Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-06-16
2003-04-08
Monshipouri, M. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S350000, C435S252300, C435S325000, C435S320100
Reexamination Certificate
active
06544766
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the production and purification of human kinesins, preferably functional, using prokaryotic systems and to human kinesins isolated from bacterial systems.
BACKGROUND OF THE INVENTION
Cancer is the second-leading cause of death in industrialized nations. Effective therapeutics include the taxanes and vinca alkyloids, agents which act on microtubules. Microtubules are the primary structural element of the mitotic spindle. The mitotic spindle is responsible for distribution of replicate copies of the genome to each of the two daughter cells that result from cell division. It is presumed that it is the disruption of the mitotic spindle by these drugs that results in inhibition of cancer cell division, and also induction of cancer cell death. However, microtubules also form other types of cellular structures, including tracks for intracellular transport in nerve processes. Therefore, the taxanes have side effects that limit their usefulness.
Mitotic kinesins are enzymes essential for assembly and function of the mitotic spindle, but are not generally part of other microtubule structures, such as nerve processes. Mitotic kinesins play essential roles during all phases of mitosis. These enzymes are “molecular motors” that translate energy released by hydrolysis of ATP into mechanical force which drives the directional movement of cellular cargoes along microtubules. The catalytic domain sufficient for this task is a compact structure of approximately 340 amino acids. During mitosis, kinesins organize microtubules into the bipolar structure that is the mitotic spindle. Kinesins mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle associated with specific phases of mitosis. Experimental perturbation of mitotic kinesin function causes malformation or dysfunction of the mitotic spindle, frequently resulting in cell cycle arrest. From both the biological and enzymatic perspectives, these enzymes are attractive targets for the discovery and development of novel anti-mitotic chemotherapeutics.
A number of kinesins have been described in the art. However, there still exists a need for kinesins which can be easily produced in large quantities. In particular, human mitotic kinesins isolated and purified from a bacterial source are desirable.
Among the kinesins which have been identified is chromokinesin. Chromokinesin is a kinesin localized to mitotic chromatin and contributes to prometaphase chromosome alignment; it is expressed primarily in proliferating tissues and is enriched in mitotic compared to interphase cells. Perturbation of a Xenopus chromokinesin causes gross defects in mitotic spindle formation, including dissociation of chromosomes from spindle microtubules, multipolar spindles, misaligned chromosomes and failure of cytokinesis. Cloning of chicken (Wang and Adler, J. Cell Biol., 128:761-8 (1995)) and human (Oh, et al., direct GenBank submission without corresponding publication, submitted Jun. 11, 1998 by Molecular Biology, Institute for Medical Sciences, San5 Wonchon Paldal, Suwon, Kyongki 442-749, Korea) chromokinesin homologs have been reported. The mouse homolog of chromokinesin, Kif4, has been expressed in Sf9 cells (bacculovirus vector) and has been reported to have motility and ATPase activity (Sekine, et al., J. Cell Biol., 127-187-201 (1994)). This same study speculated that Kif4 may participate in the transport of membraneous organelles in neuronal and other cell types.
Another kinesin reported to be associated with chromosomes is Kid. Kid is reported as unrelated to other known kinesins. The C-terminal 260 amino acids of Kid expressed in bacteria and purified reportedly binds directly to DNA in vitro. The same study reported that when fused to a transcriptional activation domain and co-transfected into mammalian cells with a reporter construct this domain can stimulate expression from the promoter on the co-transfected construct in living cells. Tokai, et al., EMBO J., 15(3):457-467 (1996). This study further reports that the amino-terminal 470 amino acids of Kid, which includes the motor domain, has been expressed in bacteria fused to glutathione-S-transferase, binds to microtubues and exhibits microtubule-stimulated ATPase activity. Kid is expressed in all human cell lines that have been examined, and is most abundant in adult human speen, thymus and testis as well as fetal liver and kidney. In cultured human cells, Kid is reportedly found associated with chromatin throughout mitosis, showing some enrichment at kinetochores.
Another mitotic kinesin of interest is MKLP1 which localizes to microtubules of the spindle midzone throughout mitosis. In vitro MKLP1 can slide antiparallel microtubules relative to each other. Microinjection of antibody directed against MKLP1 into mammalian cells induces mitotic arrest with subtle defects in microtubule organization. Genetic data from both Drosophila and
C. elegans
clearly show that MKLP1 homologues are required for organization of the interzonal microtubules of the anaphase spindle and for formation of a functional contractile ring. MKLP1 associates with a kinase of the polo family in both Drosophila and mammals. Cloning of human (Nislow, et al., Nature, 359:543-7 (1992)), hamster (Kuriyama, et al., J. Cell Sci., 107(Pt 12):3485-99 (1994)), Drosophila (Adams, et al., Gene Dev., 12:1483-94 (1998)), and C. elegans (Raich, et al., Mol. Biol. Cell, 9:2037-49 (1998)) homologs of MKLP1 have been reported. Nislow, et al., supra, reported on expressed full-length human MKLP1 in bacteria, however there was relatively poor expression, and the polypeptide was not purified. Using this crude bacterial lysate, microtubule bundling and sliding activity were reported on. Kuriyama, et al., supra, reported on expressed hamster MKLP1 in Sf9 cells (baculovirus vector), but the protein was not purified.
KSP is also of interest. KSP belongs to an evolutionarily conserved kinesin subfamily of plus end-directed microtubule motors that assemble into bipolar homotetramers consisting of antiparallel homodimers. During mitosis KSP associates with microtubules of the mitotic spindle. Microinjection of antibody directed against KSP into human cells prevents spindle pole separation during prometaphase, giving rise to monopolar spindles and causing mitotic arrest. KSP and related kinesins bundle antiparallel microtubules and slide them relative to one another, thus forcing the two spindle poles apart. KSP may also mediate in anaphase B spindle elongation and focussing of microtubules at the spindle pole. Cloning of human (Blangy, et al., Cell, 83:1159-69 (1995); Whitehead, et al., direct GenBank submission without corresponding publication, submitted September 29, 1995 by Medical Biochemistry, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta TN 4N1, Canada), Drosophila (Heck, et al., J Cell Biol, 123:665-79 (1993)) and Xenopus (Le Guellec, et al., Mol. Cell Biol., 11(6):3395-8(1991)) homologs of KSP have been reported. Drosophila KLP61F/KRP130 has reportedly been purified in native form (Cole, et al.,
J. Biol. Chem
., 269(37):22913-6 (1994)), expressed in
E. coli
, (Barton, et al., Mol. Biol. Cell, 6:1563-74 (1995)) and reported to have motility and ATPase activities (Cole, et al., supra; Barton, et al., supra). Xenopus Eg5 was expressed in
E. coli
and reported to possess motility activity (Sawin, et al., Nature, 359:540-3 (1992); Lockhart and Cross, Biochemistry, 35(7):2365-73 (1996); Crevel, et al, J. Mol. Biol., 273:160-170 (1997) and ATPase activity (Lockhart and Cross, supra; Crevel et al., supra).
CENP-E, also of interest, is a plus end-directed microtubule motor essential for achieving metaphase chromosome alignment. CENP-E accumulates during interphase and is degraded following completion of mitosis. Microinjection of antibody directed against CENP-E or overexpression of a dominant negative mutant of CENP-E causes mitotic arrest with prometaphase chromosomes scattered on a bipolar spindle. The tail domain of CENP-E mediate
Beraud Christophe
Ohashi Cara
Sakowicz Roman
Vaisberg Eugeni
Wood Ken
Beyer Weaver & Thomas LLP
Cytokinetics Inc.
Monshipouri M.
Stevens, Esq. Lauren L.
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