preparation of cus tom grid-screen crystallization trays using standardized pipetting maps is presented. J. Appl. Cryst. 38, 847–850.
Hendrickson, W. (1999) Maturation of MAD phasing for the determination of mac-romolecular structures. J. Synchrotron. Radiat. 6, 845–851.
Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326.
Terwilliger, T. C., and Berendzen, J. (1999) Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861.
Morris, R. J., Perrakis, A., and Lamzin, V. S. (2003) ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 229–244.
CCP4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763.
Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graph ics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
Aagaard, A., Listwan, P., Cowieson, N., Huber, T., Ravasi, T., Wells, C. A., Flanagan, J. U., Kellie, S., Hume, D. A., Kobe, B., and Martin, J. L. (2005) An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relation ship to cystatins and the tumor suppressor TIG1. Structure 13, 309–317.
Forwood, J. K., Thakur, A. S., Guncar, G., Marfori, M., Mouradov, D., Meng, W., Robinson, J., Huber, T., Kellie, S., Martin, J. L., Hume, D. A., and Kobe, B. (2007) Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation. Proc. Natl. Acad. Sci. U.S.A. 104, 10382–10387.
Hunt, M. C., and Alexson, S. E. (2002) The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41, 99–130.
Faergeman, N. J., and Knudsen, J. (1997) Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling. Biochem. J. 323, 1–12.
Yamada, J. (2005) Long-chain acyl-CoA hydrolase in the brain. Amino Acids 28, 273–278.
Acknowledgments
This work was supported by Australian Research Council (ARC; to JLM and BK). BK is an ARC Federation Fellow and a National Health and Medical Research Council (NHMRC) Honorary Research Fellow. JMH is the recipient of an RD Wright Biomedical Career Development Award from the NHMRC. MP thanks DEST for the Australia-Asia Fellowship.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Meng, W. et al. (2008). Overview of the Pipeline for Structural and Functional Characterization of Macrophage Proteins at the University of Queensland. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_38
Download citation
DOI: https://doi.org/10.1007/978-1-60327-058-8_38
Publisher Name: Humana Press
Print ISBN: 978-1-58829-809-6
Online ISBN: 978-1-60327-058-8
eBook Packages: Springer Protocols