Cell-Free Protein Synthesis for Structure Determination by X-ray Crystallography
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.
Key wordsStructural genomics Toxic protein Wheat germ Restriction enzyme Selenomethionine SAD
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.
- 4.Kobayashi, I. (2004) Restriction – modification systems as minimal forms of life. In Restriction Endonucleases (Pingoud, A., ed.), Berlin, Springer. pp. 19–62.Google Scholar
- 7.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.CrossRefPubMedGoogle Scholar
- 9.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.CrossRefPubMedGoogle Scholar
- 14.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.Google Scholar
- 18.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.CrossRefPubMedGoogle Scholar
- 22.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.Google Scholar