CN1230993A - Expression vector, cell and method for preparing thrombopoietin polypeptide - Google Patents

Abstract

The invention discloses a thrombopoietin polypeptide, a carrier, a cell and a method for producing the polypeptide. These polypeptides are characterized by an amino acid backbone comprising C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker containing 1 or 2 amino acid residues, provided that X alone Or no dibasic amino acid pair is provided in combination with C or B; and B is a polypeptide comprising residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and up to 35% of B Amino acid residues can be replaced by other amino acid residues, respectively. These polypeptides are useful in methods of stimulating proliferation or development of hematopoietic cells in vivo and in vitro.

Description

Expression vector, cell and method for preparing thrombopoietin polypeptide

Background of the invention

Hematopoiesis is the process by which pluripotent stem cells in the bone marrow develop and differentiate into blood cells. This process involves a complex interaction of polypeptide growth factors (cytokines) that act through membrane-bound receptors on target cells. Cytokine action results in cell proliferation and differentiation in response to particular cytokines, often germline-specific and/or stage-specific. The development of single cell types such as platelets from stem cells requires a complex coordination of cytokines acting in the proper order.

Known cytokines include interleukins, such as IL-1, IL-2, IL-3, IL-6, IL-8, etc.; and colony-stimulating factors, such as G-CSF, M-CSF, GM-CSF, Erythropoietin (EPO), etc. Typically, interleukins act as mediators of immune and inflammatory responses. Colony-stimulating factors stimulate the proliferation of myeloid-derived cells, activate mature leukocytes, and form an integral part of the host response to inflammation, infection, and immune attack.

Various cytokines have been developed as therapeutic agents. For example, erythropoietin, which stimulates the development of red blood cells, has been used to treat anemia caused by kidney failure. Several colony-stimulating factors have been used in combination with cancer chemotherapy to speed recovery of the patient's immune system. Interleukin-2, alpha-interleukin and C-interleukin are used in the treatment of certain cancers. Active substances that stimulate megakaryocytopoiesis and thrombopoiesis have been identified in the body fluids of platelet-producing animals and are referred to in the literature as "thrombopoietins" (recently by McDonald, Experimental Hematology 16:201-205, 1988 and McDonald, USA Journal of Pediatric Hematology-Oncology 14:8-21, 1992 review). Work to purify and characterize this active substance led to the cloning of a protein that binds to the cellular Mpl receptor and stimulates megakaryocytopoiesis and thrombopoiesis. See de Sauvage et al., Nature 369:533-538, 1994; Lok et al., Nature 369:565-568, 1994; Kaushansky et al., Nature 369:568-571, 1994; Wendling et al., Nature 369:571-574, 1994; Bartley et al., Cell 77:1117-1124, 1994; and WIPO Publication WO 95/21920. This Mpl receptor binds a cytokine called thrombopoietin.

Amino acid sequence analysis indicated that mature human TPO extends from amino acid residue 1 (Ser) to amino acid residue 332 (Gly) of SEQ ID NO:2. TPO is susceptible to proteolysis and has been isolated in heterologous or degraded forms (de Sauvage et al., Nature 369:533-538, 1994; Bartley et al., Cell 77:1117-1124, 1994). Small molecule types of 25 kD have been found to be active in vitro (Bartley et al., ibid), and recombinant human TPO polypeptides of 153 amino acids (deSauvage et al., ibid) and 174 amino acids (Bartley et al., ibid) have been reported to be active in vitro , the expression product of the full-length human cDNA encoding the initial translation product of 353 amino acids (Bartley et al., ibid) was also active in vitro.

There is a need in the art for methods of efficiently producing thrombopoietin in large quantities; there is also a need for methods of producing thrombopoietin in eukaryotic microorganisms such as yeast; Full-length molecules are less susceptible to proteolysis than full-length molecules. The present invention fulfills these needs and has other related advantages.

Summary of the invention

In one aspect, the invention provides expression vectors capable of replicating in eukaryotic host cells. The vector includes the following operably linked elements: (a) transcriptional promoter; (b) the first DNA fragment encoding the secretory leader peptide; (c) the second DNA fragment encoding the thrombopoietin (TPO) polypeptide containing C-X-B , wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker containing 1 or 2 amino acid residues, with the proviso that X alone or in combination with C or B does not provide a pair of dibasic amino acids; and B is a polypeptide comprising residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and up to 35% of the amino acid residues in B can be replaced by other amino acid residues, respectively; and (d) transcription terminator. In one embodiment of the invention, the expression vector is replicable in yeast. In another embodiment, the secretory leader is the alpha factor secretory leader of Saccharomyces cerevisiae. In another embodiment, B does not include the Arg-Arg dipeptide. In other embodiments, residue 4 of B is Thr or Asp; y is at least 10 and residue 10 of B is Arg or Glu; and y is at least 14 and residue 14 of B is Val or Ala. In another embodiment, y is at least 14, the 4th residue of B is Thr or Asp, the 10th residue of B is Arg or Glu, and the 14th residue of B is Val or ala. In other embodiments, X is a peptide bond or a single amino acid residue.

Another aspect of the invention provides a cultured eukaryotic cell comprising an expression vector as disclosed above, wherein the cell synthesizes and secretes a TPO polypeptide. In a preferred embodiment the cells are yeast cells.

The third aspect of the invention provides a thrombopoietin polypeptide characterized by a C-X-B amino acid backbone, wherein C is a human thrombopoietin cytokine domain polypeptide; x is a peptide bond or a linker containing 1 or 2 amino acid residues, The proviso is that x alone or in combination with C or B does not provide a dibasic amino acid pair; and B is a polypeptide comprising residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein Up to 35% of the residues in B may be substituted by other amino acid residues, respectively.

In a fourth aspect of the invention, there is provided a method for producing a TPO polypeptide, comprising culturing a eukaryotic host cell transfected or transformed with the expression vector disclosed above, and the connected first and second DNA fragments in the vector are expressed by the host cell to Synthesizing the TPO polypeptide, and recovering the TPO polypeptide.

The fifth aspect of the invention discloses a method for increasing the number of platelets in a mammal, comprising administering to the mammal the TPO polypeptide disclosed above in combination with a pharmaceutically acceptable carrier.

These and other aspects of the invention will become apparent upon reference to the following detailed description. Detailed description of the invention

Before describing the present invention in detail, some terms used herein are defined first:

The term "allelic variation" is used herein to refer to altered forms of a gene produced by mutation, or to an altered polypeptide encoded by a mutated gene. A genetic mutation can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence.

An "expression vector" is a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest operably linked to other segments for transcription. Such fragments include promoter and terminator sequences. Expression vectors may also contain one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. The term "operably linked" means that the segments are arranged to function in concert for their intended purpose, eg, transcription begins at a promoter and passes through the coding segment to a terminator. Expression vectors can replicate independently in the host organism or by integrating into the host genome.

The term "characterized by" is used to refer to a feature or element with a qualification. For example, a polypeptide is characterized by an amino acid backbone of a given sequence comprising the recited amino acid sequence but also comprising other amino acid residues. Moreover, the polypeptide may also contain sugar chains or other post-translational modifications.

A "secretory leader peptide" is a polypeptide that directs and facilitates the delivery of proteins through the secretory pathway of a host cell. A secretory leader peptide is also sometimes referred to as a presequence. Secretory leader peptides are characterized by a core of hydrophobic amino acids and are usually, but not exclusively, at the amino terminus of newly synthesized proteins. Secretory leader peptides are often cleaved from the mature protein in one or more cleavage events during secretion. These secretory leaders contain processing sites that allow the secretory leader to be cleaved from the mature protein as it passes through the secretory pathway.

A "promoter" is that portion of a gene where RNA polymerase binds and mRNA synthesis begins.

"Thrombopoietin" (or "TPO") is a protein characterized by the ability to specifically bind the Mpl receptor of the same strain and to stimulate platelet production in vivo. In normal experimental animals, TPO increased platelet levels by 100% or more within 10 days of starting daily dosing. Full-length TPO comprises an amino-terminal cytokine domain and a carboxy-terminal ("C-terminal") domain. Referring to SEQ ID NO:2, the cytokine domain is located between the 7th and 151st cysteine residues.

The term "thrombopoietin polypeptide" includes both the full-length thrombopoietin molecule and biologically active portions thereof, which are fragments of thrombopoietin that exhibit qualitative biological activities of the intact molecule (receptor binding and stimulation of platelet production in vivo).

SEQ ID NO: 1 represents a representative cDNA encoding full-length human thrombopoietin. Those skilled in the art will recognize that the DNA of SEQ ID NO: 1 and its encoded amino acid sequence (SEQ ID NO: 2) represent a single allele of the human TPO gene and that allelic variation is expected. The allelic variation of SEQ ID NO: 1 can be obtained by cloning from cells, tissues or nucleic acids prepared from different individuals and sequencing the resulting clones.

As used herein, the term "thrombopoietin cytokine domain polypeptide" refers to this core polypeptide (residues 7-151 or the corresponding regions of SEQ ID NO:2 and allelic variations of SEQ ID NO:2), which can be further Including short N-terminal (e.g. residues 1-6 of SEQ ID NO:2) and/or C-terminal (e.g. residue 152 of SEQ ID NO:2) extensions which do not disrupt the essential biological properties of the molecule active. Considerable sequence variation is allowed in these stretches. The C-terminal domain of human TPO extends from residue 155 (Ala) to residue 332 (Gly) of SEQ ID NO:2. This domain contains potential O- and N-linked glycosylation sites. This domain can be deleted completely or partially without complete loss of biological activity. These two domains are separated by the Arg-Arg dipeptide (residues 153 to 154 of SEQ ID NO: 2). Although this dipeptide is postulated to be the processing site for cleavage during TPO maturation (eg de Sauvage et al., ibid), studies conducted by the assignee of the present invention indicated that no significant cleavage occurs at this site.

As used herein, the phrase "part of the C-terminal domain of thrombopoietin" includes 1 amino acid of the TPOC-terminal domain to amino acids including the entire TPO C-terminal domain. Typically, part of the C-terminal domain is a contiguous segment of the naturally occurring TPO C-terminal domain, containing the first amino acid residue of the corresponding complete TPO C-terminal domain as its first (amino-terminal) amino acid residue base (e.g., the amino acid residue corresponding to residue 155 of SEQ ID NO: 2). The portion of the C-terminal domain used in the invention is preferably 5 to 18 amino acid residues in length, more preferably at least 9 residues in length, most preferably 14 to 18 residues in length.

The present invention provides improved methods of producing thrombopoietin polypeptides, as well as expression vectors and cells useful in the methods. The present invention is based in part on the discovery that certain amino acid changes in TPO polypeptides result in increased secretion by eukaryotic host cells. In the present invention, the secretion of TPO polypeptide is enhanced by deleting or replacing the Arg-Arg dipeptide in the native molecule that separates the cytokine domain from the C-terminal domain. Although TPO polypeptides do not cleave at this Arg-Arg dipeptide as speculated in the literature (eg, de Sauvage et al., ibid), this dipeptide was found to inhibit secretion. The present invention therefore provides TPO polypeptides characterized by the elimination of the dibasic amino acid pair immediately C-terminal to the cytokine domain. These TPO polypeptides are thus characterized by a C-X-B structure, where C is the human thrombopoietin cytokine domain polypeptide, X is a peptide bond or a linker containing 1 or 2 amino acid residues, and B is derived from the C-terminal domain of thrombopoietin. In part, with the proviso that X alone or together with C or B does not form a pair of basic amino acid residues.

Thus in the protein of the invention the dibasic sequence present at the junction of the cytokine and C-terminal domains of wild-type thrombopoietin (eg residues 153 and 154 of SEQ ID NO: 2) is absent. Preferably, no such dibasic sequence is present elsewhere in the molecule, especially in B, either.

The TPO polypeptides of the invention most commonly comprise the amino acid sequence of a naturally occurring human TPO cytokine domain, and this sequence is linked by a peptide bond to a portion of the C-terminal domain of mammalian, preferably human, thrombopoietin. In a preferred embodiment of the invention, B comprises 5 to 18 contiguous residues of the C-terminal domain starting from the amino-terminal residue of the C-terminal domain, wherein up to 35% of the amino acid residues in B may be substituted by other amino acid residues, respectively. The TPO cytokine domain may also contain from 1 to about 15, preferably no more than 10, more preferably no more than 7 amino acid substitutions. Amino acid substitutions occur at non-essential amino acid residues, ie those residue substitutions that do not seriously affect the biological activity of the molecule. Methods of identifying non-essential amino acid residues are known in the art and include alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502 , 1991), wherein a single alanine mutation was introduced at each residue of the molecule, and the biological activity of the resulting mutant molecule was assayed. The effect of multiple amino acid substitutions on protein activity can be assessed as disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. National Academy of Sciences USA 86:2152-2156, 1989). Briefly, these authors disclose the method of simultaneously randomly testing 2 or more sites in a polypeptide and screening for functional polypeptides, followed by sequencing the mutagenized polypeptides to determine permissible substitutions at each site scope. Other methods that can be used include phage display (e.g., Lowman et al., Biochemistry 30:10832-10837, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al. Gene 46:145, 1986; Ner et al., DNA 7:127, 1988). In addition to testing the biological activity of the mutagenized polypeptide, the expression vector encoding the polypeptide can also be transformed into cultured cells to detect the effect of the mutation on the secretion of the polypeptide.

Amino acid substitutions in B relative to the naturally occurring TPO sequence can be designed to preserve or enhance the beneficial effect of this portion of the molecule on secretion. Preferred substitutions include replacing charged residues with uncharged residues. Other preferred substitutions include substitution of alanine for valine (residue 168 of SEQ ID NO:2), substitution of glutamic acid for arginine (residue 164 of SEQ ID NO:2), and substitution of Aspartic acid was substituted for threonine (residue 158 of SEQ ID NO: 2). These substitutions may be made alone or in any combination. Preferably no more than 25% of the residues in B are substituted compared to the corresponding naturally occurring sequence. SEQ ID NO:4 to SEQ ID NO:7 represent typical C-terminal domain sequences.

Without being bound by theory, the Thr-Thr dipeptide may provide an attachment site for O-linked sugar chains. Thus in one embodiment of the invention B comprises a Thr-Thr dipeptide. In a particularly preferred embodiment, the N-terminal 5 amino acid residues of B are Ala-Pro-Pro-Thr-Thr (SEQ ID NO: 8).

Secretion levels can be further increased by adding N-linked carbohydrate attachment sites (Asn-X-Ser/Thr). However, the presence of such sequences may lead to undesired hyperglycosylation in certain host cells such as Saccharomyces cerevisiae.

Those skilled in the art will recognize that certain amino acid substitutions may be host cell specific. The effect of such substitutions should therefore preferably be tested in the host cell type used to synthesize the polypeptide.

The expression vector capable of replicating in eukaryotic host cells of the present invention comprises a transcriptional promoter and a transcriptional terminator, which are operable with the first DNA fragment encoding the secretory peptide and the second DNA fragment encoding the TPO polypeptide as described above ground connection. Thus the second DNA fragment encodes a TPO polypeptide comprising C-X-B, wherein C, X and B are as defined above. In a preferred embodiment of the invention, B is a polypeptide comprising residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein at most 35%, preferably not more than 25%, of said Residues may be substituted by other amino acid residues, respectively.

By "DNA segment encoding a C-X-B-containing TPO polypeptide" is meant that the nascent polypeptide comprising repeat elements is read from amino-terminus to carboxy-terminus. The term "coding" is then used to refer to the direct product of transcription and translation of a DNA segment. Those skilled in the art will understand that such polypeptides may undergo post-translational processing, wherein additional components such as sugar chains may be added thereto. The precise nature of such post-translational modifications may be determined in part by the type of host cell in which the polypeptide is synthesized.

Mouse and human TPO DNAs can be cloned as disclosed in WIPO Publication WO 95/21920, the entirety of which publication is incorporated herein by reference. Plasmid pZGmpl-1081 containing the mouse TPO DNA sequence was deposited with the American Type Culture Collection, 12301 Parklaw Drive, Rockville, MD on February 14, 1994 under the terms of the Budapest Treaty and received accession number 69566. Plasmid pZGmpl-124 containing human TPO cDNA has been deposited in the American Type Culture Collection on May 4, 1994 as an Escherichia coli DH10b transformant with a deposit number of 69615. These mouse and human cDNAs can be used as probes to isolate other TPO-encoding DNAs, including genomic DNAs, allelic variations and DNAs from other species.

Suitable host cells for use in the present invention include any eukaryotic cell type that can be manipulated to express heterologous DNA, can be grown in culture, and has a secretory pathway.

To direct a TPO polypeptide into the secretory pathway of a host cell, a DNA sequence encoding a secretory leader peptide is used in conjunction with a DNA sequence encoding a TPO polypeptide. The secretory leader peptide may be the leader peptide of TPO or other secreted proteins such as tissue plasminogen activator (t-PA) or the leader peptide of the conjugative pheromone alpha-factor of Saccharomyces cerevisiae. When the heterologous secretion leader peptide is used to combine with the TPO polypeptide, the corresponding DNA fragments are connected in the correct reading frame respectively, so that the connected fragments encode the fusion protein. A proteolytic cleavage site is typically added at the junction of the secretory leader peptide and the TPO polypeptide for cleavage of the secretory leader peptide from the TPO polypeptide during secretion. However, one skilled in the art will recognize that the fusion protein can be recovered and subsequently processed to release the TPO polypeptide.

Yeast cells, especially cells of the genus Saccharomyces, are preferred hosts of the invention. Yeast cells have long been used in the production of products for human consumption and are relatively inexpensive to culture. Methods for transforming yeast cells with exogenous DNA and producing recombinant proteins therefrom have been disclosed, see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743 and Murray et al., US Patent No. 4,845,075, incorporated herein by reference. Transformed cells are selected by a phenotype determined by a selectable marker, usually drug resistance or viability in the absence of a particular nutrient such as leucine. A preferred vector system for use with yeast is the POTI vector system as disclosed by Kauasaki et al. (US Pat. No. 4,931,373), which selects transformed cells by growth on glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092, incorporated herein by reference) and the promoter and terminator of the alcohol dehydrogenase gene. See also US Patent Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, incorporated herein by reference. Transformation systems for other yeasts are known in the art, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maize, Pasteur Pichia pastoris, Pichia mongoliana and Candida maltosa. See, eg, Gleeson et al., J. Microbial Genetics 132:3459-3465, 1986; Cregg, US Patent No. 4,882,279; and Stroman et al., US Patent No. 4,879,231.

Preferably, host strains selected to secrete TPO polypeptides at high levels should be used. The parental strain, whose genotype is suitable for fermentation and protein synthesis, is subjected to mutagenesis by conventional methods such as ultraviolet irradiation or chemical mutagenesis using, for example, ethyl methanesulfonate or nitrosoguanidine. Use conventional detection methods such as filter colony detection to screen the protein secretion level of surviving cells. Cells are covered with nitrocellulose filters in filter colony assays, and colonies are subsequently probed with antibodies in Western blot analysis. Other assays such as activity assays can also be used.

Other fungal cells are also suitable as host cells. For example, Aspergillus cells can be utilized according to the methods of Mcknight et al., US Patent No. 4,935,349, incorporated herein by reference. Methods for transforming Acremonium chrysogenum are disclosed in Sumino et al., US Patent No. 5,162,228, incorporated herein by reference. Methods for transforming Neurospora cells are disclosed in Lambowitz, US Patent No. 4,486,533, incorporated herein by reference.

Methods for introducing foreign DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456,1973), electroporation (Neumann et al., EMBO J.1:841-845,1982), DEAE-dextran-mediated transfection (Ausubel et al., eds., Methods in Modern Molecular Biology, John Wiley and Sons, NY, 1987) and cationic lipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993), incorporated herein by reference. Synthesis of recombinant proteins in cultured mammalian cells has been disclosed, see, e.g., Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; Mulvihill et al., U.S. Patent No. 5,486,471; Foster et al., US Patent No. 5,358,932; and Mulvihill et al., US Patent No. 5,385,831, incorporated herein by reference. Preferred cultured mammalian cells include COS-1 (ATCC No.CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCC No.CRL 1632), BHK570 (ATCC No.CRL 10314), 293 (ATCC No. . CRL1573; Graham et al., J. Viral Genet. 36:59-72, 1977) and Chinese hamster egg cell lines (eg CHO-KI; ATCC No. CCL61). Other suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcriptional promoters, such as those from SV-40 or cytomegalovirus, are preferred. See, eg, US Patent No. 4,956,288. Other suitable promoters include those from the metallothionein gene (US Patent Nos. 4,579,821 and 4,601,978, incorporated herein by reference) and the adenovirus major late promoter.

Drug selection is often used to select cultured mammalian cells into which foreign DNA has been inserted. These cells are often referred to as "transfectants". Cells that have been cultured in the presence of a selection agent and are capable of passing the gene of interest to progeny are termed "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Screening is performed in the presence of neomycin-type drugs such as G-418, etc. Selection systems can also be used to increase the expression level of a gene of interest, a process known as "amplification." Amplification is performed by culturing transfectants in the presence of low levels of selection reagent, followed by increasing amounts of selection reagent to select for cells that synthesize high levels of the introduced gene product. A preferred amplified selectable marker is dihydrofolate reductase, which confers resistance to methotrelins. Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and synthesis of foreign proteins therein are disclosed in Guarino et al., U.S. Patent No. 5,162,222; Bang et al., U.S. Patent No. 4,775,624; and WIPO Publication WO 94/06463, incorporated herein by reference. Expression of genes in plant cells using Agrobacterium rhizogenes as a vector is discussed in Sinkar et al., Bangalore 11:47-58, 1987.

Transformed or transfected host cells are cultured according to conventional methods in a medium containing nutrients and other components required for the growth of the selected host cells. Various suitable media, including defined media and complex media, are known in the art and generally contain carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. The medium also contains components such as growth factors or plasma, if necessary. Growth media will be selected for cells containing the exogenously introduced DNA by drug selection or lack of essential nutrients supplemented by a selectable marker carried by the expression vector or co-transfected into the host cell.

TPO polypeptides prepared according to the present invention are selectively recovered by methods well known in the art, such as affinity purification and isolation based on protein size, charge, solubility, and other characteristics. When the polypeptide is synthesized in cultured mammalian cells, the cells should preferably be grown in serum-free medium to limit the amount of contaminating protein. Collect and fractionate the medium. Preferred fractionation methods include affinity chromatography, such as on solid phase Mpl receptor protein or its ligand-binding portion or by the use of affinity tags such as polyhistidine, substance P or other antibody or other specific binding agent polypeptide or protein) affinity chromatography. A specific cleavage site can be provided between the protein of interest and the affinity tag. Other chromatographic methods such as cation exchange chromatography, anion exchange chromatography and hydrophobic interaction chromatography may also be used.

TPO polypeptides prepared according to the present invention may be used in treatments requiring enhanced cell proliferation in the bone marrow, such as in the treatment of cytopenias such as those caused by aplastic anemia, myeloplastic syndrome, chemotherapy or congenital cytopenias. TPO polypeptides are also useful for increasing platelet production, such as in the treatment of thrombocytopenia. Thrombocytopenia is associated with different diseases and clinical conditions, which may act alone or synergistically to produce disease. Decreased platelet counts can be caused by, for example, defective platelet synthesis, abnormal platelet distribution, dilutional loss due to massive transfusion, or abnormal destruction of platelets. For example, chemotherapeutic drugs used in cancer treatment may inhibit the development of platelet progenitor cells in the bone marrow, and the resulting thrombocytopenia limits chemotherapy and necessitates blood transfusions. In addition, certain malignancies impair platelet synthesis and platelet distribution. Radiation therapy used to kill malignant cells also kills pre-platelet cells. Thrombocytopenia can also be caused by various platelet autoimmune disorders induced by drugs, neonatal alloimmunization, or platelet transfusion alloimmunization. TPO polypeptide can reduce or eliminate the need for blood transfusion, thereby reducing the occurrence of platelet autoimmunity. Abnormal destruction of platelets may result from: (1) increased platelet consumption in vascular grafts or injured tissue; or (2) in connection with, for example, drug-induced thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), autoimmune disease, blood Immunological mechanisms associated with disorders such as leukemias and lymphomas or metastatic cancers involving the bone marrow. Other indications for TPO include aplastic anemia and drug-induced myelosuppression caused by eg chemotherapy or treatment of HIV infection with AZT.

Thrombocytopenia manifests as increased bleeding such as bleeding from the mucous membranes of the mucous membranes in the nasal-mouth area or gastrointestinal tract, and exudate from wounds, ulcers, or injection sites.

For pharmaceutical use, TPO polypeptides are formulated for parenteral, especially intravenous or subcutaneous delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a period of 1 to several hours. Typically, pharmaceutical formulations comprise TPO polypeptide in combination with pharmaceutically acceptable carriers such as saline, buffered saline, 5% dextrose in water, and the like. The formulation may further include one or more excipients, preservatives, co-solvents, buffers, albumin to prevent protein loss on the surface of the vial, and the like. In addition, TPO polypeptides can bind to other cytokines, especially early acting cytokines such as stem cell factor, IL-3, IL-6, IL-11 or GM-CSF. When using such combination therapy, the cytokines can be combined in a single formulation or administered in separate formulations. Formulation methods are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro ed., Mack Publishing Company, Easton PA, 1990, incorporated herein by reference. The therapeutic dose of TPO is generally 0.1 to 100 micrograms per kilogram of patient body weight per day, preferably 0.5-50 micrograms per day. The precise dosage will be determined by the clinician based on accepted criteria, taking into account the nature and severity of the disease being treated and patient characteristics. In certain cases, such as when the treated patient demonstrates increased sensitivity or requires prolonged treatment, a dosage range of 0.1-20 micrograms/kg body weight per day may be prescribed. Determination of dosage is within the level of ordinary skill in the art. TPO polypeptides are typically administered for a period of up to 28 days following chemotherapy or bone marrow transplantation or until the platelet count reaches greater than 20,000/mm3, preferably greater than 50,000/mm3. More generally, TPO polypeptides are administered for about a week, usually 1 to 3 days. Typically, a therapeutically effective amount of a TPO polypeptide is an amount sufficient to cause a clinically significant increase in proliferation and/or differentiation of lymphoid or myeloid prophase cells, which will manifest as increased circulating levels of mature cells (eg, platelets or neutrophils). Treatment of platelet disorders will therefore be continued until the platelet count reaches at least 20,000/mm3, preferably 50,000/mm3. TPO polypeptides can also be administered in combination with other cytokines such as IL-3, IL-6 and IL-11; stem cell factor; erythropoietin; G-CSF and GM-CSF. In combined regimens, the daily doses of other cytokines are usually: EPO, ≤150 U/kg body weight; GM-CSF, 5-15 μg/kg body weight; IL-3, 1-5 μg/kg body weight; and G - CSF, 1-25 μg/kg body weight. For example, combination therapy with EPO is suitable for anemic patients with low EPO levels.

TPO polypeptides are also useful tools for studying hematopoietic cell differentiation and development in vitro, such as for elucidating cell differentiation mechanisms and determining mature cell lineages, and have also been found to be useful as proliferating agents in cell culture.

TPO polypeptides can also be used in vitro, such as in autologous bone marrow cultures. Briefly, bone marrow is removed from the patient prior to chemotherapy and treated with TPO polypeptide, selectively in combination with one or more cytokines. The treated bone marrow is then returned to the patient after chemotherapy to speed up the recovery of the bone marrow. In addition, TPO polypeptides can also be used for ex vivo expansion of bone marrow or peripheral blood primitive (PBPC) cells. Stimulation of bone marrow with stem cell factor (SCF) or G-CSF prior to chemotherapy releases early prophase cells into the peripheral circulation. These procells can be collected and concentrated from peripheral blood, and then one or more TPO polypeptides can be used to selectively bind one or more cytokines, including but not limited to SCF, G-CSF, IL-3, GM- CSF, IL-6, or IL-11 are treated in culture to differentiate and proliferate into high-density megakaryocyte cultures that can then be returned to the patient following high-dose chemotherapy.

The invention is further illustrated by the following non-limiting examples. Example Example 1

Two expression vectors were constructed to compare the expression of the truncated human TPO polypeptide in Saccharomyces cerevisiae in the presence or absence of the 153-154 Arg-Arg dipeptide of SEQ ID NO:2. These two vectors are from the plasmid pDPOT (ATCC #68001; disclosed in US Patent No. 5,128,321). These two vectors contain a TPO containing the S. cerevisiae triose phosphate isomerase (TPI1) gene promoter (see U.S. Patent No. 4,599,311, incorporated herein by reference), the S. cerevisiae MFα1 presequence, the TPO sequence and the S. cerevisiae TPI1 terminator expression cassette. The vector further included the Schizosaccharomyces pombe triose phosphate isomerase (POT) gene (to allow selection in media containing glucose), the ampicillin resistance gene for E. coli selection, and the Leu2-d selectable marker. Vector pD85 encodes the wild-type human TPO sequence from amino acid residue 1 to residue 172 of SEQ ID NO: 2, wherein residue 168 (Val) is replaced by Ala. Vector pD79 encodes a mutated TPO 1-172 sequence, in which the 153 and 154 arginine residue codons are deleted, and the valine codon (residue base 168).

Full-length human TPO DNA was modified by polymerase chain reaction (PCR) to add 5 codons of the MFα1 pre-sequence at the 5' end and a stop codon at the 3' end. PCR was performed using primers ZC7623 (SEQ ID NO: 9) and ZC 7627 (SEQ ID NO: 10). PCR was completed using Taq polymerase and about 20 ng of template DNA at 94°C for 1 minute; 53°C for 1 minute; 72°C for 1.5 minutes for 25 cycles. The modified TPO sequence was isolated as a 479 bp HindIII-XbaI fragment and ligated into pUC18 which had been cut with the same enzymes. The resulting plasmid was named pHB76.

The TPO sequence in pHB76 was modified to introduce BbeI and SaII sites at the 5' and 3' ends of the sequence encoding the cytokine domain, respectively. PCR was performed using primers ZC7868 (SEQ ID NO: 11) and ZC7870 (SEQ ID NO: 12). Using Taq polymerase and about 20 ng of template DNA, perform 1 cycle at 95°C for 1 minute; 68°C for 10 minutes; then 9 cycles at 95°C for 1 minute; 68°C for 4 minutes to complete PCR. The TPO sequence was recovered as a 482 bp HindIII-EcoRI fragment and ligated into pUC19 cut with the same enzymes. The resulting plasmid was named pTPOGN2.

Then by ligating the 457 bp HindIII-SalI fragment from pTPOGN2 with a synthetic fragment constructed from oligonucleotides ZC8488 (SEQ ID NO: 13) and ZC8489 (SEQ ID NO: 14) in a three-part ligation and digested with HindIII+XbaI pUC19 was combined to construct a variant TPO sequence. The resulting plasmid was named pTPOGN6. A 539 bp HindIII-XbaI fragment encoding the last residues of the alpha-factor propeptide and the truncated TPO polypeptide was isolated from pTPOGN6. This fragment was ligated with pDPOT (cut by BamHI and treated with alkaline phosphatase), 1230 bp BglII-HindIII fragment containing TPI1 promoter and MFα1 pre-sequence, and 680 bp XbaI-BamHI fragment containing TPI1 terminator in a four-part ligation. The resulting plasmid was named pD79.

Terminated by pDPOT (cleaved by BamHI and treated with alkaline phosphatase), the 1230 bp BglII-HindIIITPⅠ1-MFα1 fragment, the 540 bp fragment encoding the last few residues of the α-factor propeptide and the truncated TPO polypeptide, and the 680 bp XbaI-BamHITPⅠ1 The subfragments were ligated in a four-part ligation to construct a control plasmid encoding residues 1 to 172 of human TPO (SEQ ID NO:2). The resulting plasmid was named pD85.

Plasmids pD79 and pD85 had to be transformed into S. cerevisiae strain JG134 (MATαΔtpil::URA3 ura3-52leu2-Δ2pep4-Δ1[cir°]) as disclosed in Hinnen et al. Transformants were screened for their ability to grow on media containing glucose as the sole carbon source.

Transformants were grown for about 60 hours in liquid medium containing 1% yeast extract, 1% peptone and 5% glucose. Cells were separated from the culture medium by centrifugation. Culture medium samples were diluted with TPO dilution buffer (supplemented with 10% fetal bovine serum, 2 mmol glutamine, 1 mmol sodium pyruvate, 50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml fresh Mycin, 0.00033% β-mercaptoethanol, RPMI 1640 of 25 millimolar Hepes) was diluted 1:100.

TPO bioactivity was tested in a mitotic assay using BaF3 cells transfected with an expression vector encoding the human MPL receptor (Vigon et al., Proc. Natl. Acad. Sci. USA 89:5640-5644, 1992) as target cells. BaF3 is an interleukin-3 dependent prolymphoid cell line from murine bone marrow (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol Cell Biol 6:4133-4135, 1986). Cells are incubated with test samples at 37°C for 16 to 19 hours in the presence ^of3H -thymidine. ³ H-thymidine incorporation into cellular DNA was quantified by comparison to a standard curve for human TPO. 10 U/ml was defined as the amount at which half of the maximal stimulation was obtained in the cell division assay. The results of the two experiments are shown in Table 1.

Table 1

  Experiments   Plasmids   TPO (unit/ml culture medium)

1 pD85 40

                                                                                         

                                                                                 Below

                                                       

A series of plasmids encoding TPO polypeptides comprising the human TPO cytokine domain (residues 22 to 152 of SEQ ID NO: 2) linked to a C-terminal fragment by a peptide bond or an Arg-Arg dipeptide was constructed. Table 2 shows the structure of the encoded polypeptide: Arg-Arg indicates the presence (+) or deletion (-) of the dipeptide; the amino acid sequence number of the C-terminal fragment refers to SEQ ID NO:3.

Table 2

Plasmid Arg-Arg C-terminal sequence

pD117 - 1-5

pD119 - 1-9

pD121 - 1-13

pD123 - 1-18

pD125 + + 1-18

With pUC19 digested into linear form through HindIII and XbaI, the 457bpHindIII-SalI fragment (Example 1) of pTPOGN2 comprising the sequence of coding part of alpha factor secretory leader peptide and part of human TPO cytokine domain (Example 1) and from oligonucleotide ZC8486( SEQ ID NO: 15) and ZC8487 (SEQ ID NO: 16) to construct the SalI-XbaI linker to construct plasmid pTPOGN8 in a three-part ligation manner.

Plasmid pD83 was constructed in a four-part ligation with the following fragments: BamHI-digested, alkaline phosphatase-treated pDPOT; the 1230 bp BglII-HindIII fragment of pHB105-4 containing the TPI1 promoter of S. The Saccharomyces cerevisiae TPI1 promoter and α-factor secretion leader linked to the human TPO cytokine domain coding sequence in the pMVR1 plasmid backbone (disclosed in U.S. Patent No. 5,155,027)]; the 540bp HindIII-XbaI fragment of pTPOGN8, which contains a part α-factor secretory leader coding sequence and the coding sequence of TPO variant, wherein the coding sequence of TPO variant comprises the cytokine structural domain connected at its C-terminus with the 18 residue polypeptide of SEQ ID NO:17; And 680bp XbaI-BamHI Saccharomyces cerevisiae TPI1 terminator fragment.

The plasmids shown in Table 2 were first constructed by inserting the BglII-EcoRI fragment of pD83 containing the TPI1 terminator and vector sequence into pUC19 (cut with SalI and EcoRI) using a pair of oligonucleotides shown in Table 3. As shown in Table 3, the resulting plasmids were named pTPOMI1, 2, 3, 4 and 5. Two BglI-SalIpD83 fragments comprising TPI1 promoter, MFα1 secretory leader peptide, 5'TPO coding region and vector sequence and EcoRI-BglI fragment comprising vector sequence were combined with pTPOMI1-5 (comprising 3'TPO coding sequence, TPI1 terminator sub and vector sequences) to construct pD117, pD119, pD121, pD123 and pD125, respectively.

table 3

Plasmids Oligonucleotides

pTPOMI1 ZC10086 (SEQ ID NO; 18)

ZC10087 (SEQ ID NO:19)

pTPOMI2 ZC10095 (SEQ ID NO:20)

ZC10096 (SEQ ID NO:21)

pTPOMI3 ZC10120 (SEQ ID NO:22)

ZC10121 (SEQ ID NO:23)

pTPOMI4 ZC10118 (SEQ ID NO:24)

ZC10119 (SEQ ID NO:25)

pTPOMI5 ZC10108 (SEQ ID NO:26)

ZC10109 (SEQ ID NO:27)

The plasmids were transformed into S. cerevisiae JG134 or M35. M35 was derived from pD79-transformed JG134 by UV mutagenesis overnight cultures. Cells were diluted 1:1000 and 50 microliters of the dilution was plated on YEPD (1% yeast extract, 2% peptone, 2% D-glucose, 0.004% adenine, 0.006% leucine). The plate was irradiated with ultraviolet light for 20 seconds in a dark room, placed in a light-tight box, and incubated at 30° C. for 2 days. Each plate was then replicated on fresh YEPD plates, covered with nitrocellulose membrane and incubated overnight at 30°C. The nitrocellulose membrane is then removed and any adhering yeast cells are washed away. Nitrocellulose membranes were developed by standard Western blot techniques using 5% milk blocking in 1X PBS and a mouse anti-human TPO antibody. Colonies showing high levels of secretion in the primary screen were further tested by Western blot and viability assays. A mutant designated GN35 consistently produced 2-4 times higher activity than the parental strain. GN35 was obtained by transforming pD79 with a 2 micron size plasmid containing the prokaryotic kanamycin resistance gene and the S. cerevisiae TPI1 gene. Transformants resistant to 2 mg/ml G418 were selected and cultured in YEPD containing G418, then in YEPGGE (0.004% adenine, 0.006% L-leucine, 1% yeast extract, 0.4% D-semi Lactose, 2% peptone, 3% glycerol, 1% ethanol). Cells were then screened for inability to grow on glucose as a carbon source, indicating loss of the triose phosphate isomerase gene on pD79. The processed cells were named M35 strain. Transformants were grown in a liquid medium containing 2% peptone, 1% yeast extract and 5% glucose under aeration for 70 hours. Plasmids pD79 and pD85 were included as controls. In addition, plasmids pHB109 (encoding the human TPO cytokine domain alone) and pBJ118 (encoding the same polypeptide as pD85 and pD125) were tested.

Media was collected and assayed as disclosed in Example 1. The test results are shown in Table 4.

Table 4

  Host   Plasmids   TPO(ng/ml)

JG134 pD117 26

                                                   

                                                   

pD123 35

pD125 3

pD79 70

pD85 3

Below the limit of pHB109

                                                                                                                      

M35 pD79 180 Example 3

A series of pDPOT-based plasmids encoding TPO polypeptides were constructed using conventional molecular biology techniques. Each encoded TPO polypeptide comprises a human TPO cytokine domain linked via a peptide bond to a C-terminal fragment derived from the human TPO C-terminal domain. The sequences of the C-terminal fragments of these polypeptides are shown in Table 5 below. Amino acids are indicated by the conventional one-letter symbols.

table 5

Plasmid C-terminal domain

pD91 APPDTAVPSRTSLVLTLN

(SEQ ID NO:5)

pD93 APPDTAVPSETSLVLTLN

(SEQ ID NO:6)

pD95 APPTTAVPSETSLVLTLN

(SEQ ID NO:7)

The plasmid was transformed into strain JG134, and transformants were grown as disclosed in Example 2. Media was harvested and assayed as disclosed in Example 1.

The results are shown in Table 6

Table 6

Plasmid TPO (unit/ml)

pDPOT 0

pD79 700

pD85 100

pD91 200

pD93 300

pD95 600

From the foregoing it will be appreciated that, while specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not to be limited except by the appended claims.

Sequence Listing (1) Basic Information (ⅰ) Applicant: ZymoGenetics, Inc.

1201 Eastlake Avenue East

Seattle, washington 98102

  United State of America(ⅱ) Title of invention: Vector, cell and method for preparing thrombopoietin polypeptide (ⅳ) Sequence number: 27(ⅳ) Contact address

(A) Recipient: ZymoGenetics, Inc.

(B) Street: 1201 Eastlake Avenue East

(C) City: Seattle

(D) State name: WA

(E) Country: United States

(F) Zip code: 98102(ⅴ) Computer readable form

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: PatentIn Release#1.0, version #1.25 (ⅵ) Current application materials:

(A) Application number:

(B) Submission date:

(C) Category: (ⅶ) Lawyer/Representative Information

(A) Name: Parker.Gary E.

(B) Registration number: 31,648

(C) Reference/Description Number: 95-34 (ⅶ) Telecom Information:

(A) Tel: 206-442-6673

(B) Fax: 206-442-6678 (2) Information about SEQ ID NO: 1 (ⅰ) Sequence identity

(A) Length: 1062 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (ⅸ) Features:

(A) Name/Key: mat-peptide

(B) Position: 64..1062 (ⅸ) Features:

(A) Name/Key: sig-peptide

(B) Position: 1..63 (ⅸ) Features:

(A) Name/Key: CDS

(B) Position: 1..1062 (ⅹⅰ) Sequence description: SEQ ID NO:1:ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA48Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu THR ALA-21 -20 -10Agg CTA ACG CTG CTG TCC AGC CCG GCG GCT CCT GCT GAC CGT GAC CGA GTC96ARG Leu Thr Leu Pro Ala Ala Cys ARG Val Val Val-5 10CTC AGTC. GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC144Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

                                                                                            

30 35 40GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC GAG ATG GAG GAG ACC AAG240Val Asp Phe Ser Leu Gly TTT Llu Trp Lys Th

45                  50                  55GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG288Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met60                  65              70                      75GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG336Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly

80 85 90CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GCC CTG AGC CTC384GLN Leu Serg Leu Leu Leu GLY Ala Leu Gln Serou

95 1005CTT GGA ACC CAG CAG CTT CCA CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT432leu GLY ThR Gln Leu Pro GLN GLY AR ALA His Lys Lys Les Les Les Lysrey

110 115 120CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG480Pro Asn Ala Ile Phe Leu Leu L Ser G Vally Gln His

125                 130                 135CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC528Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala140                 145                 150                 155CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG576Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu

160 165 170AAC GAAC CTC CCA AGG AGG AGG AGG ACT GGA TTG GAG GAG ACA ACTC Act624asn Glu PREU

175 180 185GCC TCA GCC AGA AGA ACT ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA672ALA Serg THR THR Gly Seru Leu Leu Leu Ln Trp Gln Getle GLN Gl Gl Gl Gl Gl G

190 195 200TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG720Phe Arg Ala Lys Ile Le Pro Gly S Leu Gl ghr Leu Asn

205                 210                 215GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA768Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly220                 225                 230                 235ACT CGT GGA CTC TTT CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG816Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro

240 245 250GAC Att TCC TCA GGA ACA TCA GAC ACA GGC CTG CCA CCC AAC CTC864ASP Ile Ser Ser Ser Ser Ser Gly Seru Pro Pro Pro ASN Leu

                                                                                                                       

270 275 280ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC960Thr Leu Phe Leu Leu Pro G Pro Val Thr Val Leu Pro Thr

285                 290                 295CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC1008His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser300                 305                 310                 315CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA1056Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu

320 325 330GGG TAA1062Gly (2) Information about SEQ ID NO: 2 (ⅰ) Sequence characteristics

(A) Length: 353 amino acids

(B) Type: amino acid

(D) Topological structure: linear (ⅹ) Molecular type: protein (ⅹⅰ) Sequence description: SEQ ID NO:2: Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala-21 -20 -15 -15 - 10arg Leu Thr Leu Serial Pro Ala Pro Pro Ala Cys ASP Leu ARG VAL-5 1 5 10LEU Serg ARG As His Val Leu His Serg Leu Ser

15 20 20 25Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55ALA GLN ASP ILE Leu Gly Ala Val Thr Leu Leu GLU GLU GLY VAL MET60 70ALA Ala ARG GLN Leu GLY Pro Thr Cyser Leu Leu Gly Gly

80 85 90Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 135arg PHE Leu Met Leu Val Gly GLY GLY SER Leu Cys Val ARG ALA140 145 150 155PRO THR THR ALA Val Pro Serg THR Leu Val Leu Thr Leu

160 165 170Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr

175 180 185Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly

190 195 200Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu

205 215ASP Gln Ile Pro Gly Tyr Leu asn ARG iLe His Glu Leu Leu asn Gly225 235thr ARG GLE PRO PRO GLY Pro Serg ARG Thr Leu Gly Ala Pro

240 245 250Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu

255 260 265Gln Pro Gly Tyr Ser Pro Ser Pro Thr His His Pro Pro Thr Gly Gln Tyr

270 275 280Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu

285 290 295HIS Pro Leu PRO ASP Pro Sera Pro THR Pro ThR Ser300 305 315pro Leu Leu asn Tyr Tyr His Gln Gln Gln Gln Gln Gln Glu

320 325 330Gly (2) Information about SEQ ID NO: 3 (ⅰ) Sequence characteristics

(A) Length: 178 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topological structure: linear (ⅱ) Molecular type: protein (ⅹⅰ) Sequence description: SEQ ID NO:3:Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr1 5 u u n u G Le 10 Pro lu 5 Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe

20 25 20Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln

35 40 45Gly Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser

50 55 60leu ASP Gln Ile Pro Gly Tyr Leu asn ARG Ile His Glu Leu Leu ASN65 70 80GLY ThR ARG GLE PRO GLE PRO GLY Pro Serg THR Leu Gly Ala

85 90 95Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn

100 105 110Leu Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln

115 120 125Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln

130 135 140leu him project

165 170 175Glu Gly (2) Information about SEQ ID NO: 4 (ⅰ) Sequence characteristics

(A) Length: 18 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topology: Linear (ⅱ) Molecular Type: Peptide (ⅹⅰ) Sequence Description: SEQ ID NO:4:Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Ala Leu Thr1 5 5 As n u 10 1 ) Information about SEQ ID NO:5 (i) Sequence Characteristics

(A) Length: 18 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topological structure: linear (ⅱ) Molecular type: peptide (ⅹⅰ) Sequence description: SEQ ID NO:5:Ala Pro Pro Asp Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr1 5 5                       Information about SEQ ID NO: 6 (i) Sequence characteristics

(A) Length: 18 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topological structure: linear (ⅱ) Molecular type: peptide (ⅹⅰ) Sequence description: SEQ ID NO:6:Ala Pro Pro Asp Thr Ala Val Pro Ser Glu Thr Ser Leu Val Leu Thr1 5 5 As As u n 10 Le 2 ) Information about SEQ ID NO: 7 (i) Sequence Characteristics

(A) Length: 18 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: peptide (ⅹ ⅰ) Sequence description: SEQ ID NO:7:Ala Pro Pro Thr Thr Ala Val Pro Ser Glu Thr Ser Leu Val Leu Thr1 5          Information about SEQ ID NO: 8 (i) Sequence Characteristics

(A) Length: 5 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: peptide (ⅹⅰ) Sequence description: SEQ ID NO: 8Ala Pro Pro Thr Thr1 5 (2) Information about SEQ ID NO: 9 (ⅹ) Sequence characteristics

(A) Length: 49 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC7623 (ⅹⅰ) Sequence description: SEQ ID NO: 9: GCGCGCAAGC TTGGACAAGA GAAGCCCGGC TCCTCCTGCT TGTGACCTC49 (2) Information about SEQ ID NO: 10 (ⅹ) Sequence characteristics

(A) Length: 51 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC7627 (ⅹⅰ) Sequence description: SEQ ID NO: 10: GCGCGCAAGC TTCTAGACT ATCAGACGCA GAGGGTGGAC CCTCCTACAA G51 (2) Information about SEQ ID NO: 11 (ⅰ) Sequence characteristics

(A) Length: 40 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC7868 (ⅹⅰ) Sequence description: SEQ ID NO: 11: GTGTGTGAAT TCTAGACTAT CAGACGCAGA GGGTCGACCC40 (2) Information about SEQ ID NO: 12 (ⅹ) Sequence characteristics

(A) Length: 35 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC7870 (ⅹⅰ) Sequence description: SEQ ID NO: 12: GTGTGTAAGC TTGGACAAGA GAAGCCCGGC GCCTC35 (2) Information about SEQ ID NO: 13 (ⅹ) Sequence characteristics

(A) Length: 82 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC8488 (ⅹ ⅹ) Sequence description: SEQ ID NO: 13: TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGCTTTG60ACTTTGAACT GATAGAGATC TT82 (2) Information about SEQ ID NO: 14 (i) Sequence characteristics

(A) Length: 82 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC8489 (ⅹ ⅹ) Sequence description: SEQ ID NO: 14: CTAGAAGATC TCTATCAGTT CAAAGTCAAA GCCAAAGAAG TTCTGGATGG AACAGCAGTG60GTTGGTGGGG CGACGCAGAG GG82 (2) Information about SEQ ID NO: 15 (ⅰ) Sequence characteristics

(A) Length: 85 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC8486 (ⅹⅰ) Sequence description: SEQ ID NO: 15: TCGACCCTCT GCGTCGGAGC TCCACCAGAC GAAGCTGTTC CAGACAGAGA CGAATTGGTT60TTGGAATTGA ACTGATAGAG ATCTT85 (2) Information about SEQ ID NO: 16 (ⅰ) Sequence characteristics

(A) Length: 85 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC8487 (ⅹⅰ) Sequence description: SEQ ID NO:16:CTAGAAGATC TCTATCAGTT CAATTCCAAA ACCAATTCGT CTCTGTCTGG AACAGCTTCG60TCTGGTGGAG CTCCGACGCA GAGGG85 (2) Information about SEQ ID NO:17 (ⅰ) Sequence characteristics

(A) Length: 18 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear (ⅹⅰ) Sequence description: SEQ ID NO:17:Ala Pro Pro Asp Glu 10 Ala Val Pro Asp Arg Asp Glu Leu Val Leu Glu1 5 5   SEQ ID: Le 8   of SE ( 1 ) Information (i) Sequence Features

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10086 (ⅹⅰ) Sequence description: SEQ ID NO:18:TCGACCCTCT GCGTCGCCCC ACCAACCACT TGATAGA37 (2) Information about SEQ ID NO:19 (ⅹ) Sequence characteristics

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10087 (ⅹⅰ) Sequence description: SEQ ID NO:19:GATCTCTATC AAGTGGTTGG TGGGGCGACG CAGAGGG37 (2) Information about SEQ ID NO:20 (ⅰ) Sequence characteristics

(A) Length: 49 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10095 (ⅹⅰ) Sequence description: SEQ ID NO:20:TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCTGATAGA49 (2) Information about SEQ ID NO:21 (ⅹ) Sequence characteristics

(A) Length: 49 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10096 (ⅹⅰ) Sequence description: SEQ ID NO:20:GATCTCTATC AGGATGGAAC AGCAGTGGTT GGTGGGGCGA CGCAGAGGG49 (2) Information about SEQ ID NO:22 (ⅹ) Sequence characteristics

(A) Length: 61 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10120 (ⅹⅰ) Sequence description: SEQ ID NO:22: TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGTGATAG60A61 (2) Information about SEQ ID NO:23 (ⅰ) Sequence characteristics

(A) Length: 61 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10121 (ⅹ ⅹ) Sequence description: SEQ ID NO: 23: GATCTCTATC ACAAAGAGT TCTGGATGGA ACAGCAGTGG TTGGTGGGGC GACGCAGAGG60G61 (2) Information about SEQ ID NO: 24 (ⅰ) Sequence characteristics

(A) Length: 76 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10118 (ⅹ ⅹ) Sequence description: SEQ ID NO: 24: TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGTTTTG60ACTTTGAACT GATAGA76 (2) Information about SEQ ID NO: 25 (ⅰ) Sequence characteristics

(A) Length: 76 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10119 (ⅹⅰ) Sequence description: SEQ ID NO:25:GATCTCTATC AGTTCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGTTGGT60GGGGCGACGC AGAGGG76 (2) Information about SEQ ID NO:26 (ⅰ) Sequence characteristics

(A) Length: 82 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Line type (ⅶ) Direct source:

(B) Clone: ZC10108 (ⅹⅰ) Sequence description: SEQ ID NO:26: TCGACCCTCT GCGTCAGGCG GGCCCCACCA ACCACTGCTG TTCCATCCAG AACTTCTTTG60GTTTTGACTT TGAACTGATA GA82 (2) Information about SEQ ID NO:27 (ⅰ) Sequence characteristics

(A) Length: 82 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: line type (ⅶ) Direct source:

(B) Clone: ZC10109 (ⅹⅰ) Sequence description: SEQ ID NO:27:GATCTCTATC AGTTCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGTTGGT60GGGGCCCGCC TGACGCAGAG GG82

Claims (21)

1. An expression vector replicable in a eukaryotic host cell comprising the following operably linked elements: (a) a transcriptional promoter; (b) a first DNA fragment encoding a secretory leader peptide; (c) a second DNA fragment encoding a thrombopoietin (TPO) polypeptide comprising C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker comprising 1 or 2 amino acid residues, The proviso is that X alone or in combination with C or B does not provide a dibasic amino acid pair; and B is a polypeptide comprising residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein B Up to 35% of said residues in each can be replaced by other amino acid residues; and (d) Transcription terminator. 2. The expression vector according to claim 1, wherein said vector is replicable in yeast. 3. The expression vector according to claim 2, wherein the secretory leader peptide is a secretory leader peptide of Saccharomyces cerevisiae alpha factor. 4. The expression vector according to claim 1, wherein y is at least 9. 5. The expression vector according to claim 1, wherein B comprises Thr-Thr dipeptide. 6. The expression vector according to claim 1, wherein B does not comprise Arg-Arg dipeptide. 7. The expression vector according to claim 1, wherein at most 25% of said residues in B are replaced by other amino acid residues, respectively. 8. The expression vector according to claim 1, wherein residues 1 to 5 of B are Ala-Pro-Pro-Thr-Thr (SEQ ID NO: 8). 9. The expression vector according to claim 1, wherein the fourth residue of B is Thr or Asp. 10. The expression vector according to claim 1, wherein y is at least 10 and the 10th residue of B is Arg or Glu. 11. The expression vector according to claim 1, wherein y is at least 14 and the 14th residue of B is Val or Ala. 12. The expression vector according to claim 11, wherein B is Ala-Pro-Pro-Thr-Thr-Ala-Val-Pro-Ser-Arg-Thr-Ser-Leu-Ala-Leu-Thr-Leu-Asn (SEQ ID NO: 4). 13. The expression vector according to claim 1, wherein B is selected from SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. 14. The expression vector according to claim 1, wherein X is a peptide bond. 15. The expression vector according to claim 1, wherein X is a single amino acid residue. 16. The expression vector according to claim 1, wherein C comprises residues 1 to 152 of SEQ ID NO:2. 17. The cultured eukaryotic cell containing the expression vector according to claim 1, wherein said cell synthesizes and secretes said TPO polypeptide. 18. Yeast cells according to claim 17. 19. The thrombopoietin polypeptide is characterized in that it comprises a C-X-B amino acid backbone, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker containing 1 or 2 amino acid residues, and the restriction is that X is alone or combined with C or B, when combined, do not provide a pair of dibasic amino acids; and B is a polypeptide comprising residues 1 to y of SEQ ID NO: 3, wherein y is an integer from 5 to 18, and at most 35% of said residues in B groups can be replaced by other amino acid residues, respectively. 20. A method of producing a TPO polypeptide, comprising: Host cells transfected or transformed with an expression vector capable of replicating in the host cell and comprising the following operably linked elements are cultured: (a) a transcriptional promoter; (b) a first DNA fragment encoding a secretory leader peptide; (c) A second DNA fragment encoding thrombopoietin comprising C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker containing 1 or 2 amino acid residues, provided that X No dibasic amino acid pairs are provided, alone or in combination with C or B; and B is a polypeptide comprising residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and up to 35% of B is Said residues of can be replaced by other amino acids, respectively; and (d) a transcription terminator; wherein the host cell expresses the linked first and second DNA fragments to synthesize the TPO polypeptide; and The TPO polypeptide is recovered. twenty one. A method of increasing the number of platelets in a mammal, comprising administering to said animal a thrombopoietin polypeptide in combination with a pharmaceutically acceptable carrier, the polypeptide being characterized by comprising an amino acid backbone of C-X-B, wherein C is a human thrombopoietin cytokine Domain polypeptide; X is a peptide bond or a linker comprising 1 or 2 amino acid residues, with the proviso that X alone or in combination with C or B does not provide a dibasic amino acid pair; and B is a residue comprising SEQ ID NO:3 A polypeptide with bases 1 to y, wherein y is an integer from 5 to 18, and up to 35% of said residues in B may be replaced by other amino acid residues, respectively.