Abstract
The South-Paris Yeast Structural Genomics Pilot Project (http://www.genomics.eu.org) aims at systematically expressing, purifying, and determining the three-dimensional structures of Saccharomyces cerevisiae proteins. We have already cloned 240 yeast open reading frames in the Escherichia coli pET system. Eighty-two percent of the targets can be expressed in E. coli, and 61% yield soluble protein. We have currently purified 58 proteins. Twelve X-ray structures have been solved, six are in progress, and six other proteins gave crystals. In this chapter, we present the general experimental flowchart applied for this project. One of the main difficulties encountered in this pilot project was the low solubility of a great number of target proteins. We have developed parallel strategies to recover these proteins from inclusion bodies, including refolding, coexpression with chaperones, and an in vitro expression system. A limited proteolysis protocol, developed to localize flexible regions in proteins that could hinder crystallization, is also described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Brenner, S. E. (2001) A tour of structural genomics. Nat. Rev. Genet. 2, 801–809.
Quevillon-Cheruel, S., Collinet, B., Zhou, C. Z., et al. (2003) A structural genomics initiative on yeast proteins. J. Synchrotron Radiat. 10, 4–8.
Goffeau, A., Barrell, B. G., Bussey, H., et al. (1996) Life with 6000 genes. Science 274, 563–567.
Nishihara, K., Kitagawa, M., Yanagi, H., and Yura, T. (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl. Environ. Microbiol. 64, 1694–1699.
Hendrickson, W. A., Horton, J. R., and LeMaster, D. M. (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9, 1665–1672.
Hofmann, K. and Stoffel, W. (1993) Tmbase: A database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 374, 166.
Gonnet, G. H., Cohen, M. A., and Benner, S. A. (1992) Ex-haustive matching of the entire protein sequence database. Science 256, 1443–1445.
Pearson, W. R. and Lipman, D. J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444–2448.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Rost B. and Liu J. (2003) The PredictProtein server. Nucleic Acids Res. 31, 3300–3304.
Bateman A., Coin L., Durbin R., et al. (2004) The Pfam protein families database. Nucleic Acids Res. 32, 138–141.
Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
Kelley, L. A., MacCallum, R. M., and Sternberg, M. J. E. (2000) Enhanced Genome Annotation using Structural Profiles in the Program 3D-PSSM. J. Mol. Biol. 299, 499–520.
Marin, A., Pothier, J., Zimmerman, K., and Gibrat, J. F. (2001) Protein structure prediction: bioinformatic approach. In: Protein Threading Statistics: An Attempt to Assess the Significance of a Fold, (Tsigelny, I., ed.), International University Line, La Jolla, CA.
Callebaut, I., Labesse, G., Durand, P., et al. (1997) Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. Cell. Mol. Life Sci. 53, 621–645.
Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.
Shuman, S. (1994) Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J. Biol. Chem. 269, 32,678–32,684.
Trésaugues, L., Collinet, B., Minard, P., et al. (2004) Refolding strategies from inclusion bodies in a structural genomics project. J. Struct. Funct. Genomics 5, 195–204.
Miroux B. and Walker J. (1996) Over-production of proteins in Escherichia coli: mutants hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.
Kigawa, T., Muto, Y., and Yokoyama, S. (1995) Cell-free synthesis and amino acidselective stable isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6, 129–134.
Kigawa, T., Yabuki, T., Yoshida, Y., et al. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15–19.
Hammarstrom, M., Hellgren, N., van Den Berg, S., Berglund, H., and Hard, T. (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 11, 313–321.
Waldo, G. S. (2003) Improving protein folding efficiency by directed evolution using the GFP folding reporter. Methods Mol. Biol. 230, 343–359.
Boettner, M., Prinz, B., Holz, C., Stahl, U., and Lang, C. (2002) High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J. Biotechnol. 99, 51–62.
Bettache, N. A., Quevillon-Cheruel, S., Bondet, V., van Tilbeurgh, H., and Blondeau, K. Determination of optimal cultivation conditions for large scale production of yeast carboxyl methyltransferase (Ppm1) in Escherichia coli., submitted.
Studts, J. M. and Fox, B. G. (1999) Application of fed-batch fermentation to the preparation of isotopically labeled or selenomethionyl-labeled proteins. Protein Expr. Purif. 16, 109–119.
Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1993) Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124.
Doublie, S. (1997) Preparation of selenomethionyl proteins for phase determination. Meth. Enzymol. 276, 523–530.
Sambrook, J., Frits, E. F., and Maniatis, T. (eds.) (1989) Preparation and transformation of competent E. coli. In: Molecular Cloning, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, NY, pp. I.74–I.84.
David, G., Blondeau, K., Renouard, M., and Lewit-Bentley, A. (2002) Crystallization and preliminary analysis of Escherichia coli YodA. Acta Crystallogr. D Biol. Crystallogr. 58, 1243–1245.
Lesley, S. A., Kuhn, P., Godzik, A., et al. (2002) Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. USA 99, 11,664–11,669.
Vincentelli, R., Bignon, C., Gruez, A., et al. (2003) Medium-scale structural genomics: strategies for protein expression and crystallization. Acc. Chem. Res. 36, 165–172.
Heinemann, U., Bussow, K., Mueller, U., and Umbach, P. (2003) Facilities and methods for the high-throughput crystal structural analysis of human proteins. Acc. Chem. Res. 36, 157–163.
Matte, A., Sivaraman, J., Ekiel, I., Gehring, K., Jia, Z., and Cygler, M. (2003) Contribution of structural genomics to understanding the biology of Escherichia coli. J. Bacteriol. 185, 3994–4002.
Smyth, D. R., Mrozkiewicz, M. K., McGrath, W. J., Listwan, P., and Kobe, B. (2003) Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 12, 1313–1322.
Uversky, V. N. (2002) Natively unfolded proteins: a point where biology waits for physics. Protein Sci. 11, 739–756.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Quevillon-Cheruel, S. et al. (2007). Cloning, Production, and Purification of Proteins for a Medium-Scale Structural Genomics Project. In: Walker, J.M., Doublié, S. (eds) Macromolecular Crystallography Protocols. Methods in Molecular Biology, vol 363. Humana Press. https://doi.org/10.1007/978-1-59745-209-0_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-209-0_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-292-6
Online ISBN: 978-1-59745-209-0
eBook Packages: Springer Protocols