EP1451204A2 - Motorische proteine und verfahren zu deren verwendung - Google Patents

Motorische proteine und verfahren zu deren verwendung

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Publication number
EP1451204A2
EP1451204A2 EP01946001A EP01946001A EP1451204A2 EP 1451204 A2 EP1451204 A2 EP 1451204A2 EP 01946001 A EP01946001 A EP 01946001A EP 01946001 A EP01946001 A EP 01946001A EP 1451204 A2 EP1451204 A2 EP 1451204A2
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European Patent Office
Prior art keywords
protein
hskifl
nucleic acid
seq
sequence
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English (en)
French (fr)
Inventor
Christophe Beraud
Richard Freedman
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Cytokinetics Inc
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Cytokinetics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention provides isolated nucleic acid and amino acid sequences of HsKifl3a, methods of detecting HsKifl3a and screening for HsKifl3a modulators using biologically active HsKifl3a, and kits for screening for HsKifl3a modulators.
  • the kinesin super/family is an extended family of related microtubule motor proteins. It can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. This family is exemplified by "true" kinesin, which was first isolated from the axoplasm of squid, where it is believed to play a role in anterograde axonal transport of vesicles and organelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)). Kinesin uses ATP to generate force and directional movement associated with microtubules (from the minus to the plus end of the microtubule, hence it is a "plus-end directed" motor).
  • Kinesin superfamily members are defined by a kinesin-like motor domain that is about 340 amino acids in size and typically shares approximately 35-45% identity with the "true" kinesin motor domain.
  • a variable cargo-binding domain bestows a variety of activities on different family members.
  • KEF Keratin Family proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. Nakagawa et al. (1997) Proc. Natl. Acad. Sci. USA 94:9654-9659 have reported on the identification of Kifl3a in the mouse. They found that the KLF13a transcript was overall ubiquitous, but dominant in brain, lung, heart, kidney, and testis. However, only a partial sequence of the motor domain was reported.
  • the present invention is based on the discovery of a new human kinesin motor protein, HsKifl 3 a, the polynucleotide encoding HsKifl 3 a, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, neurological disorders, and disorders of vesicular transport.
  • the invention provides an isolated nucleic acid sequence encoding a kinesin superfamily motor protein, wherein the motor protein has the following properties: (i) the protein's activity includes microtubule stimulated ATPase activity; and (ii) the protein has a sequence that has greater than 70% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. In one embodiment, the protein further specifically binds to polyclonal antibodies raised against SEQ ID NO:2.
  • the nucleic acid encodes HsKifl 3 a or a fragment thereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2, or SEQ LD NO:4. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3.
  • the nucleic acid comprises a sequence, which encodes an amino acid sequence that has one or more of the following characteristics: greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 85% or 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
  • the nucleic acid comprises a sequence that has one or more of the following characteristics: greater than 55 or 60% sequence identity with SEQ ID NO:l, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO: 1.
  • the nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence or complementary sequence ofSEQ TD NO:l.
  • the invention provides an expression vector comprising a nucleic acid encoding a kinesin superfamily motor protein, wherein the motor protein has the following properties: (i) the protein's activity includes microtubule stimulated ATPase activity; and (ii) the protein has a sequence that has greater than 70% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm.
  • the invention further provides a host cell transfected with the vector.
  • the invention provides an isolated kinesin superfamily motor protein, wherein the protein has one or more of the properties described above.
  • the protein specifically binds to polyclonal antibodies generated against a motor domain, tail domain or other fragment of HsKifl3a.
  • the protein comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • the protein provided herein comprises an amino acid sequence that has one or more of the following characteristics: greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
  • the invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof and more particularly, the motor domain of the amino acid sequence of SEQ ID NO:2.
  • the invention provides a method for screening for modulators of HsKifl 3 a, the method comprising the steps of: (i) contacting biologically active motor protein having at least one of properties described above, with at least one candidate agent at a test and control concentration and detecting whether a change in the activity of the motor protein occurs between the test and control concentration, wherein a change indicates a modulator of the motor protein.
  • the activity is selected from the group consisting of microtubule stimulated ATPase activity and microtubule binding activity.
  • the method further comprises the step of isolating biologically active HsKifl 3 a from a cell sample.
  • the biologically active HsKifl 3 a is recombinant.
  • the mvention provides a kit for screening for modulators of HsKifl 3 a, the kit comprising; (i) a container holding biologically active HsKifl 3 a; and (ii) instructions for assaying for HsKifl 3 a activity, wherein the HsKifl 3 a activity is microtubule binding activity or microtubule stimulated ATPase activity.
  • Figures 1A, IB, 1C, and ID show an embodiment of a nucleic acid sequence encoding HsKifl 3 a.
  • Figures 2 A and 2B shows the predicted amino acid sequence of HsKifl 3 a.
  • Figure 3 shows an embodiment of a nucleic acid sequence encoding HsKifl 3a motor domain fragment.
  • Figure 4 shows the predicted amino acid sequence of HsKifl 3 a motor domain fragment.
  • an "agricultural compound” as used herein refers to a chemical or biological compound that has utility in agriculture and functions to foster food or fiber crop protection or yield improvement.
  • one such compound may serve as a herbicide to selectively control weeds, as a fungicide to control the spreading of plant diseases, as an insecticide to ward off and destroy insect and mite pests.
  • one such compound may demonstrate utility in seed treatment to improve the growth environment of a germinating seed, seedling or young plant as a plant regulator or activator.
  • Amplification primers are oligonucleotides comprising either natural or analogue nucleotides that can serve as the basis for the amplification of a select nucleic acid sequence. They include, e.g., polymerase chain reaction primers and ligase chain reaction oligonucleotides.
  • Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulm genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the term antibody also includes antibody f agments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
  • an “anti-HsKifl3a” antibody is an antibody or antibody f agment that specifically binds a polypeptide encoded by the HsKifl3a gene, cDNA, or a subsequence thereof.
  • Biologically active HsKifl 3a refers to HsKifl 3a that has microtubule stimulated ATPase activity, as tested, e.g., in an ATPase assay. Biological activity can also be demonstrated in a microtubule gliding assay or a microtubule binding assay.
  • ATPase activity refers to ability to hydrolyze ATP.
  • Bio sample as used herein is a sample of biological tissue or fluid that contains HsKifl 3 a or a fragment thereof or nucleic acid encoding a HsKifl 3 a protein or a fragment thereof. Biological samples may also include sections of tissues such as frozen sections taken for histologic purposes.
  • a biological sample comprises at least one cell, preferably plant or vertebrate. Embodiments include cells obtained from a eukaryotic organism, preferably eukaryotes such as fungi, plants, insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
  • a “comparison window' includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 25 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra.).
  • HSPs high scoring sequence pairs
  • M return score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the default parameters of the BLAST programs are suitable.
  • the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.
  • the TBLATN program uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix, (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. NatT. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). As a general rule, PileUp can align up to 500 sequences, with any single sequence in the final alignment restricted to a maximum length of 7,000 characters.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. A series of such pairwise alignments that includes increasingly dissimilar sequences and clusters of sequences at each iteration produces the final alignment.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCT all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each degenerate codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Cytoskeletal component denotes any molecule that is found in association with the cellular cytoskeleton, that plays a role in maintaining or regulating the structural integrity of the cytoskeleton, or that mediates or regulates motile events mediated by the cytoskeleton.
  • cytoskeletal polymers e.g., actin filaments, microtubules, myosin fragments, filaments
  • molecular motors e.g., cytoskeleton associated regulatory proteins
  • Cytoskeletal function refers to biological roles of the cytoskeleton, including but not limited to the providing of structural organization (e.g., microvilli, mitotic spindle) and the mediation of motile events within the cell (e.g., muscle contraction, mitotic contractile ring, pseudopodal movement, active cell surface deformations, vesicle formation and translocation.)
  • structural organization e.g., microvilli, mitotic spindle
  • motile events within the cell e.g., muscle contraction, mitotic contractile ring, pseudopodal movement, active cell surface deformations, vesicle formation and translocation.
  • a “diagnostic” as used herein is a compound, method, system, or device that assists in the identification and characterization of a health or disease state.
  • the diagnostic can be used in standard assays as is known in the art.
  • An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • HsKifl3a is a member of the kinesin superfamily of microtubule motor proteins.
  • nucleic acid when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid.
  • High stringency conditions may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll/0.1% polyvinylpyrrolidone / 50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,
  • High throughput screening refers to an assay that provides for multiple candidate agents or samples to be screened simultaneously.
  • examples of such assays may include the use of microtiter plates and nucleic acid or protein arrays which are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • host cell is meant a cell that contains an expression vector and supports the replication or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, or plant cells. Both primary cells and tissue cultures cells are included in this definition.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.05 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50 or 100 amino acids in length.
  • This definition also refers to the complement of a test sequence, provided that the test sequence has a designated or substantial identity to a reference sequence.
  • the percent identity exists over a region of the sequence that is at least about 25 nucleotides in length, more preferably over a region that is 50 or 100 nucleotides in length.
  • sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g,. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. The scoring of conservative substitutions can be calculated according to, e.g., the algorithm of Meyers & Millers, Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • isolated refers to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In an isolated HsKifl 3a, nucleic acid is separated from open reading frames that flank the HsKifl 3a gene and encode proteins other than HsKifl 3 a.
  • purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • label is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include fluorescent proteins such as green, yellow, red or blue fluorescent proteins, radioisotopes such as P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used m an ELIS A), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptide of SEQ ID NO:2 can be made detectable, e.g., by incorporating a radio-label into the peptide, and used to detect antibodies specifically reactive with the peptide).
  • a "labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker, or through ionic, van der Waals, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
  • Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • Modulators refer to modulatory molecules identified using in vitro and in vivo assays for HsKifl 3 a activity. Such assays include ATPase activity, microtubule gliding, microtubule depolymerizing activity, and binding activity such as microtubule binding activity or binding of nucleotide analogs. Samples or assays that are treated with a candidate agent at a test and control concentration. The control concentration can be zero. If there is a change in HsKifl 3 a activity between the two concentrations, this change indicates the identification of a modulator.
  • a change in activity which can be an increase or decrease, is preferably a change of at least 20% to 50%, more preferably by at least 50% to 75%, more preferably at least 75% to 100%, and more preferably 150% to 200%, and most preferably is a change of at least 2 to 10 fold compared to a control. Additionally, a change can be indicated by a change in binding specificity or substrate.
  • Molecular motor refers to a molecule that utilizes chemical energy to generate mechanical force. According to one embodiment, the molecular motor drives the motile properties of the cytoskeleton.
  • motor domain refers to the domain of HsKifl 3 a that confers membership in the kinesin superfamily of motor proteins through a sequence identity of approximately 35-45% identity to the motor domain of true kinesin.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608 (1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98 (1994)).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • Nucleic acid probe or oligonucleotide is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, C, or T) or modified bases.
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labeled with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
  • polypeptide polypeptide
  • peptide protein
  • a HsKifl 3a polypeptide comprises a polypeptide demonstrated to have at least microtubule stimulated ATPase activity and that binds to an antibody generated against HsKifl 3 a.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes, the one-letter symbols recommended by the IUPAC-IUB Biochemical
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA box element.
  • a promoter also optionally includes distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription.
  • a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is under environmental or developmental regulation.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
  • the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding moieties typically have an affinity for one another of at least 10 6 M "1 .
  • affinities such as 10 , 10 , 10 or 10 M " .
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to HsKifl 3 a with the amino acid sequence encoded in SEQ ID NO:2 can be selected to obtain only those antibodies that are specifically immunoreactive with HsKifl 3 a and not with other proteins, except for polymorphic variants, orthologs, alleles, and closely related homologues of HsKifl 3 a. This selection may be achieved by subtracting out antibodies that cross react with molecules, for example, such as C. elegans unc-104 and human KiflA.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid- phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • Test composition refers to a molecule or composition whose effect on the interaction between two or more cytoskeletal components it is desired to assay.
  • the “test composition” can be any molecule or mixture of molecules, optionally in a carrier.
  • a “therapeutic” as used herein refers to a compound that is believed to be capable of modulating the cytoskeletal system in vivo which can have application in both human and animal disease.
  • Modulation of the cytoskeletal system would be desirable in a number of conditions including, but not limited to: abnormal stimulation of endothelial cells (e.g., atherosclerosis), solid and hematopoietic tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, pyogenic granulomas, vascular malfunctions, abnormal would healing, inflammatory and immune disorders such as rheumatoid arthritis, Behcet's disease, gout or gouty arthritis, abnormal angiogenesis accompanying: rheumatoid arthritis, psoriasis, diabetic retinopathy, and other ocular angiogenic disease such as, macular degeneration, corneal graft rejection, corneal overgrowth, glaucoma, Osier Webber syndrome, cardiovascular diseases such as hypertension, cardiac ischemia and systolic and diastolic dysfunction and fungal diseases such as aspergillosis, candid
  • the present invention provides for the first time a nucleic acid encoding HsKifl3a.
  • This protein is a member of the kinesin superfamily of motor proteins and demonstrates microtubule stimulated ATPase activity.
  • HsKifl 3a can be defined by having at least one or preferably more than one of the following functional and structural characteristics. Functionally, HsKifl3a has a microtubule-stimulated ATPase activity, and microtubule motor activity that is ATP dependent. HsKifl 3 a activity can also be described in terms of its ability to bind microtubules.
  • the novel nucleotides sequences provided herein encode HsKifl 3 a or fragments thereof.
  • the nucleic acids provided herein are defined by the novel proteins provided herein.
  • the protein provided herein comprises an amino acid sequence which has one or more of the following characteristics: greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
  • the sequence identity can be the same percentages or slightly lower due to the degeneracy in the genetic code.
  • the invention also includes fragments of the nucleotide sequence shown in Fig.
  • nucleotide sequences shown in the Figures or sequence can refer to the sequence shown, its perfect complement or a duplex of the two strands.
  • HsKifl 3 a comprises an amino-terminal, kinesin-like microtubule "motor" domain (see Fig. 4).
  • Portions of the HsKifl 3 a nucleotide sequence may be used to identify polymorphic variants, orthologs, alleles, and homologues of HsKifl3a. This identification can be made in vitro, e.g., under stringent hybridization conditions and sequencing, or by using the sequence information in a computer system for comparison with other nucleotide sequences. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below, with PILEUP as a preferred algorithm.
  • any of the peptides provided herein can be routinely confirmed by the assays provided herein such as those which assay ATPase activity or microtubule binding activity.
  • polymorphic variants, alleles, and orthologs, homologues of HsKifl 3 a are confirmed by using a ATPase or microtubule binding assays as known in the art.
  • HsKifl 3 a The isolation of biologically active HsKifl 3 a for the first time provides a means for assaying for modulators of this kinesin superfamily protein.
  • Biologically active HsKifl3a is useful for identifying modulators of HsKifl3a or fragments thereof and kinesin superfamily members using in vitro assays such as microtubule gliding assays, ATPase assays (Kodama et al, J. Biochem. 99:1465-1472 (1986); Stewart et al, Proc. Nat'lAcad. Sci. USA 90:5209-5213 (1993)), and binding assays including microtubule binding assays (Vale et al, Cell 42:39-50 (1985)).
  • in vitro assays such as microtubule gliding assays, ATPase assays (Kodama et al, J. Biochem. 99:1465-1472 (1986
  • HsKifl3a Some portions or fragments of HsKifl3a include at least 7, 10, 15, 20, 35, 50, 100, 250, 300, 350, 500, or 1000 contiguous amino acids from the sequence shown in Fig. 2. Some fragments contain fewer than 1000, 500, 250, 100 or 50 contiguous amino acids from the sequence shown in Fig. 2. For example, exemplary fragments include fragments having 15-50 amino acids or 100-500 amino acids. Some fragments include a motor domain. Such fragments typically include the span from amino acid residue 1 to 352 of Fig. 2 or an active portion thereof. Some fragments include a ligand binding domain of HsKifl 3 a.
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • bp base pairs
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from mass spectroscopy, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphorarnidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Nan Devanter et al, Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 225:137-149 (1983).
  • sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al, Gene 16:21-26 (1981).
  • nucleic acid sequences encoding HsKifl 3 a and related nucleic acid sequence homologs are cloned from cD ⁇ A and genomic D ⁇ A libraries or isolated using amplification techniques with oligonucleotide primers.
  • expression libraries can be used to clone HsKifl 3 a and HsKifl 3 a homologues by detected expressed homologues immunologically with antisera or purified antibodies made against HsKifl 3 a that also recognize and selectively bind to the HsKifl 3 a homologue.
  • amplification techniques using primers can be used to amplify and isolate HsKifl 3 a from D ⁇ A or R ⁇ A.
  • Amplification techniques using degenerate primers can also be used to amplify and isolate HsKifl 3a homologues.
  • Amplification techniques using primers can also be used to isolate a nucleic acid encoding HsKifl 3a. These primers can be used, e.g., to amplify a probe of several hundred nucleotides, which is then used to screen a library for full-length HsKifl 3 a.
  • Appropriate primers and probes for identifying the gene encoding homologues of HsKifl3a in other species are generated from comparisons of the sequences provided herein.
  • antibodies can be used to identify HsKifl3a homologues.
  • antibodies made to the motor domain of HsKifl 3 a or to the whole protein are useful for identifying HsKifl 3a homlogs.
  • HsKifl3a a source that is rich in the mRNA of choice, e.g., HsKifl3a.
  • HsKifl3a mRNA is expressed in testes, bone marrow, fetal liver, brain, salivary gland, heart, thyroid, kidney, adrenal gland, spleen, pancreas, liver, ovary, colon, uterus, lung, prostate, small intestine, skin, muscle, peripheral blood lymphocytes, stomach, and placenta.
  • the mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and introduced into a recombinant host for propagation, screening and cloning.
  • Methods for making and screening cDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25: 263-269); Sambrook et al., supra; Ausubel et al., supra).
  • the DNA is extracted from the tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro. Recombinant phage are analyzed by plaque hybridization as described in Benton & Davis, Science 196:180-182 (1977). Colony hybridization is read out as generally described in Grunstein et al, Proc. Natl. Acad. Sci. USA, 72:3961-3965 (1975).
  • An alternative method of isolating HsKifl 3 a nucleic acid and its homologues combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A guide to Methods and Applications (Innis et al., eds. 1990)).
  • Methods such as polymerase chain reaction and ligase chain reaction can be used to amplify nucleic acid sequences of HsKifl 3 a directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • Degenerate oligonucleotides can be designed to amplify HsKifl 3a homologues using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of HsKifl 3 a encoding mRNA in physiological samples, for nucleic sequencing or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • HsKifl3a can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A + RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, quantitative PCR, and the like.
  • Synthetic oligonucleotides can be used to construct recombinant HsKifl 3 a genes for use as probes or for expression of protein. This method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and nonsense strands of the gene. These DNA fragments are then annealed, ligated and cloned. Alternatively, amplification techniques can be used with precise primers to amplify a specific subsequence of the HsKifl3a gene. The specific subsequence is then ligated into an expression vector.
  • the gene for HsKifl 3 a is typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • the intermediate vectors are typically prokaryote vectors or shuttle vectors.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the HsKifl 3 a encoding nucleic acid in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding HsKifl 3 a and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the nucleic acid sequence encoding HsKifl 3a may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell.
  • Such signal peptides would include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc or histidine tags.
  • Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SN40 vectors, cytomegalovirus vectors, papilloma virus vectors, and vectors derived from Epstein Bar virus.
  • exemplary eukaryotic vectors include pMSG, pAN009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSNE, and any other vector allowing expression of proteins under the direction of the SN40 early promoter, SN40 late promoter, CMN promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
  • markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
  • high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a HsKifl 3 a encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
  • the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
  • the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the D ⁇ A in eukaryotic cells, if necessary.
  • Standard transfection or transformation methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of HsKifl 3 a protein, which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, inMethods in Enzymology, vol. 182 (Deutscher ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bac , 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology, 101:347-362 (Wu et al, eds, 1983).
  • Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing HsKifl 3 a.
  • the transfected cells are cultured under conditions favoring expression of HsKifl 3 a, which is recovered from the culture using standard techniques identified below.
  • HsKifl 3 a Protein Either naturally occurring or recombinant HsKifl 3 a can be purified for use in functional assays.
  • HsKifl3a may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).
  • a preferred method of purification is use of Ni-NTA agarose (Qiagen).
  • HsKifl 3 a A number of procedures can be employed when recombinant HsKifl 3 a is being purified. For example, proteins having established molecular adhesion properties can be reversibly fused to HsKifl 3a. With the appropriate ligand, HsKifl 3 a can be selected adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, HsKifl 3 a could be purified using immunoaffinity columns. A. Purification of HsKifl3a from recombinant bacteria
  • Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with TPTG is a preferred method of expression.
  • Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.
  • HsKifl 3 a it is possible to purify HsKifl 3 a from bacteria periplasm.
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art.
  • the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • HsKifl 3 a or fragments thereof can also be prepared according to the procedures set forth in U.S. Patent Appln. No. 09/295,612, which is incorporated herein for all purposes.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
  • the preferred salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins.
  • the precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
  • the precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.
  • Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
  • the molecular weight of HsKifl 3 a can be used to isolated it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes).
  • membranes of different pore size for example, Amicon or Millipore membranes.
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be cbromatographed as described below.
  • HsKifl 3a can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands.
  • antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
  • HsKifl 3 a genes and gene expression using nucleic acid hybridization technology
  • immunoassays can be used to qualitatively or quantitatively analyze HsKifl 3 a. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988). A. Antibodies to HsKifl3a
  • Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science 246:1275-1281 (1989); Ward et al, Nature 341:544-546 (1989)).
  • Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861 (incorporated by reference for all purposes).
  • Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to HsKifl3a or fragments thereof. Human antibodies against HsKifl3a can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus.
  • Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies.
  • Human polyclonal antibodies can also be provided in the form of serum from human immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using HsKifl 3 a as an affinity reagent.
  • HsKifl 3a comprising immunogens may be used to produce antibodies specifically reactive with HsKifl3a.
  • recombinant HsKifl3a or a antigenic fragment thereof such as the motor domain is isolated as described herein.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Naturally occurring protein may also be used either in pure or impure form.
  • the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
  • mice e.g., BALB/C mice
  • rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to HsKifl 3 a.
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1215-1281 (1989).
  • Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • an immunoassay for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non- HsKifl3a proteins or even other homologous proteins from other organisms (e.g., C. elegans unc-104 or human KiflA), using a competitive binding immunoassay.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a K ⁇ j of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • HsKifl3a can be detected by a variety of immunoassay methods.
  • immunoassay methods see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991).
  • the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio ed., 1980); and Harlow & Lane, supra.
  • Antibodies can be used for treatment or to identify the presence of HsKifl3a having the sequence identity characteristics as described herein. Additionally, antibodies can be used to identify modulators of the interaction between the antibody and HsKifl 3a as further described below. While the following discussion is directed toward the use of antibodies in the use of binding assays, it is understood that the same general assay formats such as those described for "non-competitive" or “competitive” assays can be used with any compound which binds to HsKifl 3 a such as microtubules or the compounds described in Serial No. 60/070,772.
  • HsKifl 3 a is detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • Methods in Cell Biology Volume 37 Antibodies in Cell Biology (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).
  • Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case the HsKifl 3 a or antigenic subsequence thereof).
  • the antibody e.g., anti-HsKifl3a
  • the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
  • the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
  • the labeling agent may be a labeled HsKifl 3 a polypeptide or a labeled anti-HsKifl3a antibody.
  • the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/HsKifl3a complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
  • the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
  • detectable moieties are well known to those skilled in the art.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 4°C to 40°C.
  • Immunoassays for detecting HsKifl3a in samples may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of antigen is directly measured.
  • the anti-HsKifl3a antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture HsKifl 3 a present in the test sample. HsKifl 3 a is thus immobilized is then bound by a labeling agent, such as a second HsKifl 3 a antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
  • the amount of HsKifl 3 a present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) HsKifl 3 a displaced (competed away) from an anti-HsKifl3a antibody by the unknown HsKifl 3a present in a sample.
  • a known amount of HsKifl 3 a is added to a sample and the sample is then contacted with an antibody that specifically binds to HsKifl 3 a.
  • the amount of exogenous HsKifl 3 a bound to the antibody is inversely proportional to the concentration of HsKifl 3 a present in the sample.
  • the antibody is immobilized on a solid substrate.
  • the amount of HsKifl 3 a bound to the antibody may be determined either by measuring the amount of HsKifl3a present in a HsKifl 3 a/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
  • the amount of HsKifl3a may be detected by providing a labeled HsKifl3a molecule.
  • a hapten inhibition assay is another preferred competitive assay.
  • the known HsKifl 3a is immobilized on a solid substrate.
  • a known amount of anti- HsKifl 3 a antibody is added to the sample, and the sample is then contacted with the HsKifl 3 a.
  • the amount of anti-HsKifl3a antibody bound to the known immobilized HsKifl 3 a is inversely proportional to the amount of HsKifl 3 a present in the sample.
  • the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
  • Immunoassays in the competitive binding format can also be used for crossreactivity determinations.
  • a protein at least partially encoded by SEQ ID NO:2 can be immobilized to a solid support.
  • Proteins e.g., C. elegans unc-104 or human Kifl A
  • the ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of HsKifl3a encoded by SEQ ID NO:2 to compete with itself.
  • the percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the added proteins listed above are selected and pooled.
  • the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologues.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps the protein of this invention, to the immunogen protein (i.e., HsKifl3a of SEQ ED NO:2).
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the protein encoded by SEQ ID NO: 2 that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to a HsKifl 3a immunogen.
  • Other assay formats are also bind to the polyclonal antibodies generated to a HsKifl 3a immunogen.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind HsKifl3a.
  • a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
  • the anti-HsKifl3a antibodies specifically bind to the HsKifl 3 a on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-HsKifl3a antibodies.
  • LOA liposome immunoassays
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well- developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.) or other labels that can be detected by mass spectroscopy, NMR spectroscopy, or other analytical means known in the art.
  • magnetic beads e.g., DYNABEADSTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • the ligands and their targets can be used in any suitable combination with antibodies that recognize HsKifl 3 a, or secondary antibodies that recognize anti-HsKifl 3 a.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • HsKifl 3 a The activity of biologically active HsKifl 3 a can be assessed using a variety of in vitro or in vivo assays known in the art, e.g., ATPase, microtubule gliding, and microtubule binding, microtubule depolymerization assays (Kodama et al, J. Biochem. 99: 1465- 1472 (1986); Stewart et al, Proc. Nat'lAcad. Sci. USA 90: 5209-5213 (1993);
  • a preferred assay for high throughput screening is an ATPase assay with colorimetric detection, e.g., malachite green for end-point detection or coupled PK LDH for continuous rate monitoring.
  • An exemplary ATPase activity assay utilizes 0.3 M PCA (perchloric acid) and malachite green reagent (8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-100). To perform the assay, 10 ⁇ L of reaction is quenched in 90 ⁇ L of cold 0.3 M PCA.
  • Phosphate standards are used so data can be converted to mM inorganic phosphate released.
  • 100 ⁇ L of malachite green reagent is added to the to relevant wells in e.g., a microtiter plate. The mixture is developed for 10-15 minutes and the plate is read at an absorbance of 650 nm. If phosphate standards were used, absorbance readings can be converted to mM Pi and plotted over time.
  • ATPase assays known in the art include the luciferase assay.
  • Solution A contains lmM ATP, 2mM phosphoenolpyruvate in a working buffer (25mM Pipes pH 6.8, 2mM MgC12, lmM EGTA, lmM DTT, 5 ⁇ M taxol, 25 ⁇ pm Antifoam, pH 6.8.
  • Solution B contains 0.6mM NADH, 0.2mg/ml BSA, 1:100 dilution of PK/LDH mixture from Sigma, 200 ⁇ g/ml microtubules, lOOnM HsKipl3a (i.e. ⁇ 2.5 ⁇ g/ml).
  • Such assays can be used to test for the activity of HsKifl 3 a isolated from endogenous sources or recombinant sources. Furthermore, such assays can be used to test for modulators of HsKifl 3 a. Modulators can increase or decrease activity of HsKifl 3 a.
  • molecular motor activity is measured by the methods disclosed in Serial No. 09/314,464, filed May 18, 1999, entitled “Compositions and assay utilizing ADP or phosphate for detecting protein modulators", which is incorporated herein by reference in its entirety. More specifically, this assay detects modulators of any aspect of a kinesin motor function ranging from interaction with microtubules to hydrolysis of ATP. ADP or phosphate is used as the readout for protein activity.
  • screens may be designed to first find candidate agents that can bind to HsKifl 3 a proteins, and then these agents, and agents already known to modulate HsKifl3a may be used in assays that evaluate the ability of the candidate agent to modulate activity of HsKifl3a.
  • screens may be designed to find candidate agents that modulate the interaction of HsKifl 3 a protein or a fragment thereof with other proteins.
  • Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion.
  • Such compounds include polypeptides, proteins, nucleic acids, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates.
  • Compounds to be screened can also be obtained from governmental or private sources, including, e.g., the National Cancer Institute's (NCI) Natural Product Repository, Bethesda, MD, the NCI Open Synthetic Compound Collection, Bethesda, MD, NCI's Developmental Therapeutics Program, or the like.
  • NCI National Cancer Institute's
  • HsKifl 3 a activity can be examined by determining modulation of HsKifl 3a in vitro using cultured cells.
  • the cells are treated with a candidate agent and the effect of such agent on the cells is then determined either directly or by examining relevant surrogate markers. For example, characteristics such as spindle assembly and metaphase arrest can be used to determine the effect.
  • the methods comprise combining a HsKifl 3 a protein and a candidate agent, and determining the effect of the candidate agent on the HsKifl 3 a protein.
  • a plurality of assay mixtures are run in parallel with ⁇ different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion.
  • Such compounds include polypeptides, proteins, nucleic acids, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates.
  • Compounds to be screened can also be obtained from governmental or private sources, including, e.g., the National Cancer Institute's (NCI) Natural Product Repository, Bethesda, MD, the NCI Open Synthetic Compound Collection, Bethesda, MD, NCI's Developmental Therapeutics Program, or the like.
  • NCI National Cancer Institute's
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • the candidate agents are organic chemical moieties, a wide variety of which are available in the literature.
  • the assays provided utilize HsKifl 3a proteins as defined herein.
  • portions of HsKifl 3 a proteins are utilized; in a preferred embodiment, portions having HsKifl 3a activity as described herein are used, hi addition, the assays described herein may utilize either isolated HsKifl 3 a proteins or cells or animal models comprising the HsKifl 3 a proteins.
  • reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • HsKifl3a and its homologues are also a useful diagnostic tool in vitro.
  • Such assays use HsKifl 3a specific reagents that specifically hybridize to HsKifl3a nucleic acid, such as HsKifl3a probes and primers, and HsKifl3a specific reagents that specifically bind to the HsKifl3a protein, e.g., anti-HsKifl3a antibodies.
  • Nucleic acid assays for the presence of HsKifl 3 a DNA and RNA in a sample are useful diagnostic assays.
  • Numerous techniques are known to those skilled in the art, including Southern analysis, northern analysis, dot blots, RNase protection, SI analysis, amplification techniques such as PCR and LCR, and in situ hybridization.
  • in situ hybridization for example, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis.
  • HsKifl 3a protein can be detected with the various immunoassay techniques described above.
  • the test sample is typically compared to both a positive control (e.g, a sample expressing recombinant HsKifl3a) and a negative control (e.g., a negative sample from Saccharomyces).
  • kits for screening for modulators of HsKifl 3 a can be prepared from readily available materials and reagents.
  • kits can comprise any one or more of the following materials: biologically active HsKifl3a, reaction tubes, and instructions for testing HsKifl3a activity.
  • the kit contains biologically active HsKifl 3 a.
  • kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.
  • the kit can be tailored for ATPase assays, microtubule gliding assays, or microtubule binding assays.
  • the kinesins of the present invention and in particular their motor domains can be used for separation of a specific ligand from a heterologous mixtures in aqueous solution as described by Stewart (U.S. Patent No. 5,830,659.
  • a kinesin motor domain is linked to a ligand binding moiety, such as streptavidin.
  • the chimeric kinesin motor domains are placed into a loading chamber containing the heterogeneous mixtures which is coupled to a receiving chamber by a channel bearing immobilized, aligned microtubules.
  • the kinesins of their invention and in particular their motor domains can also be used in the field of nanotechnology.
  • Molecular motors such as kinesin have widespread application in the construction of nanoscale machines; for a review of the general utility of biomolecular motors in nanotechnology see ⁇ http://clinton4.nara.gov/media/pdf/ch7.pdf>.
  • Biomolecular motors have real- world application in the emerging nanotechnological arts. For example, a 1999 NASA study identifies multiple applications for nanoscale motors - and kinesin in particular - in the aerospace field. See ⁇ http://www.nas.nasa.gov/ ⁇ globus/papers/NanoSpacel999/paper.html>.
  • Kinesin motor domains can be used in the construction of rotors and other mechanical components (for review see Limberis and Stewart, Nanotechnology 11 :47-51 (2000)) as well as light-operated molecular shuttles useful for nanoscale switches and pumps (see ⁇ h11p://www.foresight.org/Conferences/MNT8/Abstracts/Nogel/>). Nucleic acids encoding the kinesins of the invention are also useful for inclusion on a GeneChipTM array or the like for use in expression monitoring (see US 6,040,138, . EP 853, 679 and WO97/27317).
  • Such arrays typically contain oligonucleotide or cDNA probes to allow detection of large numbers of mRNAs within a mixture.
  • Many of the nucleic acids included in such arrays are from genes or ESTs that have not been well characterized. Such arrays are often used to compare expression profiles between different tissues or between different conditions of the same tissue (healthy vs. diseased or drug-treated vs. control) to identify differentially expressed transcripts. The differentially expressed transcripts are then useful e.g., for diagnosis of disease states, or to characterize responses of drugs.
  • the nucleic acids of the invention can be included on GeneChipTM arrays or the like together with probes containing a variety of other genes.
  • the present nucleic acids are particularly useful for inclusion in GeneChipTM arrays for analyzing the cell cycle or proliferation state of cells.
  • Nucleic acids encoding hsKifl3a can be combined with nucleic acids encoding other kinesin molecules and/or nucleic acids from other genes having roles in DNA replication, cell division or other cell cycle function.
  • Such arrays are useful for analyzing and diagnosing cells in a proliferating state, and diseases such as cancer characterized by presence of the same.
  • Kifl3a is expressed in most tissues, predominantly in the heart, adrenal tissue and skeletal muscle.
  • Such arrays are also useful for analyzing candidate drugs for roles in modulation of the cell cycle and proliferation. The efficacy of such drugs can be assayed by determining the effect of the drug on the expression profile of genes affecting proliferation and the cell cycle.

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