{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T06:02:08Z","timestamp":1791352928030,"version":"4.3.1"},"reference-count":28,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5364","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,5]]},"abstract":"<jats:p>\n            Anthrax lethal toxin, produced by the bacterium\n            <jats:italic>Bacillus anthracis,<\/jats:italic>\n            is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.\n          <\/jats:p>","DOI":"10.1126\/science.280.5364.734","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:35:26Z","timestamp":1027762526000},"page":"734-737","source":"Crossref","is-referenced-by-count":883,"title":["Proteolytic Inactivation of MAP-Kinase-Kinase by Anthrax Lethal Factor"],"prefix":"10.1126","volume":"280","author":[{"given":"Nicholas S.","family":"Duesbery","sequence":"first","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Craig P.","family":"Webb","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen H.","family":"Leppla","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valery M.","family":"Gordon","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kurt R.","family":"Klimpel","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Terry D.","family":"Copeland","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie G.","family":"Ahn","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marianne K.","family":"Oskarsson","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenji","family":"Fukasawa","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken D.","family":"Paull","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George F.","family":"Vande Woude","sequence":"additional","affiliation":[{"name":"N. S. Duesbery, C. P. Webb, T. D. Copeland, M. K. Oskarsson, G. F. Vande Woude, Advanced BioScience Laboratories\u2013Basic Research Program, National Cancer Institute\u2013Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702, USA."},{"name":"S. H. Leppla, V. M. Gordon, K. R. Klimpel, National Institute of Dental Research\u2013National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA."},{"name":"N. G. Ahn, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Campus Box 215, Boulder, CO 80309, USA."},{"name":"K. Fukasawa, Department of Cell Biology, University of Cincinnati, College of Medicine, P.O. Box 670521, Cincinnati, OH 45267, USA."},{"name":"K. D. Paull, Division of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_2_2_2","unstructured":"S. H. Leppla in Handbook of Natural Toxins J. Moss B. Iglewski M. Vaughan A. T. Tu Eds. (Dekker New York 1995) vol. 8 pp. 543\u2013572."},{"key":"e_1_3_2_3_2","doi-asserted-by":"crossref","first-page":"20607","DOI":"10.1016\/S0021-9258(17)32036-7","volume":"269","author":"Milne J. C.","year":"1994","unstructured":"V. M. Gordon, S. H. Leppla, E. L. Hewlett, Infect. Immun. 56, 1066 (1988); Milne J. C., Furlong D., Hanna P. C., Wall J. S., Collier R. J., J. Biol. Chem. 269, 20607 (1994).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.79.10.3162"},{"key":"e_1_3_2_5_2","doi-asserted-by":"crossref","first-page":"3472","DOI":"10.1128\/iai.59.10.3472-3477.1991","volume":"59","author":"Pezard C.","year":"1991","unstructured":"Pezard C., Berche P., Mock M., Infect. Immun.59, 3472 (1991).","journal-title":"Infect. Immun."},{"key":"e_1_3_2_6_2","first-page":"761","volume":"45","author":"Ezzell J. W.","year":"1984","unstructured":"Ezzell J. W., Ivins B. E., Leppla S. H., ibid45, 761 (1984).","journal-title":"ibid"},{"key":"e_1_3_2_7_2","doi-asserted-by":"crossref","first-page":"7123","DOI":"10.1016\/S0021-9258(17)38364-3","volume":"261","author":"Friedlander A. M.","year":"1986","unstructured":"Friedlander A. M., J. Biol. Chem.261, 7123 (1986).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_2_8_2","unstructured":"Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_2_9_2","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1111\/j.1365-2958.1994.tb00500.x","volume":"13","author":"Klimpel K. R.","year":"1994","unstructured":"Klimpel K. R., Arora N., Leppla S. H., Mol. Microbiol.13, 1093 (1994).","journal-title":"Mol. Microbiol."},{"key":"e_1_3_2_10_2","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1042\/bj3200687","volume":"320","author":"Menard A.","year":"1996","unstructured":"S. K. Kochi, G. Schiavo, M. Mock, C. Montecucco, FEMS Microbiol. Lett. 124, 343 (1994); Menard A., Papini E., Mock M., Montecucco C., Biochem. J. 320, 687 (1996).","journal-title":"Biochem. J."},{"key":"e_1_3_2_11_2","first-page":"5211","volume":"56","author":"Koo H.-M.","year":"1996","unstructured":"J. N. Weinstein, et al., Science 275, 343 (1997); Koo H.-M., et al., Cancer Res. 56, 5211 (1996).","journal-title":"Cancer Res."},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.92.17.7686"},{"key":"e_1_3_2_13_2","doi-asserted-by":"crossref","first-page":"584","DOI":"10.1016\/S0076-6879(97)83046-7","volume":"283","author":"Murakami M. S.","year":"1997","unstructured":"For reviews, see T. Moriguchi, Y. Gotoh, E. Nishida, Adv. Pharmacol. 36, 121 (1996); Murakami M. S., Vande Woude G. F., Methods Enzymol. 283, 584 (1997); W. T. Matten and G. F. Vande Woude, Sem. Dev. Biol. 5, 173 (1994).","journal-title":"Methods Enzymol."},{"key":"e_1_3_2_14_2","unstructured":"N. S. Duesbery and G. F. Vande Woude unpublished data."},{"key":"e_1_3_2_15_2","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1038\/349132a0","volume":"349","author":"Glotzer M.","year":"1991","unstructured":"Glotzer M., Murray A. W., Kirschner M. W., Nature349, 132 (1991).","journal-title":"Nature"},{"key":"e_1_3_2_16_2","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1016\/0092-8674(95)90507-3","volume":"80","author":"White M. A.","year":"1995","unstructured":"White M. A., et al., Cell80, 533 (1995).","journal-title":"Cell"},{"key":"e_1_3_2_17_2","doi-asserted-by":"crossref","first-page":"29039","DOI":"10.1016\/S0021-9258(19)62010-7","volume":"269","author":"Singh Y.","year":"1994","unstructured":"Singh Y., Klimpel K. R., Arora N., Sharma M., Leppla S. H., J. Biol. Chem.269, 29039 (1994).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.8052857"},{"key":"e_1_3_2_19_2","unstructured":"Wild-type MAPKK1 (0.5 \u03bcg) was incubated with LF (0.2 \u03bcg) as described in Fig.  2 A for 30 min at 30\u00b0C. Proteins were separated by SDS\u2013polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membrane in CAPS transfer buffer {3-[cyclohexylamino]-1 propanesulfonic acid (10 mM pH 11) and 10% methanol} at 300 mA constant current for 30 min. After transfer membranes were quickly stained with Ponceau S solution (0.1% Ponceau S and 5% acetic acid) and rinsed with distilled deionized water. The appropriate band was cut from the membrane and subjected to automated Edman degradation in an Applied Biosystems 477A gas-phase sequencer and phenylthiohydantoin derivatives were identified online with a 120 phenylthiohydantoin analyzer."},{"key":"e_1_3_2_20_2","doi-asserted-by":"crossref","first-page":"15529","DOI":"10.1021\/bi961854s","volume":"35","author":"Mansour S. J.","year":"1996","unstructured":"Mansour S. J., Candia J. M., Matsuura J. E., Manning M. C., Ahn N. G., Biochemistry35, 15529 (1996).","journal-title":"Biochemistry"},{"key":"e_1_3_2_21_2","doi-asserted-by":"crossref","first-page":"1901","DOI":"10.1093\/emboj\/16.8.1901","volume":"16","author":"Fukuda M.","year":"1997","unstructured":"Fukuda M., Gotoh Y., Nishida E., EMBO J.16, 1901 (1997).","journal-title":"EMBO J."},{"key":"e_1_3_2_22_2","unstructured":"S. E. Hammond and P. C. Hanna abstract presented at the 97th General Meeting of the American Society of Microbiology Miami Beach FL (1997)."},{"key":"e_1_3_2_23_2","doi-asserted-by":"crossref","first-page":"9165","DOI":"10.1073\/pnas.94.17.9165","volume":"94","author":"Duesbery N. S.","year":"1997","unstructured":"Duesbery N. S., et al., Proc. Natl. Acad. Sci. U.S.A.94, 9165 (1997).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_2_24_2","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/S0076-6879(88)65019-1","volume":"165","author":"Leppla S. H.","year":"1988","unstructured":"Leppla S. H., Methods Enzymol.165, 103 (1988).","journal-title":"Methods Enzymol."},{"key":"e_1_3_2_25_2","doi-asserted-by":"crossref","first-page":"3963","DOI":"10.1002\/j.1460-2075.1992.tb05490.x","volume":"11","author":"Shibuya E. K.","year":"1992","unstructured":"Shibuya E. K., Boulton T. G., Cobb M. H., Ruderman J. V., EMBO J.11, 3963 (1992).","journal-title":"EMBO J."},{"key":"e_1_3_2_26_2","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1038\/355649a0","volume":"355","author":"Yew N.","year":"1992","unstructured":"Yew N., Mellini M. L., Vande Woude G. F., Nature355, 649 (1992).","journal-title":"Nature"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)53507-9"},{"key":"e_1_3_2_28_2","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1093\/oxfordjournals.jbchem.a124524","volume":"116","author":"Mansour S. J.","year":"1994","unstructured":"A MAPKK1 construct containing a seven\u2013amino acid NH2-terminal deletion was generated by polymerase chain reaction using primer pairs A and B and pKH-1, encoding His6-tagged human wild-type MAPKK1 [Mansour S. J., et al., J. Biochem. 116, 304 (1994)], as a template. The amplified fragment was digested with Xho I and Hind III and introduced into pRSETA (Invitrogen). The primers used were 5\u2032-AAGAAGCCGCTCGAGATCCAGCTGAACCCG-3\u2032 (A) and 5\u2032-CTTTGTTAGCAGCCGGATCAAGCTTCGAAT-3\u2032 (B). Constructs containing MAPKK1 mutations Pro5 or 7 to Ala were generated by introducing the mutations into pKH-1 with the use of the Quickchange Site-Directed Mutagenesis Kit (Stratagene). The primers used were 5\u2032-ATGCCCAAGAAGAAGGCGACGCCCATCCAGCTGAACCCG-3\u2032 for P5A, 5\u2032-CCCAAGAAGAAGCCGACGGCCATCCAGCTGAACCCGGCC-3\u2032 for P7A, and their respective complementary sequences. The sequences of these constructs were confirmed by direct DNA sequencing. To produce recombinant protein, Escherichia coli strain TOP10F\u2032 was transformed with these constructs. Cultures were grown at 37\u00b0C to an optical density at 600 nm of 0.3 in 750 ml of SOB and ampicillin (100 \u03bcg\/ml) and then incubated for 1 hour in the presence of 1 mM isopropyl-\u03b2- d -thiogalactopyranoside, at which point M13\/T7 helper phage was added to a concentration of three plaque-forming units per cell. Cells were harvested by centrifugation 3 hours later, resuspended in 36 ml of extraction buffer (EB) [50 mM potassium phosphate (pH 8.0); 10% (v\/v) glycerol; 0.25% (v\/v) Tween-20; and 1 mM dithiothreitol (DTT), containing protease inhibitors (EDTA-free Complete tablets; Boehringer Mannheim)], and lysed by four sonication\/freeze\/thaw cycles. Soluble protein was separated by centrifugation (at 14,000 rpm for 30 min with an SS-34 rotor), and the supernatant was sterile-filtered and adjusted to 1% Tween-20, 1 M KCl, and 20% glycerol. The supernatant was then loaded on a Ni2+-charged HiTrap Chelating column (Pharmacia), washed with EB containing 10 mM imidazole, and eluted with EB containing 200 mM imidazole. Fractions containing recombinant protein were then pooled and desalted and concentrated in buffer containing 50 mM tris (pH 8.0), 0.1 M NaCl, 10% glycerol, 0.01% Triton X-100, and 1 mM DTT by centrifugation with a Centriprep-30 centrifugal concentrator (Amicon, Beverely, MA, XX).","journal-title":"J. Biochem."},{"key":"e_1_3_2_29_2","unstructured":"This paper is dedicated to the memory of Ken Paull whose untimely death is a significant loss to NCI and the cancer research community. The authors thank A. Cline and R. Frederickson for preparing the manuscript L. Miller for technical assistance H.-M. Koo for helpful discussions M. Jeffers and S. Koochekpour for critical comments on the manuscript and A. Murray for providing \u039490 cyclin. This research was sponsored in part by NCI Department of Health and Human Services under contract with Advanced BioScience Laboratories. By acceptance of this article the publisher or recipient acknowledges the right of the U.S. government and its contractors and agents to retain a nonexclusive royalty-free license in and to any copyright covering the article."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.280.5364.734","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:16:14Z","timestamp":1705122974000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.280.5364.734"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,5]]},"references-count":28,"journal-issue":{"issue":"5364","published-print":{"date-parts":[[1998,5]]}},"alternative-id":["10.1126\/science.280.5364.734"],"URL":"https:\/\/doi.org\/10.1126\/science.280.5364.734","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,5]]}}}