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WO2004035749A2 - Sequence nucleotidique modifiee codant pour une proteine de kinesine kip3 de c. albicans - Google Patents

Sequence nucleotidique modifiee codant pour une proteine de kinesine kip3 de c. albicans Download PDF

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WO2004035749A2
WO2004035749A2 PCT/US2003/032963 US0332963W WO2004035749A2 WO 2004035749 A2 WO2004035749 A2 WO 2004035749A2 US 0332963 W US0332963 W US 0332963W WO 2004035749 A2 WO2004035749 A2 WO 2004035749A2
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protein
nucleic acid
sequence
seq
activity
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WO2004035749A3 (fr
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Mary Maxon
Penelope Chua
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Cytokinetics Inc
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Cytokinetics Inc
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • the present invention relates to an isolated nucleic acid encoding a
  • CaKip3 kinesin protein that has been corrected for optimal expression in an Escherichia species.
  • the kinesin superfamily 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.
  • Kip3 is a novel kinesin-related protein which is required for nuclear migration to the bud site in preparation for mitosis. Kip3 is also required to achieve and maintain proper spindle positioning. Loss of Kip3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position (see DeZwaan et al, J Cell Biol. 138(5): 957-60; 1997). Epitope-tagged Kip3 localizes to astral and central spindle microtubules, but is also present throughout the cytoplasm and nucleus.
  • Candida albicans is one of the most commonly encountered human fungal pathogens, causing a wide variety of infections ranging from mucosal infections in generally healthy persons to life-threatening systemic infections in individuals with impaired immunity. Those at particular risk for such infections are those with AIDS; those having undergone bone marrow or organ transplants; those receiving chemotherapy; and others who have had debilitating illness, severe injury, prolonged hospitalization, or long-term treatment with antibacterial drugs.
  • the present invention is based on the modification of the sequence encoding the Kip3 kinesin protein in Candida albicans, whereby expression of the protein in E.coli or other organisms is optimized.
  • the invention provides a codon- corrected Candida albicans Kip3 sequence, which when transformed into E.coli, provides improved expression.
  • the invention provides an isolated nucleic acid sequence encoding a kinesin motor protein, wherein the motor protein (i) has ATPase activity; and (ii) comprises an amino acid sequence of SEQ ID NO: 2, 4, 6, or 8; and wherein the nucleic acid sequence comprises at least one codon that is modified from the Candida albicans nucleotide sequence to optimize usage in an Escherichia species.
  • the encoded protein specifically binds to polyclonal antibodies raised against SEQ ID NO: 2, 4, 6, or 8.
  • the nucleic acid has a nucleotide sequence of SEQ ID NO: 1
  • the sequence comprises at least one codon that is modified from the Candida albicans nucleotide sequence to optimize usage in an Escherichia species, and the nucleic acid also encodes a protein having ATPase activity.
  • the invention provides an isolated protein comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, wherein the protein has ATPase activity.
  • the invention provides an expression vector, wherein the vector has one or more of the properties described above.
  • the present invention provides a transformed host cell transformed with the expression vector aforementioned.
  • the transformed host cell is Escherichia coli.
  • the present invention provides a method for producing the modified kinesin protein, comprising the steps of: providing an expression vector, transforming compatible E. coli host cells with the vector, thereby obtaining a transformant; culturing the transformed cells under conditions suitable for growth; and recovering the protein from the host cell culture.
  • the present invention provides a method of identifying a compound as a modulator of an activity of the protein. The method comprises contacting the protein with a compound at a first concentration and determining a level of activity of the target protein. The method further comprises contacting the protein with a compound at a second concentration, and then determining a level of activity of the protein. A change in the level of activity between the protein contacted with the first concentration and the second concentration indicates that the compound modulates an activity of the protein.
  • modulators of the protein including agents for the treatment of fungal infection as common in conditions of immunocompromise or impaired immunity, including AIDS, bone marrow or organ transplants, chemotherapy, or prolonged antibacterial therapy.
  • agents and compositions provided herein can be used in a variety of applications and formulations.
  • Allele refers to any of two or more alternative forms of a gene occupying the same chromosomal locus.
  • ADP refers to adenosine diphosphate and also includes ADP analogs, including, but not limited to, deoxyadenosine diphosphate (dADP) and adenosine analogs.
  • Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin 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 fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
  • Variant 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.
  • the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which 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 which encodes a polypeptide is implicit in each described sequence.
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to about 20 amino acids, although considerably longer insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases, deletions may be much longer. [0020] Substitutions, deletions, and insertions or any combinations thereof may be used to arrive at a final derivative. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger characteristics may be tolerated in certain circumstances.
  • Isoleucine Isoleucine
  • Leucine L
  • Methionine M
  • Valine V
  • 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, intermediate filaments, myosin fragments
  • molecular motors e.g., kinesins, myosins, dyneins
  • cytoskeleton associated regulatory proteins e.g., tropomysin, alpha-actinin
  • cytoskeletal associated binding proteins e.g., microtubules associated proteins, actin binding 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 chromosome movements, contractile ring formation and function, 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 chromosome movements, contractile ring formation and function, 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.
  • High stringency conditions may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.2 times SSC and
  • a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH
  • 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 cultured cell lines are included in this definition.
  • 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 gene, the nucleic acid of interest is separated from open reading frames which flank the gene of interest and encode proteins other than the protein of interest.
  • 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.
  • Modulators refer to modulatory molecules identified using in vitro and in vivo assays for target protein 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 target protein 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.
  • the phrase "motor domain” refers to the domain of a target protein 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 streptavidine 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 and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • a target protein comprises a polypeptide demonstrated to have at least microtubule stimulated ATPase activity.
  • 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, i.e., the one-letter symbols recommended by the IUPAC-IUB.
  • 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 which 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 Target Protein is The Target Protein
  • the present invention provides for the modification of the nucleic acid sequence encoding a CaKip3 kinesin protein, corrected for optimal expression in an Escherichia species or other organism.
  • the corrected codon is the CUG codon.
  • Candida albicans differs from most yeast strains in that it does not use the same genetic code.
  • the codon CUG which in the universal code is read as leucine, is decoded as a serine in Candida. Replacement of leucine by serine at CUG encoded residues has been shown to often result in the production of inactive proteins.
  • CaKip3 can be defined by having at least one or preferably more than one of the following functional and structural characteristics. Functionally, CaKip3 will have microtubule-sti ulated ATPase activity, and microtubule motor activity that is ATP dependent. CaKip3 activity can also be described in terms of its ability to bind microtubules.
  • the nucleic acids provided herein are defined by the proteins 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 or SEQ ID NO:4, 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 or SEQ ID NO:4.
  • the sequence identity may be slightly lower due to the degeneracy in the genetic code.
  • Also included within the definition of the target proteins are amino acid sequence variants of wild-type target proteins.
  • Portions of the modified CaKip3 nucleotide sequence may be used to identify polymorphic variants, orthologs, alleles, and homologues of CaKip3. 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.
  • the target proteins can be made in a variety of ways, including both synthesis de novo and by expressing a nucleic acid encoding the protein.
  • Target proteins of the present invention may also be modified in a way to form chimeric molecules comprising a fusion of a target protein with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the amino or carboxyl terminus of the target protein. Provision of the epitope tag enables the target protein to be readily detected, as well as readily purified by affinity purification.
  • Various tag epitopes are well known in the art. Examples include polyhistidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (see, Field et al. (1988) Mol. Cell.
  • Tag polypeptides include the Flag-peptide (see, Hopp, et al. (1988) BioTechnology 6:1204); the Kt3 epitope peptide (see, Martine et al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner (1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag (see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393).
  • any of the peptides provided herein can be routinely confirmed by assays such as those which assay ATPase activity or microtubule binding activity.
  • assays such as those which assay ATPase activity or microtubule binding activity.
  • polymorphic variants, alleles, and orthologs, homologues of the modified CaKip3 are confirmed by using ATPase or microtubule binding assays as known in the art.
  • the isolation of the biologically active modified CaKip3 provides a means for assaying for modulators of this kinesin superfarnily protein.
  • Biologically active modified CaKip3 is useful for identifying modulators of CaKip3 or fragments thereof and kinesin superfarnily members using in vitro assays such as microtubule gliding assays, ATPase assay (Kodama et al., J. Biochem. 99;1465-1472 (1986); Stewart et al, Proc. Nat'l Acad. Sci USA 90: 5209-5213 (1993)), and binding assays including microtubule binding assays (Vale et al., Cell 42: 39-50 (1985)).
  • This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2 nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994).
  • nucleic acids sizes are given in either kilobases (kb) or base pairs
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1962 (1981), using an automated synthesizer, as described in Van 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. Clxrom. 225:137-149 (1983).
  • the modified nucleic acid sequences encoding CaKip3 and related nucleic acid sequence homologs are cloned from cDNA and genomic DNA libraries or isolated using amplification techniques with oligonucleotide primers.
  • expression libraries can be used to clone the modified CaKip3 and CaKip3 homologues by detected expressed homologues immunologically with antisera or purified antibodies made against CaKip3 that also recognize and selectively bind to the CaKip3 homologue.
  • amplification techniques using primers can be used to amplify and isolate CaKip3 from DNA or RNA.
  • Amplification techniques using degenerate primers can also be used to amplify and isolate CaKip3 homologues.
  • Amplification techniques using primers can also be used to isolate a nucleic acid encoding CaKip3. 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 CaKip3.
  • Appropriate primers and probes for identifying the gene encoding homologues of CaKip3 in other species are generated from comparisons of the sequences provided herein.
  • antibodies can be used to identify CaKip3 homologues. For example, antibodies made to the motor domain of CaKip3 or to the whole protein are useful for identifying CaKip3 homologues.
  • tissue source that is rich in the mRNA of choice.
  • 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 generally described in Granstein et al, Proc. Natl. Acad. Sci. USA, 72:3961-3965 (1975).
  • An alternative method of isolating CaKip3 modified nucleic acid and its homologues combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Pat. Nos. 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 CaKip3 directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • Degenerate oligonucleotides can be designed to amplify CaKip3 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 CaKip3 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.
  • Gene expression of CaKip3 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 CaKip3 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 CaKip3 gene. The specific subsequence is then ligated into an expression vector.
  • the gene for the modified CaKip3 protein 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.
  • a cloned gene such as those cDNAs encoding the modified sequence of the CaKip3 protein
  • Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al.
  • Bacterial expression systems for expressing the CaKip3 protein are available in, e.g., E.
  • Kits for such expression systems are commercially available.
  • Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
  • the pET expression system (Novagen) is a preferred prokaryotic expression system.
  • 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 stall 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 CaKip3 encoding the modified nucleic acid in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding modified CaKi ⁇ 3 and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the modified nucleic acid sequence encoding CaKip3 may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell.
  • 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.
  • 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 i 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 DNA 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 the modified CaKip3 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, in Methods in Enzymology, vol. 182 (Deutscher ed., 1990)).
  • Transformation of the prokaryotic E. coli host cell is performed according to standard techniques (see, e.g., Morrison, J.
  • 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 the modified CaKip3.
  • E. coli a nutrient medium such as LB medium or a minimal medium such as M9 medium is used with the addition of a carbon source, a nitrogen source, a vitamin source, etc.
  • the transformant is cultured at approximately 16 to 42 degrees C. for 5-168 hours. The culture conditions may vary. E. coli cultures will be aerated on a shaker. [0063] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the modified CaKip3, which is recovered from the culture using standard techniques.
  • E. coli Numerous strains of E. coli exist which may serve as a host.
  • Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31, 446); E. coli XI 776 (ATCC 31,537); E. coli Strain W3110 (ATCC 27,325); and K5 772 (ATCC 53,635).
  • Preferred bacterial strains include E. coli BL21 (D ⁇ 3), BL21 9DE3), pLysS, BL21 (DE3) pLysEIV.
  • derivatives of BL21 (DE3) codon plus (Stratagene), Rosetta (Novagen), and star strains (Stratagene). Codon plus and Rosetta express rare codons, resulting in better expression of human proteins in E. coli.
  • the star strains have an RNAse gene deleted for higher mRNA stability and therefore, higher protein expression. Purification of the Modified CaKip3 Protein
  • the modified CaKip3 protein may be purified for use in functional assays.
  • the protein is purified for use in assays to provide substantially pure samples.
  • the protein need not be substantially pure as long as the sample comprising the target protein is substantially free of other components that can contribute to the production of ADP or phosphate.
  • the protein may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse- phase HPLC chromatography, chromatofocussing, selective precipitation with such substances as ammonium sulfate; and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).
  • the target protein can be purified using a standard anti-target antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • a preferred method of purification is use of Ni-NTA agarose (Qiagen).
  • the expressed protein can be purified by standard chromatographic procedures to yield a purified, biochemically active protein.
  • the activity of 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.
  • Biologically active target protein is useful for identifying modulators of target protein or fragments thereof and kinesin superfarnily 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'l Acad. Sci. USA 90:5209-5213 (1993)), and binding assays including microtubule binding assays (Vale et al., Cell 42:39-50 (1985)), as described in detail below.
  • Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG 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.
  • 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.sub.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.
  • 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 the modified CaKip3 protein can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or MiUipore membranes).
  • membranes of different pore size for example, Amicon or MiUipore 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 chromatographed as described below.
  • the modified CaKip3 protein 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).
  • 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)).
  • a number of CaKip3 protein comprising immunogens may be used to produce antibodies specifically reactive with CaKip3.
  • recombinant CaKip3 or an 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. [0076] Methods of production of polyclonal antibodies are known to those of skill in the art.
  • 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 CaKip3.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see Harlow & Lane, supra).
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art.
  • 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.
  • 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 246:1275-1281 (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.
  • polyclonal antisera with a titer of 10.sup.4 or greater are selected and tested for their cross reactivity against non-CaKip3 proteins or even other homologous proteins from other organisms (e.g., C.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a K.sub.d of at least about 0.1 mM, more usually at least about 1 .mu.M, preferably at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M or better.
  • CaKip3 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 modified CaKip3 protein having the sequence identity characteristics as described herein. Additionally, antibodies can be used to identify modulators of the interaction between the antibody and CaKip3 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 the modified CaKip3 such as microtubules or the compounds described in Ser. No. 60/070,772.
  • the modified CaKip3 protein is detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see also Methods in Cell Biology Volume 37: Antibodies in Cell Biology (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.
  • Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case the CaKip3 or antigenic subsequence thereof).
  • the antibody e.g., anti-CaKip3
  • 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.
  • Western blot (immunoblot) analysis is used to detect and quantify the presence of modified CaKip3 protein in the sample.
  • 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 the modified CaKip3.
  • the anti-CaKip3 antibodies specifically bind to the CaKip3 on the solid support.
  • 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-CaKip3 antibodies.
  • labeled antibodies e.g., labeled sheep anti-mouse antibodies
  • Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al, Amer. Clin. Prod. Rev. 5:34-41 (1986)).
  • Assays that can be used to test for modulators of the target protein include a variety of in vitro or in vivo assays, e.g., microtubule gliding assays, binding assays such as microtubule binding assays, microtubule depolymerization assays, and ATPase assays (Kodama et al., J. Biochem. 99: 1465-1472 (1986); Stewart et al, Proc. Nat'l Acad. Sci. USA 90: 5209-5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115 (1995); (Vale et al., Cell 42:39-50 (1985)).
  • in vitro or in vivo assays e.g., microtubule gliding assays, binding assays such as microtubule binding assays, microtubule depolymerization assays, and ATPase assays
  • Modulation is tested by screening for candidate agents capable of modulating the activity of the target protein comprising the steps of combining a candidate agent with the target protein, as above, and determining an alteration in the biological activity of the target protein.
  • the candidate agent should both bind to the target protein (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods and in vivo screening of cells for alterations in cell cycle distribution, cell viability, or for the presence, morphology, activity, distribution, or amount of mitotic spindles, as are generally outlined above.
  • molecular motor activity is measured by the methods disclosed in U. S. Patent 6, 410,254 ( Finer, J., et al..), 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. In one embodiment, ADP or phosphate is used as the readout for protein activity.
  • kinase reactions such as pyruvate l ⁇ nases are known. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31 (1970). This is a preferred method in that it allows the regeneration of ATP.
  • the level of activity of the enzymatic reaction is determined directly.
  • the level of activity of the enzymatic reaction which uses ADP as a substrate is measured indirectly by being coupled to another reaction.
  • the method further comprises a lactate dehydrogenase reaction under conditions which normally allow the oxidation of NADH, wherein said lactate dehydrogenase reaction is dependent on the pyruvate kinase reaction.
  • Measurement of enzymatic reactions by coupling is known in the art.
  • there are a number of reactions which utilize phosphate. Examples of such reactions include a purine nucleoside phosphorylase reaction. This reaction can be measured directly or indirectly.
  • a particularly preferred embodiments utilizes the pyruvate kinase/lactate dehydrogenase system.
  • the detection of the ADP or phosphate proceeds non- enzymatically, for example, by binding or reacting the ADP or phosphate with a detectable compound.
  • phosphomolybdate based assays may be used which involve conversion of free phosphate to a phosphomolybdate complex.
  • One method of quantifying the phosphomolybdate is with malachite green.
  • a fluorescently labeled form of a phosphate binding protein such as the E. coli phosphate binding protein, can be used to measure phosphate by a shift in its fluorescence.
  • target protein activity can be examined by determining modulation of target protein 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 whether directly or by examining relevant surrogate markers. For example, characteristics such as mitotic spindle morphology and cell cycle distribution can be used to determine the effect.
  • the methods comprise combining a target protein and a candidate, and determining the effect of the candidate on the target 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.
  • the components may be added in buffers and reagents to assay target protein activity and give optimal signals. Since the methods allow kinetic measurements, the incubation periods can be optimized to give adequate detection signals over the background.
  • an antifoam or a surfactant is included in the assay mixture.
  • Suitable antifoams include, but are not limited to, antifoam 289 (Sigma).
  • Suitable surfactants include, but are not limited to, Tween, Tritons, including Triton X- 100, saponins, and polyoxyethylene ethers.
  • the antifoams, detergents, or surfactants are added at a range from about 0.01 ppm to about 10 ppm.
  • ком ⁇ онент may be done by combining the target protein and a compound in a first sample in a first concentration. A level of activity is then determined for the protein. The protein is further contacted with the compound at a second concentration, and a level of activity of the protein is determined. A difference between the level of activity of the protein contacted with the first concentration of the compound and the level of activity of the protein contacted with the second concentration of the compound indicates that the compound is a modulator of protein activity.
  • the assays provided utilize target protein as defined herein.
  • portions of target protein are utilized; in a preferred embodiment, portions having target protein activity as described herein are used.
  • the assays described herein may utilize either isolated target proteins or cells or animal models comprising the target proteins.
  • a variety of other 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.
  • a variety of cell-based assays may be used to determine activity. Among these are growth inhibition, halo assays, disc plate diffusion, and inhibition of fungal hyphae length. These assays utilize standard techniques that are well-known in the art ((R.N. Jones et al, Manual of Clinical Microbiology, 4 th ed., (1985); and M.A. Pfaller et al, Antimicrobial Agents and Chemotherapy, 34 (1990)).
  • the methods of the invention are used to identify compounds useful in the treatment of systemic fungal infections in mammals.
  • Candidate agents having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a patient, as described herein.
  • the compounds may be formulated in a variety of ways as discussed below.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt. %.
  • the agents maybe administered alone or in combination with other treatments, i.e., radiation, or other chemotherapeutic agents.
  • the pharmaceutical compositions are in a water soluble form, such as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically- active compounds.
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as a variety of formulations.

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Abstract

La présente invention concerne un acide nucléique isolé codant pour une protéine de kinésine Kip3 de C. albicans, la séquence de l'acide nucléique isolé ayant été corrigée en vue de l'expression optimale dans une espèce d'Escherichia ou dans un autre organisme. L'invention concerne également des acides nucléiques, des vecteurs d'expression, des transformants obtenus par la transformation à l'aide de vecteurs d'expression, des méthodes de production de la protéine modifiée à l'aide du transformant, ainsi que des méthodes de criblage de modulateurs de protéines.
PCT/US2003/032963 2002-10-17 2003-10-16 Sequence nucleotidique modifiee codant pour une proteine de kinesine kip3 de c. albicans Ceased WO2004035749A2 (fr)

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