Abstract
Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhang, Q. and Wang, Y. (2008) High mobility group proteins and their post-translational modifications. Biochim. Biophys. Acta 1784, 1159–1166.
Hay, R.T. (2005) SUMO: a history of modification. Mol. Cell 18, 1–12.
Kobayashi, I. (2001) Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Nucleic Acids Res. 29, 3742–3756.
Kobayashi, I. (2004) Restriction – modification systems as minimal forms of life. In Restriction Endonucleases (Pingoud, A., ed.), Berlin, Springer. pp. 19–62.
Orlowski, J. and Bujnicki, J.M. (2008) Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses. Nucleic Acids Res. 36, 3552–3569.
Chinen, A., Uchiyama, I. and Kobayashi, I. (2000) Comparison between Pyrococcus horikoshii and Pyrococcus abyssi genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms. Gene 259, 109–121.
Ishikawa, K., Watanabe, M., Kuroita, T., Uchiyama, I., Bujnicki, J.M., Kawakami, B., Tanokura, M. and Kobayashi, I. (2005) Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi. Nucleic Acids Res. 33, e112.
Miyazono, K., Watanabe, M., Kosinski, J., Ishikawa, K., Kamo, M., Sawasaki, T., Nagata, K., Bujnicki, J.M., Endo, Y., Tanokura, M. and Kobayashi, I. (2007) Novel protein fold discovered in the PabI family of restriction enzymes. Nucleic Acids Res. 35, 1908–1918.
Roberts, R.J., Belfort, M., Bestor, T., Bhagwat, A.S., Bickle, T.A., Bitinaite, J., Blumenthal, R.M., Degtyarev, S.Kh., Dryden, D.T., Dybvig, K., Firman, K., Gromova, E.S., Gumport, R.I., Halford, S.E., Hattman, S., Heitman, J., Hornby, D.P., Janulaitis, A., Jeltsch, A., Josephsen, J., Kiss, A., Klaenhammer, T.R., Kobayashi, I., Kong, H., Krüger, D.H., Lacks, S., Marinus, M.G., Miyahara, M., Morgan, R.D., Murray, N.E., Nagaraja, V., Piekarowicz, A., Pingoud, A., Raleigh, E., Rao, D.N., Reich, N., Repin, V.E., Selker, E.U., Shaw, P.C., Stein, D.C., Stoddard, B.L., Szybalski, W., Trautner, T.A., Van Etten, J.L., Vitor, J.M., Wilson, G.G. and Xu, S.Y. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31, 1805–1812.
Sawasaki, T., Ogasawara, T, Morishita, R. and Endo, Y. (2002) A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. U.S.A. 99, 14652–14657.
Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.
Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326.
Weeks, C. and Miller, R. (1999) The design and implementation of SnB version 2.0. J. Appl. Cryst. 32, 120–124.
Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. and Paciorek, W. (2003) Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D Biol. Crystallogr. 59, 2023–2030.
Terwilliger, T. (2003) Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D Biol. Crystallogr. 59, 38–44.
Vagin, A. and Teplyakov, A. (1997) MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022-1025.
Murshudov, G. N., Vagin, A. A. and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255.
Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. and Warren, G. L. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921.
McRee, D. E. (1999) XtalView/Xfit – a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165.
Madin, K., Sawasaki, T., Ogasawara, T. and Endo, Y. (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. U.S.A. 97, 559-564
Hanes, J. and Pluckthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. U.S.A. 94, 4937–4942.
Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., Kamura, N., Ogasawara, T. and Endo Y. (2002) A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Lett. 514, 102–105.
Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y. and Alakhov, Y. B. (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–1164.
Sawasaki, T., Gouda, M.D., Kawasaki, T., Tsuboi, T., Tozawa, Y., Takai, K. and Endo, Y. (2005) The wheat germ cell-free expression system: methods for high-throughput materialization of genetic information. Methods Mol. Biol. 310, 131–144.
Matthews, B. W. (1968) Solvent content of protein crystals. J. Mol. Biol. 33, 491–497.
Collaborative, Computational, Project, Number, 4. (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763.
Acknowledgments
The study of PabI was carried out in collaboration with Jan Kosinski, Ken Ishikawa, Masayuki Kamo, Koji Nagata, and Janusz M. Bujnicki. We are grateful to Kazuyuki Takai for encouragements and patience. IK was supported, during writing, by twenty-first century COE program “Genome and Language” from MEXT and “Grants-in-Aid for Scientific Research” (19657002, 21370001) from JSPS.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Watanabe, M., Miyazono, Ki., Tanokura, M., Sawasaki, T., Endo, Y., Kobayashi, I. (2010). Cell-Free Protein Synthesis for Structure Determination by X-ray Crystallography. In: Endo, Y., Takai, K., Ueda, T. (eds) Cell-Free Protein Production. Methods in Molecular Biology, vol 607. Humana Press. https://doi.org/10.1007/978-1-60327-331-2_13
Download citation
DOI: https://doi.org/10.1007/978-1-60327-331-2_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-330-5
Online ISBN: 978-1-60327-331-2
eBook Packages: Springer Protocols