Definitions

• This invention relates to a novel protein which has an activity to stimulate or increase platelet production in vivo in a specific manner or to enhance proliferation and differentiation of megakaryocyte progenitor cells, to a DNA sequence coding for said protein, and to a process for the production of the same.
• Megakaryocytes are large cytoplasm-rich multinucleate cells which produce platelets and can be found mainly in bone marrow. Megakaryocytes originate from a pluripotent hematopoietic stem cell in bone marrow. A primitive pluripotent stem cell differentiates to some degree into megakaryocyte progenitor cells, which are committed to the megakaryocytic lineage. Megakaryocyte progenitor cells proliferate and differentiate into megakaryocytes. Megakaryocytes ⁇ further undergo polyploidization and cytoplasmic maturation and finally release their anuclear cytoplasmic fragments, namely platelets, into the circulation. Two to four thousands of platelets are on an average formed from one mature megakaryocyte.
• megakaryocytes are typically localized on the albuminal surface of the sinus endothelium in the bone marrow and produce cytoplasmic processes that extend into the sinusoid where they undergo fragmentation of platelets.
• a platelet the most important function of a platelet is the production of a thrombus in a hemostatic mechanism. If the hemostatic mechanism does not function properly due to a decrease in the platelet number, a tendency towards hemostasis results.
• the presence of a specific regulatory mechanism has been suggested with regard to megakaryocytopoiesis and thrombopoiesis. Platelets are maintained in effective numbers in healthy people and normal animals. However, it is known that, when an anti-platelet antibody is administered to a normal animal, only the number of platelets decreases sharply within a short period of time, starts to increase thereafter and temporarily exceeds the normal level, but finally returns to the normal level.
• Platelets Most important function of platelets is the formation of blood clot in the hemostatic mechanism. Bleeding tendency occurs when normal function of the hemostatic mechanism is spoiled by thrombocytopenia. In the field of radiotherapy and chemotherapy of cancers, thrombocytopenia caused by bone marrow suppression is a mortal complication, and platelet transfusion is applied to such patients in order to prevent bleeding tendency. Platelet transfusion is also applied to patients after bone marrow transplantation or of aplastic anemia.
• Platelets for use in such platelet transfusion are prepared by plateletpheresis from blood of healthy blood donors, but such platelets for transfusion use have a short shelf life and a possibility of causing contamination by bacterial infection.
• the platelet transfusion also has a possible danger of exposing patients to dangerous viruses such as human immunodeficiency virus (HIV) or various hepatitis viruses, of inducing antibodies specific for a major histocompatibility antigen (HLA) of the transfused platelets or of causing graft versus host disease (GVHD) due to contaminated lymphocytes in the platelets for transfusion use.
• HIV human immunodeficiency virus
• HLA major histocompatibility antigen
• GVHD graft versus host disease
• Megakaryocyte colony stimulating factor (Meg-CSF) is a regulatory factor which stimulates proliferation and differentiation of CFU-MK to form megakaryocyte colonies in a semi-solid culture medium.
• the other regulatory factor called megakaryocyte potentiating factor (Meg-Pot), megakaryocyte stimulating factor, thrombopoiesis stimulating factor or the like, acts mainly upon immature or mature megakaryocytes thereby enhancing their differentiation and maturation.
• the Meg-Pot is detectable in combination with Meg-CSF activity in some cases.
• TPO thrombopoietin
• cytokines whose genes have been cloned were examined for their abilities to stimulate megakaryocytopoiesis and thrombopoiesis.
• Human IL-3 stimulated formation of human megakaryocyte colonies (Bruno et al., Exp. Hematol., vol.16, pp.371 - 377, 1988) and, at least in monkey, increased platelet counts (Donahue et al., Science, vol.241 , p.1820, 1988).
• IL-3 is a factor which exerts its effects upon proliferation and differentiation of all hematopoietic cells, it can be distinguished from specific regulatory factors controlling megakaryocytopoiesis and platelet production.
• Human IL-6 did not show Meg-CSF activity, but acted upon immature megakaryocytes and then enhanced their differentiation into mature megakaryocytes (Williams et al., Exp. Hematol., vol.18, p.69, 1990).
• In vivo administration of IL-6 induced platelet production and promoted both maturation and shifted to higher ploidy of bone marrow megakaryocytes in primates, but also caused side effects such as reduction of body weight, induction of acute phase protein (Asano et al., Blood, vol.75, pp.1602 - 1605, 1990; Stahl et al., Blood, vol.78, pp.1467 - 1475, 1991 ).
• Human IL-11 showed no Meg-CSF activity, but exerted Meg-Pot activity and promoted platelet production in mice (Neben et al., Blood, vol.81 , pp.901 - 908, 1993).
• human LIF significantly increased platelet counts in primates (Mayer et al., Blood, vol.81 , pp.3226 - 3233, 1993), but its in vitro action upon megakaryocytes was weak (Burstein et al., J. Cell. Physiol., vol.153, pp.305 - 312, 1992).
• cytokines Although clinical application of these cytokines as platelet increasing factors is expected, their functions are not specific for the megakaryocyte lineage and they cause side effects. Therefore, development of a platelet increasing factor which is specific for the megakaryocyte-platelet system and causes less side effects has been called for in the clinical field.
• Meg-CSF, Meg-pot or TPO activity has been found in serum, plasma or urine of thrombocytopenic patients or animals, or in culture supernatant of certain human cultured cell lines. However, whether these activities are due to the presence of a single factor or a combination of several factors or whether they are different from known cytokines is presently unknown.
• Meg-CSF activity has been found in sera of cancer patients receiving intensive cytotoxic chemotherapy and bone marrow-transplant patients (Mazur et al., Exp. Hema tol., vol.12, pp.624 - 628, 1984; de Alarcon and Schmieder, Prog. Clin. Bio. Res., vol.215, pp.335 - 340, 1986). Hoffman et al. have reported that Meg-CSF with an apparent molecular weight of 46,000 was purified from plasma of patients with hypomegakaryocytic thrombocytopenia (Hoffman et al., J. Clin.
• Meg-CSF and TPO-like activities have also been detected in urine samples of patients with aplastic anemia and severe ITP (Kawakita et al., Br. J. Haematol., vol.48, pp.609 - 615, 1981 ; Kawakita et al., Blood, vol.556 - 560, 1983).
• Kawakita et al. have further reported that the Meg-CSF activity found in the urine extract of aplastic anemia patients showed an apparent molecular weight of 45,000 by gel filtration under a dissociation condition (Kawakita et al., Br. J. Haematol., vol.62, pp.715 - 722, 1986).
• Meg-CSF megakaryocyte stimulating factor
• Turner ef al. have purified a megakaryocyte stimulating factor (MSF) having Meg-CSF activity from the urine of bone marrow transplant patients and cloned its gene (Turner et al., Blood, vol.78, p.1106 279a, 1991 , (abstr., supple. 1)).
• MSF megakaryocyte stimulating factor
• This MSF has a molecular weight of 28,000 to 35,000. Identity of this factor with the Meg-CSF so far detected in serum and plasma samples of thrombocytopenic patients and its platelet increasing activity remain to be elucidated.
• a substance having a TPO-like activity with a molecular weight of 32,000 has been purified from the culture supernatant of a human embryonal kidney-derived cell line (HEK cells) and its biological and biochemical properties have extensively been examined, but its structure is still unknown (McDonald et al., J. Lab. Clin. Med., vol.106, pp.162 - 174, 1985; McDonald, Int. J. Cell Cloning, vol.7, pp.139 - 155, 1989).
• Tayrien and Rosenberg have also purified a factor having an apparent molecular weight of 15,000 from thrombocytopenic rabbit plasma and culture supernatant of HEK cells, which stimulates production of platelet factor 4 in a rat megakaryocytic cell line, but with no information on its structure (Tayrien and Rosenberg, J. Biol. Chem., vol.262, pp.3262 - 3268, 1987).
• Nakeff has found a Meg-CSF activity in serum of thrombocytopenic mice induced by anti-platelet administration (Nakeff, "Experimental Hematology Today” ed. ' by Baum and Ledney, Springer-Verlag, NY, pp.111 - 123, 1977).
• sera from thrombocytopenic rabbits enhanced maturation of megakaryocytes (Keller ef al., Exp. Hematol., vol.16, pp.262 - 267, 1988; Hill et al., Exp.
• Miura et al. have detected a Meg-CSF activity in plasma of rats rendered thrombocytopenic by sublethal whole body irradiation (Miura ef al., Blood, vol.63, pp.1060 - 1066, 1984), and suggested that induction of the Meg-CSF activity in vivo is related to decreased megakaryocytes, but not decreased platelets, because this activity did not change by platelet transfusion (Miura et al., Exp. Hematol., vol.16, pp.139 - 144, 1988).
• Objects of the present invention are to isolate a TPO protein from natural sources and ' identify it, the TPO protein having an activity to stimulate or increase platelet production i n vivo and/or to enhance proliferation and differentiation of megakaryocyte progenitor cells (hereinafter, referred to as "TPO activity"); and to isolate a gene coding for the TPO protein and to provide a process for the production of said protein in a homogeneous quality and in a large quantity with recombinant DNA techniques. Success in accomplishing such objects will lead to the substitution or frequency- reduction of the currently used platelet transfusion, and such a novel protein will also be used for treatment and diagnosis of platelet disorders.
• TPO activity an activity to stimulate or increase platelet production i n vivo and/or to enhance proliferation and differentiation of megakaryocyte progenitor cells
• the present invention therefore, relates to (i) a purified and isolated DNA sequence encoding a protein having TPO activity, which is selected from the group consisting of:
• the present invention further relates to a pharmaceutical composition comprising an effective amount of said protein having TPO activity, and to a method for treating platelet disorders, especially thrombocytopenia, comprising administering said protein to patients having the disorders.
• Figure 1 shows Sephacryl S-200HR gel filtration chromatography of Phenyl Sepharose 6 FF/LS F2 derived from XRP.
• Figure 2 shows Capcell Pak C1 reverse phase chromatography of YMC-pack CN-AP TPO-active fraction derived from low molecular TPO sample (Sephacryl S-200HR F3) of XRP.
• Figure 3 shows a SDS-PAGE analysis of Capcell Pak C1 TPO-active fraction (FA) from low molecular TPO sample of XRP.
• Figure 4 shows peptide maps on C18 reverse phase HPLC of rat TPO isolated by SDS-PAGE.
• the peptide fragments were obtained by systematic hydrolysis with three proteases.
• Figure 5 shows TPO activity derived from XRP in the rat CFU-MK assay system.
• Figure 6 shows a construction of expression vector pEF18S.
• Figure 7 shows TPO activity in the culture supernatant of COS1 cells into which pEF18S-A2 ⁇ was introduced in the rat CFU-MK assay system.
• Figure 8 shows TPO activity in the culture supernatant of COS1 cells into which pEF18S-HL34 was introduced in the rat CFU-MK assay system.
• Figure 9 shows TPO activity in the culture supernatant of COS1 cells into which pHT1 -231 was introduced in the rat CFU-MK assay system.
• Figure 10a shows TPO activity in the culture supernatant of COS1 cells into which pHTF1 was introduced in the rat CFU-MK assay system.
• Figure 10b shows TPO activity in the culture supernatant of COS1 cells into which pHTF1 was introduced in the M-07e assay system.
• Figure 11 shows a restriction map of the ⁇ H G T 1 clone and construction of pHGT1 and pEFHGTE.
• Figure 12a shows TPO activity in the culture supernatant of COS1 cells into which pEFHGTE was introduced in the rat CFU-MK assay system.
• Figure 12b shows TPO activity in the culture supernatant of COS1 cells into which pEFHGTE was introduced in the M-07e assay system.
• Figure 13a shows TPO activity in the culture supernatant of COS1 cells into which pHT1 -21 1 #1 , pHT1 - 191#1 , or pHT1 -171 #2 was introduced in the rat CFU-MK assay system.
• Figure 13b shows TPO activity in the culture supernatant of COS1 cells into which pHT1 -163#2 was introduced in the rat CFU-MK assay system.
• Figure 14 shows TPO activity in the culture supernatant of COS1 cells into which pHT1 -21 1 #1 , pHT1 - 191#1 , pHT1 -171 #2, or pHT1 -163#2 was introduced in the M- 07e assay system.
• Figure 15 is a chromatogram of a reverse phase chromatography (Vydac C4 column) for purification of human TPO from the culture supernatant of CHO cells into which pDEF202-hTPO-P1 has been introduced and allowed the TPO to be expressed.
• Figure 16 is a photograph showing the SDS-PAGE separation of the human TPO purified from the culture supernatant of CHO cells into which pDEF202-hTPO-P1 has been introduced and allowed the TPO to be expressed.
• Figure 17 is a chromatogram of a reverse phase chromatography for purification of human TPO from J cfiii into which pCFM536/h6T(1 -163) has been introduced and allowed the TPO to be expressed.
• Figure 18 is a photograph showing the SDS-PAGE separation of the variant human TPO, h6T(1-163) isolated and purified from E ⁇ cj ⁇ into which pCFM536/h6T(1 -163) has been introduced . and allowed the TPO to be expressed.
• Figure 19 shows an elution pattern of hTP0163 on Superdex 75 pg column with regard to the purification of the hTPO 163 from the culture supernatant, as a starting material, prepared by transfection of the human TPO expression plasmid pDEF202-hTPO163 into CHO cells.
• the protein amount was determined at 220 nm.
• Figure 20 shows an SDS-PAGE analysis of the standard hTP0163 eluted from Superdex 75 pg column upon the purification of the hTP0163 from the culture supernatant, as a stating material, prepared by transfection of the human TPO expression plasmid pDEF202-hTPO163 into CHO cells.
• the hTP0163 was silver-stained on the gel.
• Figure 21 shows a structure of the expression vector pSMT201.
• Figure 22 shows a graph showing the TPO activity as determined by M-07e assay, in the culture supernatant of the COS7 that ⁇ GL-TPO, N3/TPO or 09/TPO has been introduced into and then expressed.
• Figure 23 is a graph showing a TPO activity, as determined by M-07e assay, in the supernatants of COS7 cell cultures that an insertion or deletion derivative of human TPO has been introduced into and expressed.
• Figure 24 shows increased platelet count in mice administered TPO via intravenous and subcutaneus injection.
• Figure 25 shows dose dependent increased platelet count in mice following subcutaneous injection of TPO.
• Figure 26 shows TPO induced increase in platelet count following treatment of mice with 5-FU to induce thrombocytopenia.
• Figure 27 shows TPO induced increase in platelet count following treatment of mice with nimustine hydrochloride to induce thrombocytopenia.
• Figure 28 shows TPO induced increase in platelet count thrombocytopenic mice following bone marrow transplant.
• Figure 29 shows TPO induced platelet count following x-ray irradiation of mice to ind uce thrombocytopenia.
• Figure 30 shows dose-dependent increase in platelet count following administration of truncated TPO (amino acids 1-163 in SEQ ID NO: 6).
• Figure 31 shows increase in platelet count following administration of truncated TPO (amino acids 1 -163 in SEQ ID NO: 6) following administration of nimustine hydrochloride to induce thrombocytopenia.
• Figure 32 shows that increasing concentrations of Mpl-X added to the human megakaryocyte culture system increasingly block megakaryocyte development.
• Figure 33 describes TPO activity, as determined by M-07e assay, of TPO derivatives [Met -2 , Lys -1 , Ala 1 , Val 3 , Alg 133 ]TPO(1-163), [Met" , Lys- 1 , Ala 1 , Val 3 , Pro 148 ]TPO(1- 163) and [Mef 2 , Lys- 1 , Ala 1 , Va
• Figure 34 describes TPO activity, as determined by M-07e assay, of TPO derivatives [Met -2 , Lys -1 , Ala 1 , Val 3 , Alg 12 9]TPO(1-163), [Mef 2 , Lys' 1 , Ala 1 , Val 3 , Arg 14 3] ⁇ P0(1 - 163), [Met- 2 , Lys- 1 , Ala 1 , Val 3 , Leu 82 ]TPO(1-163), [Mef 2 , Lys- Ala 1 , Val 3 , Leu 146 ]TPO(1-163) and [Met "2 , Lys '1 , Ala 1 , Val 3 , Arg 59 ]TPO(1-163) expressed in E. coli.
• thrombopoietin (TPO) polypeptides having the biological activity of specifically stimulating or increasing platelet production and comprising the amino acid sequence 1 to 332 of SEQ ID NO: 6 or derivatives thereof.
• Illustrative polypeptides include those which consist of the amino acid sequences 1 to 163 of SEQ ID NO: 6; 1 to 232 of SEQ ID NO: 6; 1 to 151 of SEQ ID NO: 6; the mature sequence of amino acids of SEQ ID NOs: 2, 4 and 6; including those having from 1 to 6 NH 2 terminal amino acids deleted.
• Additional illustrative polypeptides of the invention include [Thr 33 , Thr 333 , Ser 334 lie 335 , Gly 336 , Tyr 337 , Pro 338 , Tyr 339 , Asp 340 , Val 341 Pro 342 , Asp 343 , Tyr 344 , Ala 345 , Gly 346 , Val 347 , His 343 His 349 , His 350 , His 351 , His 352 , His 353 ]TPO, [Asn 25 , Lys 231 Thr 333 , Ser 334 , lie 335 , Gly 336 , Tyr 337 , Pro 338 , Tyr 339 Asp 3 0, Val 341 , Pro 342 , Asp 343 , Tyr 344 , Ala 345 , Gly 34 ⁇ Val 347 , His 348 , His 349 , His 350 , His 351 , His 352 , His 353 ]TPO [Asn 25 ]TPO and
• polypeptides of the invention include derivatives of a TPO polypeptide [ ⁇ His ]TPO(1-163), [ ⁇ Arg 11 7]TPO(1-163), [ ⁇ GIy 1 6]TPO(1 - 163), [His 33 , Thr 33' , Pro 34 ]TPO(1-163), [His 33 , Ala 33 ', Pro 34 ]TPO(1-163), [His 33 , Gly 33 ', Pro 34 , Ser 33 ]TPO(1-163), [Gly 11 6, Asn 1 6', Arg 117 ]TPO(1-163), [Gly 11 6, Ala 1 6'_ Arg 11 7]TPO(1-163), [Gly 1 6, Arg 1 ?]TPO(1 -163), [Ala 1 , Val 3 , Arg 1 9]TPO(1-163), [Ala 1 , Val 3 , Arg 133 ]TPO(1 - 163), [Ala 1 , Val 3
• the TPO polypeptides of the invention also include those which are covalently bonded to a polymer, preferably polyethylene glycol.
• the TPO polypeptides of the invention may further comprise the amino acids [Met ⁇ 2 -Lys ⁇ 1 ], [Met -1 ] or [Gly' 1 ].
• DNAs provided by the invention include those which encode the TPO polypeptide and derivatives described above and are suitably provided as cDNA, genomic DNA and manufactured DNAs.
• Also provided by the invention are processes for producing a TPO polypeptide as described above comprising the steps of expressing a polypeptide encoded by a DNA of the invention in a suitable host, and isolating said TPO polypeptide.
• the TPO polypeptide expressed is a Met "2 - Lys "1 polypeptide
• processes can further include the step of cleaving Met "2 -Lys *1 from said isolated TPO polypeptide.
• TPO polypeptides such as glutathione-S-transferase (GST) fusion polypeptides.
• GST glutathione-S-transferase
• DNA encoding an amino terminal GST polypeptide, a thrombin recognition peptide and a TPO polypeptide is introduced into a suitable host, the fusion polypeptide is isolated and the GST moiety removed by treatment with thrombin.
• Resulting TPO polypeptides are of the [Gly -1 ] structure.
• procaryotic or eucaryotic host cells transformed or transfected with a DNA sequence according to the invention in a manner enabling said host cell to express a polypeptide having the biological activity of specifically stimulating or increasing platelet production.
• compositions of the invention comprise an effective amount of a TPO polypeptide or derivative in combination with a pharmaceutically acceptable carrier and are susceptible to use in treatment of platelet disorders, particularly the treatment of thrombocytopenia, such as is induced by chemotherapy, radiotherapy or bone marrow transplantation. Corresponding treatment methods are provided by the invention.
• the present invention provides antibodies specifically immunoreactive with TPO polypeptides and derivatives thereof as described above. Such antibodies are useful in methods for isolation and quantification of TPO polypeptides of the invention.
• DNA sequence of the present invention includes a DNA sequence which encodes the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 of the Sequence Listing attached hereto.
• DNA sequence of the present invention is a DNA sequence which encodes a partly modified (substitution, deletion, insertion or addition) version of the aforementioned amino acid sequence shown in SEQ ID NO: 2, 4 or 6, provided that such modifications do not spoil the TPO activity. That is, DNA sequences coding for TPO derivatives are also included in the present invention.
• the DNA sequence of the present invention includes DNA sequences which encode protein molecules whose amino acid sequences are substantially the amino acid sequences shown in SEQ ID NO: 2, 4 or 6.
• amino acid sequences are substantially the amino acid sequences shown in SEQ ID NO: 2, 4 or 6 as used herein means that said amino acid sequences include those represented by SEQ ID NO: 2, 4 or 6 as well as those represented SEQ ID NO: 2, 4 or 6 which have partial modification therein such as substitution, deletion, insertion, addition or the like, provided that such modifications do not spoil the TPO activity.
• DNA sequence of the present invention consists essentially of a DNA sequence encoding a protein having a TPO activity.
• DNA sequence which encodes the amino acid sequence includes all the DNA sequences which may have degeneracy in nucleotide sequences.
• the DNA sequence of the present invention also comprises the following sequences:
• DNA sequence of the present invention also comprises the following sequences:
• stringent hybridization conditions are those employed in the examples addressing PCR amplification of DNAs of the invention using degenerate and/or unique sequence oligonucleotide primers (probes). See also for example, Chapters 11 and 14 of Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) and Unit 2.10 in Current Protocols in Molecular Biology, Ausubel et al., Eds., Current Protocols, USA, 1993).
• DNA sequence of the present invention is a DNA sequence which encodes a protein having TPO activity and comprises the nucleotide sequence encoding positions 1-163 of the amino acid sequence represented by SEQ ID NO: 6.
• DNA sequences may be supplemented with a restriction enzyme cleavage site and/or an additional DNA sequence at the initiation, termination or intermediate site which facilitate construction of readily expressed vectors.
• a preferred codon for the gene expression in the host may be incorporated.
• An example of the DNA sequence of the present invention is a cDNA molecule which is obtained by preparing mRNA from cells of mammals, including human, and then screening the cDNA in the usual way from a cDNA library prepared by a known method.
• Sources of the mRNA of this case include cells of a rat hepatocyte-derived cell line McA- RH8994, HTC cells, H4-II-E cells, rat liver, kidney, brain and small intestine human liver and the like.
• genomic DNA molecule which is obtained by screening it in the usual way from a genomic library prepared by a known method from cells of mammals including human.
• Sources of the genomic DNA of this case include chromosomal DNA preparations obtained from human, rat, mouse and the like.
• a DNA sequence which encodes a TPO derivative may be obtained from the thus obtained cDNA sequence that encodes a protein having TPO activity, by modifying the cDNA sequence making use of the known site-directed mutagenesis thereby effecting partial modification of the corresponding amino acid sequence.
• a DNA sequence which encodes a partially modified amino acid sequence can be obtained easily by chemical synthesis.
• the DNA sequence of the present invention is a useful material for the large scale production of a protein having TPO activity making use of various recombinant DNA techniques.
• the DNA sequence of the present invention is useful as a labeled probe for the isolation of a gene which encodes a TPO-related protein, as well as cDNA and genomic DNA coding for TPO of other mammalian species. It is also useful in the gene therapy of human and other mammalian species.
• the DNA sequence of the present invention is useful for the development of a transgenic mammalian species which can be used as a eucaryotic host for large scale production of TPO (Palmiter et al., Science, vol.222, pp.809 - 814, 1983).
• Also provided by the present invention are a vector in which the aforementioned DNA sequence coding for a protein having TPO activity is integrated, host cells transformed with said vector and a process for the production of a protein having TPO activity which comprises culturing said host cells and separating and purifying the expressed protein having TPO activity.
• Examples of host cells useful in this case include cells of prokaryotes, such as, E. coli and the like, and of eukaryotes, such as yeasts, insects, mammals and the like.
• Illustrative examples of mammalian cells include COS cells, Chinese hamster ovary (CHO) cells, C-127 cells, baby hamster kidney (BHK) cells and the like.
• yeasts include, a baker's yeast (Saccharomyces cerevisiae), a methanol assimilating yeast (Pichia pastoris) and the like.
• Illustrative examples of insect cells include, silkworm cultured cells and the like.
• pKC30 Shiatake H. and M. Rosenberg, Nature, 292, 128 - 132, 1981
• pTrc99A Mann E. et al., Gene, 69, 301 - 315, 1988
• pCAGGS Newa et al., Gene, 108, 193 - 200, 1991
• pcDL-SR ⁇ 296 Takebe et al., Mol. Cell.
• Biol., 8, 466 - 472, 1988) or the like may be used.
• yeast cells pG-1 (Schena M. and Yamamoto K.R., Science, 241 , 965 - 967, 1988) or the like may be used.
• a transfer vector for recombinant virus construction for example, pAc373 (Luckow et al., Bio/Technology, 6, 47 - 55, 1988) may be used for the transformation of silkworm cells.
• each of these vectors may contain a replication origin, selection marker(s), a promoter and the like, as well as an RNA splice site, a polyadenylation signal and the like in the case of vectors for use in eukaryotic cells.
• a sequence derived from, for example, SV40, adenovirus, bovine papilloma virus or the like may be used in vectors for mammalian cells.
• a sequence derived from ColEI , R factor, F factor or the like may be used in vectors for E. coli cells.
• a sequence derived from 2 ⁇ m DNA, ARS1 or the like may be used in vectors for yeast cells.
• promoters for gene expression those which are derived from, for example, retrovirus, polyoma virus, adenovirus, SV40 and the like may be used in vectors for mammalian cells.
• a promoter derived from bacteriophage ⁇ for example, trp, Ipp, lac or tac promoter, may be used in vectors for E. coli cells.
• ADH, PH05, GPD, PGK or MAF ⁇ promoter may be used in vectors for baker's yeast cells, and AOX1 promoter or the like in vectors for methanol assimilating yeast cells.
• a promoter derived from a nuclear polyhedrosis virus may be used in vectors for silkworm cells.
• selection markers useful in vectors for mammalian cells include a neomycin (neo) resistance gene, a thymidine kinase (TK) gene, a dihydrofolate reductase (DHFR) gene, an E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene and the like.
• Illustrative examples of selection markers useful in vectors for E. coli cells include a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene and the like, and those for yeast cells include Leu2, Trp1 , Ura3 and the like genes.
• Production of a protein having TPO activity making use of appropriate combinations of these host-vector systems may be performed by transforming appropriate host cells with a recombinant DNA obtained by inserting the gene of the present invention in an appropriate site of the aforementioned vector, culturing the resulting transformant and then separating and purifying the polypeptide of interest from the resulting cells, or culture medium or filtrate. Commonly used means and procedures may be applied to this process in combination.
• the original signal sequence When expressing the gene of interest, the original signal sequence may be modified or replaced by a signal sequence derived from another protein in order to obtain homogeneous N-terminal of the expressed product. Homogenization of the N-terminal may be carried out by modification (substitution or addition)- of the amino acid residues in the N-terminal or its vicinity.
• lysine residue may be further supplemented in addition to methionine residue.
• novel protein of the present invention having TPO activity includes proteins each of which comprising the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
• TPO derivatives whose amino acid sequences are partially modified are also included in the present invention, provided that the TPO activity is not spoiled by the modification.
• the protein of the present invention includes protein molecules whose amino acid sequences are substantially the amino acid sequences shown in SEQ ID NO: 2, 4 or 6.
• amino acid sequences are substantially the amino acid sequences shown in (or represented by) SEQ ID NO: 2, 4 or 6" as used herein means that said amino acid sequences include those shown in SEQ ID NO: 2, 4 or 6 as well as those shown in SEQ ID NO: 2, 4 or 6 which have partial modification therein such as substitution, deletion, insertion, addition and the like, provided that such modifications do not spoil the TPO activity.
• the protein of this invention includes a protein containing the positions 7-151 of the amino acid sequence shown in SEQ ID NO: 6 and having a TPO activity. Also included in the protein of the present invention is a protein which has TPO activity and comprises positions 1 -163 of the amino acid sequence represented by SEQ ID NO: 6.
• TPO derivatives of the present invention include a derivative whose stability and durability in vivo were improved by amino acid modification (substitution, ' deletion, insertion or addition), a derivative in which at least one potential glycosylation was changed by deletion or addition, a derivative in which at least one cysteine residue was deleted or substituted by other amino acid residue (alanine or serine residue for example).
• the protein of the present invention is characterized in that it is separated and purified from host cells transformed with a recombinant vector containing a cDNA molecule, a genomic DNA molecule or a DNA fragment obtained by chemical synthesis.
• a protein in which an initiation methionine residue is added to the N-terminal side of a protein molecule having TPO activity is obtained, which is also included in the present invention.
• the produced protein having TPO activity may or may not be glycosylated, and each of such cases is included in the protein of the present invention.
• the protein of the present invention also includes naturally occurring TPO-active proteins purified and isolated from natural sources such as cell culture medium having TPO activity or human urine, serum and plasma.
• a process for the purification of TPO from such natural sources is also included in the present invention.
• Such a purification process may be performed by employing one of or a combination of usually used protein purification steps such as an ion exchange chromatography, a lectin affinity chromatography, a triazine dye affinity chromatography, a hydrophobic interaction chromatography, a gel filtration chromatography, a reverse phase chromatography, a heparin affinity chromatography, a sulfated gel chromatography, a hydroxylapatite chromatography, isoelectric focussing chromatography, a metal chelating chromatography, a preparative electrophoresis, an isoelectric focussing gel electrophoresis and the like.
• TPO is the ligand for Mpl (de Sauvage et al., Nature 369: 533-538 (1994); Bartley et al., Cell 77: 1 1 17-1 124 (1994); Kaushansky et al., Nature 369: 565-568 (1994)), by which the TPO may be purified using an affinity gel column that the Mpl has been coupled to a resin.
• an example of the column is an Mpl-X column which is prepared by coupling a resin with the extracellular region of Mpl (Mpl-X) produced by recombinant DNA techniques using CHO cell as a host (Bartley et al. (1994), supra ⁇ .
• the TPO polypeptides of this invention are further characterized by the ability to bind to the Mpl receptor and specifically to the extracellular (soluble) domain thereof.
• a protein encoded by a DNA moiety which is complementary to the protein-coding strand of a human cDNA or genomic DNA sequence of TPO gene namely a "complementary inverted protein” disclosed by Tramontano ef al. (Nucl. Acids Res. , vol.12, pp.5049 - 5059, 1984).
• a protein of the present invention which is labeled with a detectable marker, for example, 1 25 l labeling or biotinylation, thus rendering possible provision of a reagent which is useful for the detection and quantification of TPO or TPO receptor- expressing cells in solid samples such as tissue and liquid samples such as blood, urine and the like.
• the biotinylated protein of the present invention is useful in the case of its binding to immobilized streptavidin in order to remove megakaryoblasts from bone marrow at the time of autogenous bone marrow transplantation. It is also useful in the case of its binding to immobilized streptavidin in order to concentrate autogenous or allogenic megakaryocytic cells at the time of autogenous or allogenic bone marrow transplantation.
• a conjugate of TPO with a toxin such as ricin, diphtheria toxin or the like or with a radioactive isotope is useful in antitumor therapy and in conditioning for bone marrow transplantation.
• the present invention also provides a nucleic acid material which is useful when labeled with a detectable marker including a radioactive marker or a non-radioactive marker such as biotin or when it is used in a hybridization procedure for the detection of the position of human TPO gene and/or its related gene family on a chromosomal map.
• a detectable marker including a radioactive marker or a non-radioactive marker such as biotin
• Such a material is also useful for the confirmation of human TPO gene disorders at the DNA level and can be used as a genetic marker for the confirmation of adjoining genes and their disorders.
• a pharmaceutical composition which contains a therapeutically effective amount of the protein of the present invention together with a useful and effective diluent, antiseptic agent, solubilizing agent, emulsifying agent, adjuvant and/or carrier.
• a useful and effective diluent, antiseptic agent, solubilizing agent, emulsifying agent, adjuvant and/or carrier means an amount which provides a therapeutic effect for specified conditions and routes of administering conditioning.
• Such a composition is used in the form of liquid, freeze-dried or dried preparation and comprises a diluent selected from various buffers (Tris-HCI, acetate and phosphate for example) having various pH values and ionic strengths, a surface adsorption preventing additive such as albumin or gelatin, a surface active agent such as Tween 20, Tween 80, Pluronic F68 or bile acid salt, a solubilizing agent such as glycerol or polyethylene glycol, an antioxidant such as ascorbic acid or sodium metabisulfite, an antiseptic agent such as thimerosal, benzyl alcohol or paraben and a vehicle or tonicity agent such as lactose or mannitol.
• a diluent selected from various buffers (Tris-HCI, acetate and phosphate for example) having various pH values and ionic strengths
• a surface adsorption preventing additive such as albumin or gelatin
• a surface active agent such as
• compositions comprising covalent bonding of the protein of the present invention to a polymer such as polyethylene glycol, chelation of the protein with metal ions, incorporation of the protein into a granular preparation, or on the surface, consisting of a polymer compound such as polylatic acid, polyglycolic acid or hydrogel, or incorporations of the protein into liposomes, microemu.lsion, micelles, single or multilayer vesicles, red cell ghosts or spheroplasts.
• a composition will exert influences upon physical conditions, solubility, stability, in vivo releasing rate and in vivo clearance of TPO. Selection of the composition may be decided depending on the physical and chemical properties of the used TPO-active protein.
• compositions in which granules are coated with a polymer such as poloxamer or poloxamine and TPO which binds to antibodies for tissue specific receptors, ligands or antigens or to tissue specific receptor ligands.
• a polymer such as poloxamer or poloxamine and TPO which binds to antibodies for tissue specific receptors, ligands or antigens or to tissue specific receptor ligands.
• Other examples of the composition of the present invention are those which are in the form of granules, have a protective coating and contain a protease inhibitor or a penetration enhancer, for use in various routes of administration such as parenteral, pulmonary, transnasal and oral administration.
• the pharmaceutical composition comprising the protein of the present invention may be administrated several times per day, usually in an amount of 0.05 ⁇ g to 1 mg/kg (as TPO protein) of body weight depending on conditions and sex of a patient, administration route and the like.
• the pharmaceutical composition containing the protein of the present invention can be administered at a dose of 25,000-500,000,000 of the active ingredient (in terms of the relative activity by the M-07e assay which will be given later) per kg of body weight for one to several times a day and for one to seven days a week depending upon the symptoms, sex and route of administration.
• the present inventors have confirmed that, with respect to the C-terminal site of the human TPO, the activity is maintained even when the amino acid residues up to the 152nd amino acid sequence shown in SEQ ID NO: 6 are deleted and that, with respect to the N-terminal site, the activity is maintained even when the amino acid residues up to the 6th amino acid sequence are deleted.
• additional hematopoietic factors such as EPO, G-CSF, GM-CSF, M-CSF, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11 , IL-12, IL-13, LIF and SCF, in addition to the protein of the present invention, is also included in the present invention.
• the protein of the present invention is useful for the treatment of various thrombocytopenic diseases, either alone or in combination with other additional hematopoietic factors.
• additional hematopoietic factors include EPO, G-CSF, GM-CSF, M-CSF, IL-1 , IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, LIF and SCF.
• thrombocytopenia due to damages in the production of platelets or reduction in life of platelets (caused by destruction or consumption of platelets), which can be treated with the protein of the present invention.
• it can be used for the enhancement of the restoration of platelets in a thrombocytopenic patient due to congenital Fanconi anemia or aplastic anemia caused by chemotherapy or irradiation, myelodysplastic syndrome, acute myelocytic leukemia, aplastic crisis after bone marrow transplantation. It is also useful in the treatment of thrombocytopenia due to diminished production of TPO.
• Thrombocytopenia due to reduction in life of platelets or megakaryocytes includes idiopathic thrombocytopenic purpura, hypoplastic anemia, acquired immunodeficiency syndrome (AIDS), disseminated intravascular coagulation syndrome and thrombotic thrombocytopenia.
• AIDS acquired immunodeficiency syndrome
• thrombotic thrombocytopenia it is also useful in the case of autotransfusion of platelets, in which TPO is administered to a patient prior to a surgical operation in order to increase the number of the patient's own platelets and the thus increased platelets are used as transfusion platelets at the time of the operation.
• TPO can be used for the purpose of enhancing release of new "intact" platelets in such patients.
• the present invention further relates to an antibody specific for TPO.
• the protein of the present invention can be used as antigens, and the corresponding antibodies include both monoclonal and polyclonal antibodies and chimeric antibodies, namely "recombinant" antibodies, prepared by conventional methods.
• human TPO itself can be used as the antigen or, alternatively, a partial peptide of human TPO can be utilized as the antigen.
• the epitope can be defined by specifying the antigen region.
• the antigen region can be clarified by analyzing the epitope of the antibody.
• these antibodies each having the thus-clarified epitope can be utilized to fractionate, detect, quantitate and purify various TPOs which differ in their properties such as the kind of the sugar chains added, the length of the peptide chains, etc.
• a TPO peptide containing the thus-selected amino acid sequence is synthesized, and bonded to a suitable carrier protein , for example, albumin, KLH (key hole limpethemocyanine) or the like, by covalent bonding to prepare an immunoreactive antigen.
• a suitable carrier protein for example, albumin, KLH (key hole limpethemocyanine) or the like
• a peptide of a multiple antigen peptide (MAP) type is produced to be an antigen, according to the Tarn's method (Proc. Natl. Acad. Sci. (USA) 85:5409-5413, 1988). Then, with the thus-prepared antigen along with an adjuvant or the like, immunized are mammals, birds, etc.
• the epitope can be defined by specifying the antigen region.
• the regions of various TPO molecules having different molecular weights are screened and identified by immunological engineering.
• a peptide-deleted region can be screened and identified.
• an antibody gene or a part of the gene is cloned from the cells expressing the intended antibody to obtain an antibody molecule that has been expressed by genetic engineering.
• a monoclonal antibody is specific for a single antigenic determinant on the antigen.
• a TPO-specific antibody is useful in improving selectivity and specificity of an antigen-antibody reaction-aided diagnosis and an analytical assay method and in performing separation and purification of TPO.
• such antibodies can be applied to the neutralization or removal of TPO from serum.
• Monoclonal antibodies are also useful for detection and quantification of TPO in, for example, serum of whole blood.
• the anti-human TPO antibody of the present invention can be used as the ligand in affinity chromatography for purification and isolation of human TPO.
• any conventional methods for fixing various enzymes can be employed.
• employable is a method of using a carrier of CNBr-activated Sepharose 4B (produced by Pharmacia Fine Chemicals Co.) or the like.
• the fixed anti-human TPO antibody is filled into a column and a human TPO-containing liquid is passed through the column. By this operation, a large amount of human TPO is adsorbed onto the carrier in the column.
• the solvent for elution for example, usable are glycine-HCI buffer (pH 2.5), sodium chloride solution, propionic acid, dioxane, ethylene glycol, chaotropic salt, guanidine hydrochloride, urea, etc.
• glycine-HCI buffer pH 2.5
• sodium chloride solution sodium chloride solution
• propionic acid dioxane
• ethylene glycol ethylene glycol
• chaotropic salt guanidine hydrochloride
• urea urea
• the antibody of the present invention can be used for determining human TPO by immunochemical quantitative assay, especially by enzyme-immunoassay to be conducted by a solid phase sandwich process.
• Monoclonal antibodies can be produced by hybridoma cells in a medium which does not contain any other immunoglobulin molecules.
• Monoclonal antibodies can be prepared from culture supernatants of hybridoma cells or from mouse ascites induced by intraperitoneal injection of hybridoma cells.
• the hybridoma technique originally disclosed by Kohler and Milstein (Eur. J. Immunol. 6:511 -519, 1976) can be used broadly for the formation of hybrid cell lines which are possessed of high level monoclonal antibodies for a number of specific antigens.
• affinity chromatography such as protein A affinity chromatography, protein G affinity chromatography, Avid gel chromatography, anti-immunoglobulin-fixed gel chromatography, etc.; as well as cation-exchange chromatography, anion-exchange chromatography, lectin affinity chromatography, dye adsorption chromatog raphy, hydrophobic interaction chromatography, gel permeation chromatography, reversed phase chromatography, hydroxyl-apatite chromatography, fluo ro-apatite ch ro matog raphy, metal chelating chromatog raphy, isoelectric poi nt ch romatog raphy, partitioning electrophoresis, isoelectric point electrophoresis, etc.) which are generally employed for purification of protein can be combined.
• affinity chromatography such as protein A affinity chromatography, protein G affinity chromatography, Avid gel chromatography, anti-immunoglobulin-fixed
• a method of antigen affinity purification can also be employed, in which a gel carrier or membrane, to which has been chemically bonded a human TPO protein itself or a peptide that contains the region of the antigen or a part of the region or, that is, a molecule capable of recognizing the intended antibody, is prepared, an antjbody-containing material is added thereto so that the intended antibody is made to be adsorbed onto the carrier or membrane, and the thus-adsorbed antibody is then eluted and recovered under suitable conditions.
• the present invention also allows use of endogenous DNA sequences encoding a TPO polypeptide for the production of large quantities of polypeptide products.
• in vitro and ex vivo homologous recombination procedures may be employed to transform host cells to provide for expression or enhanced expression of polypeptides.
• Preferred host cells include human cells (e.g., liver, bone marrow and the like) wherein a promoter or enhancer sequence is inserted employing flanking sequences that are homologous to a target region in a cellular genome with the result that TPO polypeptide expression is accomplished or enhanced. See, e.g. , U,S. Letters Patent 5,272,071 , published PCT WO 90/14092, WO 91/06666 and WO 91/09955.
• Also provided by the invention are processes for producing a TPO polypeptide as described above comprising the steps of expressing a polypeptide encoded by a DNA of the invention in a suitable host, and isolating said TPO polypeptide.
• the TPO polypeptide expressed is a Met "2 - Lys '1 polypeptide
• processes can further include the step of cleaving Met ⁇ 2 -Lys ⁇ 1 from said isolated TPO polypeptide.
• TPO polypeptides having [Gly " ' ' ] structure comprising the steps of introducing into a suitable host cell a DNA encoding a glutathione-S-transferase (GST) polypeptide 5' to TPO polypeptide encoding sequences, the GST and TPO polypeptide encoding sequences separated by DNA encoding a thrombin recognition polypeptide, isolating the GST-TPO expression product and treating the expressed polypeptide with thrombin to remove GST amino acids.
• GST-TPO expression product and treating the expressed polypeptide with thrombin to remove GST amino acids.
• the resulting TPO polypeptides have a [Gly -1 ] structure.
• the inventors of the present invention have firstly attempted to purify a protein (rat TPO) which has an activity to enhance proliferation and differentiation of rat CFU-MK.
• rat TPO a protein which has an activity to enhance proliferation and differentiation of rat CFU-MK.
• many trial and error attempts were made on the purification procedures, such as selection of various natural supply sources, selection of gels for chromatographies and separation modes.
• the present inventors have succeeded in purifying a protein having a TPO activity from blood plasma of thrombocytopenic rats induced by X-ray or ⁇ -ray irradiation, making use of the TPO activity as a marker based on a rat CFU-MK assay which will be described later in ⁇ Reference Example>, and in determining partial amino acid sequences of the purified protein ⁇ Examples 1 and 2>.
• a blood plasma sample was prepared from about 1 ,100 thrombocytopenic rats induced by X-ray or ⁇ - ray irradiation and subjected to a Sephadex G-25 chromatography, an anion exchange chromatography (Q-Sepharose FF) and a lectin chromatography (WGA-Agarose) in that order, thereby obtaining a WGA-Agarose-adsorbed TPO-active fraction.
• Q-Sepharose FF anion exchange chromatography
• WGA-Agarose lectin chromatography
• each of the TPO- active fractions F2 and F3 was concentrated to obtain a high molecular weight TPO sample F2 and a low molecular weight TPO sample F3 which were used separately in the subsequent purification steps.
• TPO sample F3 was subjected to a preparative reverse phase chromatography (YMC-Pack PROTEIN-RP), a reverse phase chromatography (YMC-Pack CN-AP) and another reverse phase chromatography (Capcell Pack C1) in that order.
• YMC-Pack PROTEIN-RP a preparative reverse phase chromatography
• YMC-Pack CN-AP reverse phase chromatography
• Capcell Pack C1 another reverse phase chromatography
• TPO-active fraction obtained by the final step reverse phase chromatography (Capcell Pack C1) was applied to SDS-PAGE and the TPO-active substance was extracted from the gel, the presence of the TPO activity was confirmed in a band corresponding to apparent molecular weights of about 17,000 to 22,000 under a non-reducing condition.
• rat TPO activities in various organs and cell culture supernatants were screened based on the biochemical and biological ' properties of the rat plasma-derived TPO.
• TPO activities almost equal to the rat plasma-derived TPO were found in the culture supernatant of rat hepatocyte- derived cell lines McA-RH8994, HTC and H4-II-E, as well as in the culture supernatant of rat primary hepatocytes ⁇ Example 4>.
• the expression vector pEF18S was constructed from 2 expression vectors pME18S and pEFBOS ⁇ Example 5>. Construction of this vector has rendered possible easy cloning of cDNA using a high efficiency expression vector having multiple cloning sites which can be used for the integration of inserts.
• pUC and pBR based plasmid vectors and ⁇ based phage vectors are mainly used for the construction of cDNA libraries.
• the McA-RH8994 cells were cultured, homogenized by adding guanidine thiocyanate solution and then subjected to CsCI density gradient centrifugation to obtain total RNA ⁇ Example 6>.
• RNA can also be made by a hot phenol method, an acid guanidium phenol chloroform method and the like.
• first strand cDNA was synthesized by the action of reverse transcriptase using oligo dT as a primer to which a restriction enzyme ⁇ /ofl recognition sequence has been added, followed by the synthesis of 2nd strand cDNA using RNase H and E.coli DNA polymerase I.
• An EcoRI adaptor was added to the thus obtained double-strand cDNA, the resulting cDNA was ligated with a mammalian cell expression vector pEF18S constructed in Example 5, which has been digested with Notl and EcoRI and then the thus ligated cDNA was transformed into competent cells of an E. coli strain DH5 to construct a cDNA library ⁇ Example 7>.
• a DNA sequence was deduced from the partial amino acid sequence of the rat TPO which has been purified from rat blood plasma, in order to synthesize degenerate primers to be used in the polymerase chain reaction (PCR).
• Primers to be used may also be obtained based on an amino acid sequence other than the position of the primers used herein. Highly degenerate primers may also be used without employing inosine.
• primers with reduced degeneracy can be designed making use of a codon having high usage in rat (Wada et al., Nucleic Acids Res., vol.18, p.2367- 2411 , 1990).
• the cDNA library prepared above was divided into pools, each containing about 10,000 clones, and plasmid DNA was extracted from each of 100 pools.
• PCR was carried out using each pool of plasmid DNA as a template and primers newly synthesized based on the nucleotide sequence of the A1 fragment, bands considered to be specific were detected in 3 pools.
• One of these 3 pools was divided into sub-pools, each containing about 900 clones, and plasmid DNA was purified from 100 sub-pools to carry out PCR in the same manner.
• a specific band was detected in 3 sub-pools.
• One of these pools was divided into sub-pools, each containing 40 clones, and finally each clone was screened by PCR in the same manner.
• a clone pEF18S-A2 ⁇ which seemed to encode the rat TPO cDNA was isolated ⁇ Examples 9 and 10>.
• TPO mRNA expression in rat tissues was analyzed by PCR, and specific expression was detected in the brain, the liver, the small intestines and the kidney ⁇ Example 12>.
• the liver was selected as the starting tissue for the cloning of human TPO cDNA.
• a cDNA library was constructed using a commercial mRNA derived from normal human liver.
• pEF-18S as a vector like the case of the rat library
• cDNA was synthesized by the same procedure as the case of rat to obtain a directionally cloned cDNA library by making use of restriction enzymes Not ⁇ and EcoRI.
• the library prepared by introducing vector-ligated cDNA into E. coli DH5 contained about 1 ,200,000 clones ⁇ Example 13>.
• this cDNA seemed to be an artificial product of the cloning, because no termination codon was found in this clone and a poly A tail-like sequence was found on its 3'-end.
• an expression vector was constructed which encodes the amino acid sequence excluding a corresponding portion to the poly A tail-like sequence.
• TPO activity was found in the resulting culture supernatant ⁇ Example 18>.
• DNA fragment in the 3'-end region of the human TPO was obtained by PCR so as to analyze the structure of a full- length cDNA.
• cloning of human TPO cDNA can be attained by colony hybridization or plaque hybridization using a rat TPO cDNA fragment as a probe, making use of a library constructed using a pUC or pBR based plasmid vector, a ⁇ phage based vector or the like.
• inosine may be used to decrease the degree of degeneracy.
• the inventors of the present invention have carried out colony hybridization of two million clones in a normal human liver cDNA library prepared in Example 13 using a rat TPO cDNA fragment as a probe, but a human TPO cDNA clone was not able to be obtained.
• Example 15 Since the clone HL34 obtained in Example 15 seemed to contain an incomplete cDNA, a cDNA library was reconstructed using a commercial normal human liver-derived poly (A) + RNA preparation to obtain a complete human TPO cDNA.
• Primers for PCR were synthesized based on the nucleotide sequence (SEQ ID NO: 3) of a partial cDNA obtained in Example 14 and the nucleotide sequence (SEQ ID NO: 196) of a complete cDNA of human TPO predicted in Example 19.
• the human liver cDNA library (hTPO-F1 ) constructed in Example 20 was divided into 3 pools (pool # 1 - 3) . PCR was carried out using the plasmid DNA prepared from each pool as a template and the synthesized primers. As the result, a DNA fragment having the expected size was amplified when the plasmid DNA prepared from the pool # 3 was used. The # 3 pool was then divided into subpools, each containing 15,000 transformants, and screening using PCR was carried out as above. As the result, amplification of DNA having the expected size was found in 6 of the 90 pools.
• a genomic library given from Prof. T. Yamamoto at the Gene Research Center, Tohoku University was inoculated onto 18 NZYM plates in such an inoculum size that one plate contained 30,000 phage particles, and two replica filters were prepared from each of the thus prepared 18 plates.
• a human TPO cDNA fragment (base position numbers 178 to 1 ,025 in SEQ ID NO: 7) was amplified by PCR and purified. Using the purified fragment .labeled with 32 P as a probe, plaque hybridization was carried out. As the result, 13 positive signals were obtained. Plaques were collected from the original plates and inoculated again onto NZYM plates in such an inoculum size that 1 ,000 plaques were formed on each plate.
• nucleotide sequence of the thus obtained clone pHGTI was determined, chromosomal DNA carried by the clone was found to contain an entire coding region of the protein predicted in Example 19, and nucleotide sequence of the coding region coincided completely with that of the predicted nucleotide sequence (SEQ ID NO: 196).
• Example 18 and 22 Preparation of human TPO deletion derivatives, their expression in COS1 cells and confirmation of TPO activity
• the results of Example 18 and 22 revealed that human TPO protein could exhibit its biological activity even after the removal of its carboxyl-terminal third.
• deletion derivative experiments were performed.
• a series of expression plasmids were prepared by PCR using the DNA of the plasmid clone pHT1 -231 obtained in Example 18 as a template and synthesized oligonucleotides as primers.
• Expression plasmids which contained DNA coding for human TPO deletion derivatives lacking the carboxyl- terminal region of the TPO protein, that is, deletion derivatives coding for positions 1 -21 1 , 1 -191 , 1 -171 and 1 - 163 amino acid sequence, were obtained.
• TPO activity was detected in each of the culture supernatant ⁇ Example 27>.
• Derivatives of a protein having its TPO biological activity can be obtained by modifying (deletion, substitution, insertion or addition) cDNA coding the protein. Methods such as PCR, site directed mutagenesis and chemical synthesis can be used for the modification.
• the TPO cDNA region in pHTP1 was used to construct a vector pDEF202-hTPO-P1 for its expression in CHO cell. ⁇ Example 31 >
• Example 32 large scale cultivation was carried out for a human TPO producing CHO cell line (CHO 28-30 cell, resistant to 25 nM MTX) which has been prepared by transfecting the human TPO expression plasmid pDEF202- hTPO-P1 into CHO cells.
• a human TPO producing CHO cell line CHO 28-30 cell, resistant to 25 nM MTX
• Example 57 human TPO was purified from the culture supernatant in Example 55 in a different manner.
• Example 33 Using the TPO cDNA region encoded in the plasmid pBLTEN prepared in Example 30, an expression vector BMCGSneo-hTPO-P1 was constructed for use in X63.6.5.3. cells. ⁇ Example 33>
• Example 35 The expression vector pHTP1 prepared in Example 30 was transfected into COS 1 cells to obtain a large quantity (a total of about 40 liters) of culture supernatant containing the expressed product. ⁇ Example 35>.
• TPO Purification of TPO was carried out from about 7 liters of the COS 1 cell serum-free culture supernatant prepared in Example 35 containing the expression vector pHTP1 -derived TPO. It was able to obtain high activity TPO through the steps of a hydrophobic interaction chromatography, a cation exchange column chromatography, a WGA column chromatography and a reverse phase column chromatography. ⁇ Example 36>
• a vector pGEX-2T/hT(1 -174) was constructed for use in the expression of a fusion protein (to be referred to as "GST-TPO(1 -174)") of glutathione-S-transferase and human TPO (amino acid residues of positions 1 to 174) in E. coli.
• GST-TPO(1 -174) glutathione-S-transferase
• human TPO amino acid residues of positions 1 to 174
• a vector pCFM536/h6T(1 -1 63) was constructed for use in the expression in E. coli of a mutation type human TPO protein (to be referred to as "h6T(1 -163") in which the 1 -position Ser residue and the 3-position Ala residue of human TPO (amino acid residues of positions 1 to 163) were respectively replaced by an Ala residue and a Val residue and, at the same time, a Lys residue and a Met residue were respectively added to the -1 and -2 positions. Entire portion of the human TPO cDNA nucleotide sequence (corresponding to 1 to 163 amino acid residues) carried by this vector was exchanged with E. coli preference codons. ⁇ Example 42>
• the h6T(1 -163) was expressed in E. coli, the resulting cells were lysed and then solubilization of h6T(1 - 163) contained in the precipitated fraction and conditions for the refolding of the protein were examined, thereby succeeding in partially purifying a protein containing a TPO amino acid sequence as designed. It was confirmed that the protein shows TPO activity in the rat CFU-MK assay system.
• a vector pCFM536/hMKT(1 -163) was constructed for use in the expression in E. coli of a mutation type human TPO protein (to be referred to as "hMKT(1-163)") in which a Lys residue and a Met residue were respectively added to the -1 and -2 positions.
• the hMKT(1-163) was expressed in E. coli in the same manner as described in Example 43, and the expression protein was subjected to SDS-PAGE, transferred on a PVDF membrane and then subjected to N-terminal amino acid sequence analysis, thereby confirming that this protein contains the designed amino acid sequence.
• the vector pCFM536/hMKT(1 -332) for expression in E. coli of a mutation type human TPO protein in which a Lys residue is added at the position-1 and Met residue at the position-2 of human TPO (amino acid positions 1 to 332) (referred to as "hMKT (1-332)") respectively was constructed.
• the hMKT (1 -332) was expressed within E. coli in the same manner as in Example 42, and its expression was confirmed by Western blotting using the anti-human TPO peptide antibody prepared in Example 45 set forth below.
• Rabbit polyclonal anti-TPO peptide antibodies were prepared ' by synthesized peptides corresponding to three partial regions of the rat TPO amino acid sequence determined in Example 10. It was confirmed that these antibodies can recognize rat and human TPO molecules. Furthermore, peptides corresponding to 6 partial regions in the amino acid sequence of human TPO shown in SEQ ID NO: 6 (or SEQ ID NO: 7) were synthesized and then employed to prepare rabbit polyclonal anti TPO peptide antibodies. The resultant antibodies were confirmed to recognize human TPO. ⁇ Example 45>
• Example 46 (R) Purification of human TPO expressed in COS 1 cells making use of anti-TPO peptide antibody column, and molecular weight and biological characteristics thereof
• Example 48 The TPO-active fraction purified in Example 36, namely the TPO sample purified up to the step of Capcell Pak Cl 300A column from a culture supernatant obtained by transfecting COS 1 cells with the expression vector pHTP1 , was checked for its biological activities to determine if it exerts a platelet number increasing action in the living body.
• T Expression of human TPO chromosomal DNA in CHO cells and confirmation of the activity
• a vector pDEF202-ghTPO was constructed for use in the expression of human TPO chromosome in CHO cells.
• a recombinant virus for the expression of human TPO in insect cells was prepared ⁇ Example 58> and expressed in the insect cell Sf 21 , and subsequently the TPO activity was identified in the culture supernatant. ⁇ Example 59>
• the vector pDEF202-hTPO163 for expression in CHO cells of the human TPO protein (referred to as "hTP0163") having amino acids 1 to 163 in the human TPO amino acid sequence shown in SEQ ID NO: 7 was constructed.
• the expression vector pDEF202-hTPO163 was transfected into CHO cells and thus-obtained hTP0163-producing CHO cell line was cultured in a large scale. From the supernatant of the culture hTP0163 was purified. ⁇ Examples 62 to 65>
• N3/TPO A derivative (referred to as "N3/TPO") wherein Arg-25 and Glu-231 of human TPO have been substituted by Asn and ' Lys, respectively, was prepared as well as a derivative (referred to as "09/TPO") wherein His-33 has been replaced by Thr.
• TPO activity an in vitro assay system
• Polyclonal antibodies are prepared using peptide fragments of human TPO ⁇ Example 69> and subsequently utilized in Western blot analysis ⁇ Example 70> and in the construction of anti-TPO antibody affinity columns ⁇ Example 71 >.
• Purified human TPO is administered to mice with induced thrombocytopenia and changes in platelet counts are monitored versus a control group.
• Pharmaceutical compositions are described as potential useful in treatment of thrombocytopenia.
• TPO activity is defined in terms of specific binding interaction with the Mpl receptor. ⁇ Example 90> through ⁇ Example 93>.
• .rat CFU-MK assav ⁇ system liquid culture system ⁇ Megakaryocytes incorporate and store serotonin in cytoplasmic dense granules through an active, energy- dependent process (Fedorko, Lab. Invest, vol.36, pp.310 - 320, 1977). This phenomenon can be observed already in small and mononuclear acetylcholinesterase-positive cells which are considered to be positioned between CFU-MK and recognizable megakaryocytes (Bricker and Zuckerman, - Exp. Hematol., vol.12, p.672, 1984). The amount of serotonin incorporated increases in response to the increment of the size of megakaryocytes (Schick and Weinstein, J. Lab. Clin.
• Advantages of this assay system are that indirect influences of contaminated cells (for example, formation of Meg-CSF activity by contaminated cells stimulated by a certain substance other than the factor of interest, or formation of a certain factor by contaminated cells, which undergoes a combined action with the factor of interest) can be reduced, because the percentage of CFU-MK in cells of Gp l l b/l l la + CFU-MK fraction is markedly high (see the following [Assay method]) while the number of contaminating cells is small.
• appropriate culture conditions can be maintained for a relatively long period, because the total number of cells seeded per one well is small.
• Another advantage is that a number of large-size mature megakaryocytes grown from CFU-MK in the presence of an active sample during a culture period can visually detected under a phase contrast microscope, thus rendering possible qualitative judgement of the presence and degree of the activity. Results of the qualitative judgement correspond well to the results of quantitative determination based on the incorporation of 1 4 C-serotonin. Therefore, reliability of the quantitative determination can be further improved by joint use of the qualitative judgement.
• femur and tibia are removed from Wister rats (male, 8 to 12 weeks of age) to prepare bone marrow cell suspension as described.
• a suspending medium a solution consisting of 13.6 mM trisodium citrate, 11.1 mM glucose, 1 mM adenosine, 1 mM theophylline, 10 mM HEPES (pH 7.25), 0.5% bovine serum albumin (to be referred to as "BSA” hereinafter) (Path-O-Cyte 4; Seikagaku Kogyo Co., Ltd.) and Ca 2+ and Mg + - free Hanks' balanced salt solution : to be referred to as "HATCH solution” hereinafter) which is a slightly modified version of the megakaryocyte separation medium reported by Levine and Fedoroko (Levine and Fedoroko, Blood, vol.50, pp.713 - 725, 1977) is used for the preparation of the bone marrow cell suspension.
• the thus prepared bone marrow cell suspension is layered over a Percoll discontinuous density gradient solution (density; 1 .050 g/ml/1 .063 g/ml/1 .082 g/ml) which is prepared by diluting Percoll solution (manufactured by Pharmacia) with HATCH solution, and centrifuged at 20 ⁇ at 400 x g for 20 minutes. After the centrifugation, cells between the 1 .063 g/ml and 1.082 g/ml density layers are recovered.
• a Percoll discontinuous density gradient solution density; 1 .050 g/ml/1 .063 g/ml/1 .082 g/ml
• IMDM culture medium Iscove's modified Dulbecco culture medium
• FCS 10% fetal calf serum
• Non adherent cells are suspended in HATCH solution and incubated for 1 hour on - 100-mm bacteriological petri dishes to which a mouse monoclonal anti-rat platelet
• Gpllb/llla antibody P55 antibody (Miyazaki et al., Thromb. Res., vol.59, pp.941 - 953, 1990) has previously been adsorbed .
• CFU-MK fraction hereinafter
• this cell fraction contains about 5 to 10% of CFU-MK based on the measurement by a colony assay system in the presence of a saturated concentration of rat IL-3.
• a hybridoma capable of producing the aforementioned P55 antibody has been deposited under the deposit No. FERM BP-4563 in National Institute of
• FCS FCS and dispensed in portions of 10 4 cells per well of a 96 well tissue culture plate. Each well is further supplied with a standard sample (will be described later in detail), or a sample to be assayed, thereby adjusting the final medium volume to 200 ⁇ l/well.
• the thus prepared plate is put in a CO2 incubator and incubated for 4 days at 37 ° C. On 4th days of incubation, 0.1 ⁇ Ci (3.7 KBq) of 1 4 C-serotonin is added to each well 3 hours before the completion of culturing, and the final incubation is continued at 37 " C .
• the plate After 3-hr incubation, the plate is centrifuged at 1 ,000 rpm for 3 minutes, and the resulting supernatant fluid is removed by suction. A 200 ⁇ l portion of PBS containing 0.05% EDTA is added to each well, and the plate is centrifuged to wash the plate by removing the resulting supernatant fluid. This washing step is repeated once again. A 200 ⁇ l portion of 2% Triton X-100 is added to the thus obtained pellet of cells in each well, and the plate is shaken on a plate mixer for approximately 5 to 10 minutes to lyse the cells thoroughly.
• a 150 ⁇ l portion of the thus obtained cell lysate is transferred into a commercial cap- shaped solid scintillator (Ready Cap; manufactured by Beckman) which is subsequently left overnight at 50 " C in an oven to dry the lysate. On the next day, the Ready Cap is put into a glass vial to measure radioactivity of 1 C by a liquid scintillation counter.
• Ready Cap manufactured by Beckman
• blood plasma of thrombocytopenic rats was prepared in the following manner, for use in the preparation of standard samples.
• the thus recovered blood plasma fraction was again centrifuged at 1 ,200 x g for 10 minutes, and the resulting blood plasma fraction was recovered, taking care to avoid the pellet consisting of cells, platelets and the like, and pooled.
• the blood plasma obtained in such a manner is referred to as "TRP" hereinafter.
• Ca- treated TRP (standard sample C), WGA-Agarose column active fraction (standard sample W) or Phenyl Sepharose 6 FF/LS column active fraction (standard sample P) obtained from TRP in accordance with the procedure of Example 1 were dialyzed extensively against a sufficient volume of IMDM culture medium and used as assay standards.
• the standard sample C was used in the early stage, but was , changed to the standard sample W halfway thereafter and then to the standard sample P.
• Specific activity of the standard sample C was defined tentatively as 1 , and relative activities of the standard samples W and P were calculated based on the definition.
• Relative activity of each sample to be assayed was determined by comparing the dose-response curve of the standard sample to those of the samples to be assayed, and relative activity of the sample to be assayed was defined as n where its activity is n times higher than that of the standard sample C.
• bone marrow cells are cultured in a semi-solid culture medium in the presence of a sample to be assayed, and the Meg-CSF activity is measured by counting the number of megakaryocyte colonies formed by the proliferation and differentiation of CFU-MK.
• agar 50 ⁇ M 2-mercaptoethanol and 0.3% agar (AGAR NOBLE, manufactured by DIFCO) was put in a 35-mm tissue culture plastic dish and, after solidification at room temperature, cultured at 37 * C in a CO2 incubator.
• the number of cells per one dish is adjusted to 2-4 x 10 5 in the case of unseparated bone marrow cells, 2-5 x 10 4 in the case of cells at the step of the Percoll density gradient or of the adherence depletion, or 0.5-2 x 1 0 3 in the case of cells of Gpllb/llla + CFU-MK fraction.
• the agar disks are detached from the dishes and placed on glass slides (76 mm x 52 mm).
• a piece of 50-_m nylon mesh and filter paper are placed, in that order, over the gel.
• the thus dried agar slides are heat-fixed for 5 minutes at 50 " C on a hot plate and soaked for 2 to 4 hours in an acetylcholinesterase staining solution prepared according the procedure of Jackson (Blood, vol.42, pp.413 - 421 , 1973).
• the agar slides are washed with water, dried, subjected to post- staining with Harris' hematoxylin solution for 30 seconds, washed with water and then air-dried.
• Megakaryocyte colonies are defined as 3 or more tightly grouped acetylcholinesterase- positive cells.
• the colony assay can be performed in the same manner as the above procedure (a) using 2-4 x 10 5 cells per one dish.
• Human bone marrow cells or human cord blood cells may be used as such, or as CFU-MK fractions enriched by the following manner.
• lymphoprep manufactured by Daiichi Kagaku Co., Ltd.
• the resulting interface leucocyte fraction is recovered.
• cells to which biotinylated monoclonal antibodies specific for human surface antigens (CD2, CD11c and CD19) bind are removed with avidin-linked magnetic beads.
• the cells removed by the magnetic beads method are mainly B cells, T cells, macrophages and a part of granulocytes.
• CD34 + DR + CFU-MK fraction Colony assay of human cells can be carried out in the same manner as the aforementioned case of the use of rat bone marrow cells, except that 3-5 x 10 3 cells of the CD34+DR+ CFU-MK fraction are used in one dish and a mixture of 12.5% human AB plasma and 12.5% FCS is used instead of 10% FCS.
• the culturing period for the formation of megakaryocyte colonies is 12 to 14 days.
• immunological staining of megakaryocytes is carried out by an alkaline phosphatase- anti-alkaline phosphatase antibody method with a mouse monoclonal antibody specific for a megakaryocyte surface antigen Gpllb/llla (for example, Teramura et al., Exp. Hematol., vol.16, pp.843 - 848, 1988), and colonies each consisting of 3 or more megakaryocytes are counted.
• M-07e cells a human megakaryoblast cell line, proliferate in response to GM-CSF, IL-3, SCF, IL-2 and the like (Avanzi et al., J. Cell. Physiol., vol.145, pp.458 - 464, 1990). Since these cells also respond to TPO, they are applicable to a substitutive assay system for the rat CFU-MK assay system.
• M-07e cells maintained in the presence of GM-CSF are recovered, washed thoroughly and then suspended in IMDM culture medium containing 10% FCS.
• the resulting M-07e cell suspension is dispensed in portions of 10 4 cells into wells of a 96 well tissue culture plate, and each well is further supplied with a standard sample or a sample to be assayed, thereby adjusting the final volume to 200 ⁇ l/well.
• the thus prepared plate is put in a 5% CO2 incubator and incubated for 3 days at 37- C. After 3 days of culture, 1 ⁇ Ci (37 KBq) of 3 H - thymidine is added to each well 4 hours before the completion of culturing. After completion of the culturing, the cells are collected on a glass fiber filter using a cell harvester to measure 3 H radioactivity with a liquid scintillation counter (for example, Beta Plate manufactured by Pharmacia).
• a liquid scintillation counter for example, Beta Plate manufactured by Pharmacia.
• BF-TE22 is capable of cell proliferation in response to not only
• IL3 and IL4 but also TPO it can be applied to an assay system as a substitute for the rat CFU-MK assay or the human megakaryoblastic cell line-aided assay (M-07e assay) is as follows. Briefly, BF-TE22 cells growing in Iscove's modified
• IL-3 are recovered, washed three times with IMDM and suspended in IMDM medium containing 10% FCS.
• the cell suspension is dispensed into wells of a 96 well tissue culture plate of a density of 1 x 10 4 cells per well, each well is further supplemented with a standard TPO solution or a sample to be tested and adjusted to a final volume of 200 ⁇ l/well.
• the resulting plate is incubated for 2 to 3 days in a 5% CO2 incubator. On the 2nd or 3rd day, 1 ⁇ Ci (37 KBq) of 3 H - thymidine is added to each well and the culturing is continued for additional 4 hours.
• the cells are collected on a glass fiber filter using a cell harvester to measure 3 H radioactivity with a liquid scintillation counter.
• a cell harvester to measure 3 H radioactivity with a liquid scintillation counter.
• almost all of cells cultured in media lacking TPO activity die and therefore do not incorporate 3 H-thymidine.
• cells cultured in media containing TPO activity show active proliferation in a TPO concentration-dependent manner and incorporate 3 H-thymidine.
• results of this assay are parallel to those of the M-07e and CFU-MK assays.
• TRP was prepared as the purification source from about 1 ,000 rats in accordance with the procedure described in the aforementioned ⁇ Reference Example> A, rat megakaryocyte precursor cell (CFU-MK) assay system.
• XRP blood plasma fraction
• XRP (ca. 8 L) prepared from a total of about 1 ,100 rats was used as the purification source.
• the blood plasma sample of about 1 ,100 rats was too large to process at one time because its total protein content reached 493,000 mg.
• the blood plasma sample was divided into 11 lots, each corresponding to about 100 rats, in the following purification steps (1 ) to (4).
• the sample was divided into 6 batches.
• a sample crudely purified from the total blood plasma of about 1 ,100 rats was used.
• typical example of each purification step is described in the case of a lot (XW9) and a batch (XB6).
• TPO activity was measured using the rat CFU-MK assay system described in ⁇ Reference Example>. Unless otherwise noted, all purification steps were carried out at 4_C, excluding the reverse phase chromatographies, and Superdex 75pg gel filtration in the presence of surfactant, which were carried out at room temperature. Protein assay was carried out by a Coomassie dye binding assay (a reagent system manufactured by PIERCE, catalog No. 23236X) or a method using bicinchoninic acid (a reagent system manufactured by PIERCE, catalog No. 23225). An outline of the purification " is shown in Table 1.
• Triazi ⁇ .e dye > TSK-gel AF-Blue 650MH
• Reverse phase > YMC CN-AP (Reverse phase > Capcell Pak C l
• XRP (742 ml; protein concentration, 54.8 mg/ml; total protein, 40,686 mg) corresponding to about 100 rats, which had been stored at -80 ° C , was thawed and dispensed into polypropylene centrifugation tubes (manufactured by Nalgene). Calcium chloride powder was added to each tube to a final concentration of 100 mM. After overnight incubation at 4 * C , the resulting mixture was centrifuged at 8,000 rpm for 60 minutes.
• TPO-active supernatant fluid (742 ml; protein concentration, 54.9 mg/ml; total protein, 40,740 mg) was added a protease inhibitor p-APMSF ((p- aminodiphenyl)methanesulfonyl fluoride hydrochloride, manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 010-10393) to a final concentration of 1 mM.
• p-APMSF protease inhibitor ((p- aminodiphenyl)methanesulfonyl fluoride hydrochloride, manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 010-10393)
• the supernatant fluid obtained after the calcium treatment of the step (1 ) (742 ml; protein concentration, 54.9 mg/ml; total protein, 40,740 mg) was applied at a flow rate of 40 to 70 ml/min to a Sephadex G-25 column (manufactured by Pharmacia Biotech, catalog No. 17-0033-03; diameter, 11 .3 cm; bed height, 47 cm) which had been equilibrated in advance with 20 mM Tris-HCI, pH 8. Elution was carried out with the same buffer. A 1 ,300 ml portion of the eluate before the elution of protein was discarded.
• TPO- active protein fraction exchanged by 20 mM Tris-HCI, pH 8 (1 ,377 ml; protein concentration, 27.56 mg/ml; total protein, 37,882 mg; protein yield, 93%). Relative activity of TPO in the fraction was found to be 2.3.
• the TPO-active fraction obtained in the above step (2) by the Sepharose G-25 treatment (1 ,377 ml; protein concentration, 27.5 mg/ml; total protein, 37,841 mg; relative activity, 2.3) was applied to a Q-Sepharose FF (manufactured by Pharmacia Biotech, catalog No. 17-0510-01 ; diameter, 5 cm; bed height, 27 cm) at a flow rate of 40 ml/min, eluted with 20 mM Tris-HCI (pH 8) and a fraction F1 which passed through the column was pooled (3 ,949 ml ; protein concentration, 0.98 mg/ml; total protein, 3,870 mg; relative activity, 0).
• the buffer was changed to 20 mM Tris-HCI (pH 8) containing 175 mM NaCl to elute a TPO-active fraction F2 (4,375 ml; protein concentration, 5.36 mg/ml).
• a fraction F3 (1 ,221 ml; protein concentration, 3.9 mg/ml; total protein, 4,783 mg; relative activity, 3.8) was eluted with 20 mM Tris-HCI (pH 8) containing 1 ,000 mM NaCl.
• Total protein in the TPO-active fraction F2 was found to be 23,440 mg, and protein yield of F2 by this step was 61.9%.
• relative activity of TPO was increased to 6.8.
• the TPO-active fraction F2 obtained in the above step (3) by the Q-Sepharose FF treatment was divided into three portions and applied to a WGA-Agarose (manufactured by Honen Corp., catalog No. 800273; diameter, 5 cm; bed height, 22.5 cm) at a flow rate of 5 ml/min, and eluted with Dulbecco's isotonic phosphate buffer (DPBS).
• DPBS Dulbecco's isotonic phosphate buffer
• the buffer was changed to 20 mM sodium phosphate buffer (pH 7.2) containing 0.2 M N-acetyl-D- glucosamine (GlcNAc, manufactured by Nacalai tesque, catalog No. 005-20), 150 mM NaCl and 0.02% sodium azide, and the resulting eluates were pooled and concentrated using an ultrafiltration unit (Omega Ultrasette, nominal molecular weight cutoff of 8,000; manufactured by Filtron), thereby obtaining a WGA-Agarose adsorbing TPO-active fraction F2 (2,993 ml; protein concentration, 0.376 mg/ml).
• an ultrafiltration unit Omega Ultrasette, nominal molecular weight cutoff of 8,000; manufactured by Filtron
• Total protein in the TPO-active fraction F2 was found to be 1 ,125 mg, and protein yield of F2 by this step was 4.8%. In addition, relative activity of TPO was increased to 101. The thus obtained F2 fraction was stored at -80 * C.
• the WGA-Agarose adsorbing TPO-active fraction of lot No. XW8 and the WGA-Agarose adsorbing TPO-active fraction F2 of lot No. XW9 obtained in the above step (4) starting from 215 rats-equivalent XRP were combined as a batch No. XB6 (5,947 ml, protein concentration, 0.388 mg/ml; total protein, 2,319 mg; relative activity, 150).
• a 5,974 ml portion of the combined sample was mixed with 0.85 moles of NaCl for 1 ,000 ml (296.76 g in total) to make a 6,132 ml of solution having a final NaCl concentration of 0.822 M, and the resulting solution was applied at a flow rate of 7 ml/min to a TSK-gel AF-BLUE 650 MH column (TOSOH CORP., catalog No. 08705; diameter, 5 cm; bed height, 23 cm) which had been equilibrated in advance with 20 mM sodium phosphate buffer (pH 7.2) containing 1 M NaCl.
• protein was eluted using 20 mM sodium phosphate buffer (pH 7.2) containing 1 M NaCl at a flow rate of 10 ml/min.
• the resulting eluates were pooled and concentrated using an ultrafiltration unit (Omega Ultrasette, nominal molecular weight cutoff of 8,000), thereby obtaining a passed-through fraction F1 (543 ml; protein concentration, 2.05 mg/ml; total protein, 1 ,1 12 mg; relative activity, 31).
• the elution buffer was changed to 2 M NaSCN to obtain an eluted TSK-gel AF-BLUE 650 MH adsorbing TPO- active fraction F2 (1 ,427 ml; protein concentration, 0.447 mg/ml) .
• Total protein in the TPO-active fraction F2 was found to be 638 mg, and protein yield of F2 by this step was 27.5%.
• relative activity of TPO was increased to 1 ,500.
• a 1 ,424 ml portion of the TSK-gel AF-BLUE 650 MH TPO-active fraction F2 (1 ,424 ml; protein concentration, 0.447 mg/ml; total protein, 638 mg; relative activity, 1 ,500) was mixed with 1.5 moles of ammonium sulfate powder per 1 ,000 ml (282.2 g in total) to make a 1 ,581 ml of solution having a final ammonium sulfate concentration of 1.35 M.
• the elution buffer was changed to 20 mM sodium phosphate buffer (pH 7.2) to obtain an eluted TPO- active fraction F2 (about 3,500 ml).
• the eluted fraction was concentrated using an ultrafiltration unit (Omega Ultrasette, nominal molecular weight cutoff of 8,000) and a sample for assay was taken out.
• protein concentration and total protein of the TPO-active fraction F2 (220 ml) were found to be 1.45 mg/ml and 319 mg, respectively, and protein yield of F2 by this step was 50.0%. Relative activity of TPO was found to be 1 ,230.
• the Phenyl Sepharose 6 FF/LS TPO-active fraction F2 (217 ml; protein concentration, 1.45 mg/ml; total protein, 315 mg; relative activity, 1 ,230) was mixed with 144.8 ml of 5 M NaCl solution to make a 362 ml solution having a final NaCl concentration of 2 M, and the resulting solution was concentrated to about 50 ml using an ultrafiltration unit with YM 3 membrane (76 mm in diameter, manufactured by Amicon Corp.).
• the concentrated sample of the TPO-active fraction F2 obtained by the Sephacryl S-200 HR is referred to as "high molecular TPO sample F2" hereinafter, and that of F3 as “low molecular TPO sample F3".
• the high molecular TPO sample F2 and the low molecular TPO sample F3 are pooled fractions of different elution areas in the gel filtration chromatography as a matter of convenience, and the term "high molecular” and "low molecular” therefore may not mean their true molecular weights.
• the low molecular TPO sample F3 and the high molecular TPO sample F2 are separately subjected to the subsequent purification steps.
• the low molecular TPO sample F3 (total protein, 50.3 mg; protein concentration, 0.184 mg/ml; relative activity, 20,000; total activity, 1 ,007,000; total volume, 274 ml) obtained in the above step (7) was mixed with a solvent A (0.025% trifluoroacetic acid (TFA)) and a solvent B (1 -propanol containing 0.025% TFA) to obtain a solution having a total volume of 508.63 ml and final propanol, TFA and protein concentrations of about 20%, 0.012% and 0.0989 mg/ml, respectively.
• a solvent A 0.025% trifluoroacetic acid (TFA)
• solvent B (1 -propanol containing 0.025% TFA
• the precipitate resulting from the centrifugation was dissolved in 20 ml of 20 mM sodium acetate (pH 5.5) containing 5 mM CHAPS (3-[3- cholamidopropyl)dimethylammonio]-1 -propane sulfonate ; manufactured by Dojindo Laboratories, catalog No. 75612-03- 3) and also applied to the column.
• a 214.9 ml portion of the low molecular TPO sample F3-derived YMC-Pack PROTEIN-RP TPO-active fraction F2 (total protein, 2.79 mg; protein concentration, 0.0130 mg/ml; relative activity, 130,000; total activity, 36,300) obtained in the above step (8) was mixed with 0.6 ml of 50% glycerol and concentrated to 1.8 ml. The concentrate was finally adjusted to a volume of 5 ml containing 20% or less of propanol and about 6% of glycerol.
• the concentrate was divided into 5 portions for use in the following column operation (0.555 mg protein and 1 ml volume for each operation). Each of the thus divided samples was applied at a flow rate of 0.6 ml/min to a column packed with YMC-Pack CN-AP (manufactured by YMC, catalog No. AP- 513; 6 mm in diameter and 250 mm in bed height) which has been equilibrated in advance with 15% B, using 0.1% TFA as the solvent A and 0.05% TFA-containing 1 -propanol as the solvent B. After the application, propanol concentration was increased from 15% B to 25% B, and 65 minutes of linear gradient from 25% B to 50% B was carried out.
• YMC-Pack CN-AP manufactured by YMC, catalog No. AP- 513; 6 mm in diameter and 250 mm in bed height
• a 43.12 ml portion of the 43.20 ml low molecular TPO sample F3-derived TPO-active fraction FA (total protein, 0.372 mg; protein concentration, 0.00863 mg/ml; relative activity, 800,000; total activity, 297,500) obtained in the above step (9) was mixed with 0.2 ml of 50% glycerol and concentrated to obtain 0.1 ml of glycerol solution.
• the thus obtained fraction was found to have a total protein of 39.6 ⁇ g, a protein concentration of 9.4 ⁇ g/ml, a relative activity of 4,890,000 and a total activity of 193,600.
• Examination of the SDS-PAGE patterns of the TPO-active fraction numbers 36 to 42 revealed the presence of a band whose staining density correlates with the activity strength.
• apparent molecular weight of this non-reduced band was found to be 17,000 to 19,000 when compared with those of the standard molecular weight proteins on the same gel, which were reduced, thus revealing that this band is a strong candidate of TPO.
• Pre-stained Low Range Marker manufactured by Bio-Rad Laboratories, Inc., 161 -0305
• DPCIII DPCIII
• gel portions to be subjected to silver staining were immediately cut out with a knife, put into a fixing solution and then stained using the aforementioned silver staining kit.
• the ultrafiltration using 300 ⁇ l of 20 mM sodium phosphate buffer was repeated twice to remove remaining SDS. Thereafter, a similar step was repeated to exchange to the assay culture medium for preparation of a sample (300 ⁇ l) which was subsequently sterilized and subjected to TPO activity measurement.
• the TPO-active protein was purified in the active fraction of Capcell Pak C1 300A column to a detectable level on the electrophoresis gel. Based on the silver-stained density of the band obtained by 15% SDS-PAGE, the amount of the candidate TPO protein (an apparent molecular weight of about 17,000 to 19,000) in the total TPO-active fraction was determined to be about 1.7 ⁇ g.
• the high molecular TPO sample F2 (total protein, 257 mg; protein concentration, 0.894 mg/ml; relative activity, 7,840; total activity, 2,015,000; total volume, 287 ml) obtained in the above step (7) was mixed with 95.8 ml (1/3 volume of the sample) of a solvent B (1 -propanol containing 0.025% TFA) to obtain a solution having a total volume of 383 ml and final propanol, TFA and protein concentrations of about 25%, 0.006% and 0.671 mg/ml, respectively.
• a solution of 0.025% TFA was used for a solvent A.
• Insoluble materials formed during the preparation of input sample were separated by centrifugation, and the resulting supernatant fluid was divided into six 62.3 ml (42.8 mg protein) portions and applied at a flow rate of 2 ml/min to a column packed with YMC-Pack PROTEIN-RP (manufactured by YMC, catalog No. A-PRRP-33-03- 15; 3 cm in diameter and 7.5 cm in bed height) which has been equilibrated in advance with 30% B.
• the precipitate resulting from the centrifugation was dissolved in 10 ml of 20 mM sodium acetate (pH 5.5) containing 5 mM CHAPS and also applied to the column.
• a 538.2 ml portion of the high molecular TPO sample F2-derived YMC-Pack PROTEIN-RP TPO-active fraction F2 (total protein, 11.3 mg; protein concentration, 0.021 mg/ml; relative activity, 227,000; total activity, 2,565,000) obtained in the above step (12) was mixed with 0.6 ml of 50% glycerol and concentrated by evaporation. To this was added 18 ml of 20 mM CHAPS, followed by stirring and a subsequent 41 hour incubation at 4 * c Thereafter, the first sample was applied to a column packed with HiLoad 26/60 Superdex 75 pg (manufactured by Pharmacia Biotech, catalog No.
• the Superdex 75 column operation was repeated 6 times by dividing the entire YMC-Pack PROTEIN-RP TPO-active fraction into 6 portions.
• the eluates of each operation were collected in 5 ml portions in 45 tubes, thus finally obtaining 45 fractions each containing 30 ml eluate after completion of the 6 column operations.
• a 24.66 ml portion of the 25.20 ml high molecular TPO sample F2-derived YMC-Pack CN-AP TPO-active fraction FA (total protein, 0.606 mg; protein concentration, 0.0246 mg/ml; relative activity, 700,000; total activity, 424,000) obtained in the above step (14) was mixed with 0.4 ml of 50% glycerol and concentrated by evaporation.
• a 2 ⁇ l portion of each fraction (1/300 fraction) was sampled for use in electrophoresis, dried by evaporation and treated at 95 ° c for 5 minutes with 10 ⁇ l of a nonreducing sample buffer for SDS-PAGE.
• the thus treated sample was subjected to SDS-PAGE using a 15-25% SDS-polyacrylamide precast gel (manufactured by Daiichi Pure Chemicals Co., Ltd.) and then stained using the aforementioned silver staining kit.
• DPCIII described in the foregoing was used as molecular weight markers.
• TPO- active fraction FA obtained in the purification step (10) of Example 1 was carried out in accordance with the procedure of Iwamatsu (Iwamatsu ef al., "Shin Kiso Seikagaku Jikken-ho (New Basic Biochemical Experiments)", vol.4, pp.33 - 84, pub. Maruzen; Iwamatsu, A, Seikagaku (Biochemistry), vol.63, no.2, pp.139 - 143, 1991 ; Iwamatsu, A., Electrophoresis, vol.13, pp.142 - 147, 1992).
• the sample was subjected to SDS-PAGE and transferred electrically onto a polyvinylidene difluoride (PVDF) membrane.
• PVDF polyvinylidene difluoride
• the thus treated protein was digested into peptide fragments by systematic and stepwise restrictive enzymatic hydrolysis in situ with three proteases, and the resulting peptide fragments on each digestion step were separated and purified by reverse phase chromatography and analyzed for their amino acid sequences by a high sensitivity amino acid sequence determination method. The following describes this process in detail.
• TPO-active fraction FA (tube numbers 36 to 42) obtained in the purification step (10) of Example 1 (total protein, 39.6 ⁇ g ; protein concentration, 9.4 ⁇ g/ml; relative activity, 4,890,000; total activity, 193,600), 4,151 ⁇ l (98.8% of the total fraction) was used in the amino acid sequence analysis.
• the total protein deduced from the chromatogram was 39.1 ⁇ g, of which about 1.6 ⁇ g was the TPO candidate protein stained with silver after SDS-PAGE and having an apparent molecular weight of about 17,000 to 19,000.
• This sample was mixed with glycerol and concentrated by evaporation to obtain 5 ⁇ l of a glycerol solution.
• a non-reducing buffer for SDS- PAGE and 1 M Tris-HCI (pH 8) in order to adjust its pH, thereby obtaining about 25 ⁇ l of sample containing 200 mM Tris-HCI (pH 8.0), 50 mM Tris-HCI (pH 6.8), 1.1% SDS, 2 mM EDTA, 0.02% bromophenol blue and 30% glycerol.
• the thus prepared sample was maintained at room temperature for 14 hours and then treated at 60 " c for 5 minutes, in order to effect proper SDS reaction without excess heating.
• Micro-slab gels (4.0% acrylamide stacking gel and 15% acrylamide separation gel) were prepared and SDS-PAGE was carried out at room temperature for 2 hours at a constant current of 12.5 mA and then at 17.5 mA.
• Pre-stained Low Range Marker Bio-Rad, 161 -0305
• DOCIII were used as molecular weight markers.
• the resulting protein molecules were transferred on a PVDF membrane (see the following step).
• a portion of the sample was also used in another electrophoresis using 15-25% polyacrylamide precast gel (Multi Gel 15/25 manufactured by Daiichi Pure Chemicals Co., Ltd., catalog No. 211072) under non-reducing conditions or after reducing the sample with dithiothreitol (DTT).
• DTT dithiothreitol
• molecular weight of the protein band expected to be TPO was about 19,000 under a reducing condition and purity of the TPO candidate protein of Capcell Pak C1 300A column was a few percent.
• the candidate protein contains at least one disulfide bond.
• Protein transfer on a PVDF membrane was carried out for 1 hour at a constant current of 160 mA (11 to 17 V) using a semi-dry transfer apparatus (Model KS-8460, manufactured by Marysol).
• a solution consisting of 0.3 M Tris and 20% methanol (pH 10.4) was used as the anolyte, a solution consisting of 25 mM Tris and 20% methanol (pH 10.4) as the transfer membrane solution and a solution consisting of 25 mM Tris, 40 mM aminocaproic acid and 20% methanol (pH 10.4) as the cathlyte.
• stepwise restrictive enzymatic hydrolysis was carried out using the following three proteases.
• sequence (K)DSFLADVK of AP12 completely coincided with an internal sequence KDSFLADVK of a rat corticosteroid-binding globulin (CBG) precursor [PIR data base accession No. A40066; Smith and Hammond; "Rat corticosteroid-binding globulin : primary structure and messenger ribonucleic acid levels in the liver under different physiological conditions.”, Mol. Endocrinol., (1989), 3, 420 - 426].
• CBG corticosteroid-binding globulin
• sequence KQYYESE (SEQ ID NO: 193) having high similarity to the sequence (K)XYYESZ (X is A, S, G, M or Q and Z is E or K) of AP3 is contained in the rat CBG amino acid sequence.
• sequences which correspond to those of AP12 and AP3 are linked to each other thereby forming an internal amino acid sequence KDSFLADVKQYYESE (SEQ ID NO: 190).
• Fig. 5 shows a dose response curve in the case of the use of a TPO sample partially purified from thrombocytopenic rat plasma (TPO-active fraction F2 from YMC Pack Protein-RP column described in the purification step (8) of Example 1-2).
• TPO-active fraction F2 from YMC Pack Protein-RP column described in the purification step (8) of Example 1-2.
• Such a process formation is called cytoplasmic process formation (Leven and Yee, Blood, vol.69, pp.1046 - 1052, 1987) or proplatelet process formation (Topp et al., Blood, vol.76, pp.912 - 924, 1990), which is considered to be a platelet precursor structure further differentiated from mature megakaryocytes and is considered to be at the final stage of megakaryocyte differentiation so far observable in vitro. Since such a morphological change was observed with a high frequency by using the TPO sample alone, it is possible that this factor alone can stimulate proliferation and differentiation of CFU-MK, can produce mature megakaryocytes and can finally release platelets.
• the rat plasma-derived TPO stimulated formation of megakaryocyte colonies.
• the TPO-induced megakaryocyte colonies are characterized in that each colony consists of smaller numbers of megakaryocytes, but each megakaryocyte is larger in size, which means advanced maturity.
• TPO since TPO produced no or few colonies of the other cell lineages, the Meg-CSF activity of TPO can be regarded as megakaryocyte-specific. On the basis of these facts, it is evident that TPO has different biological properties from those of other cytokines such as rat IL-3, mouse GM-CSF and human EPO and exerts unique Meg-CSF activity.
• the TPO sample partially purified from blood plasma of thrombocytopenic rats also exerted its Meg-CSF activity on CD34 + ,DR + cell fractions derived from human bone marrow cells or human cord blood cells and induced significant numbers of human megakaryocyte colonies, indicating that this factor has no species specificity.
• rat TPO-producing organs were carried out in order to ensure a mRNA source for use in the cloning of a rat TPO gene.
• bone marrow, lungs, livers and spleens were taken periodically from rats rendered thrombocytopenic by P55 antibody administration, their cells (organ sections in the case of lungs and livers) were cultured and activities in the resulting culture supernatant were examined by the rat CFU-MK assay system. This initial attempt, however, did not yield distinctive results.
• H4- ll-E cells ATCC deposit No. CRL1548; Pitot et al., Nat. Cancer Inst. Monogr., vol.13, pp.229 - 245, 1964; purchased from Dainippon Pharmaceutical Co., Ltd.
• HTC cells Thimpson et al., Proc. Natl. Acad. Sci. USA, vol.56, pp.296 - 303, 1966; purchased from Dainippon Pharmaceutical Co., Ltd.).
• McA-RH8994 cells were suspended in alpha-MEM(-) liquid culture medium containing 10% FCS and transferred into a 175 cm 2 plastic tissue culture flask at 1 x 10 6 cells/flask. After 3 days of culturing at 37 * c in a 5% CO2 incubator, the medium was exchanged with IMDM culture medium containing
• H4-II-E cells were suspended in Dulbecco's modified Eagle liquid culture medium (glucose 4.5 g/l, to be referred to as "DMEM" hereinafter) containing 10% FCS and transferred into a 175 cm 2 plastic tissue culture flask at 5 x 10 5 cells/flask. After 3 days of culturing at 37 ° c in a 5% CO2 incubator, the medium was exchanged with IMDM culture medium containing 5% FCS and the culturing was continued for additional 3 days to recover the resulting culture supernatant.
• DMEM Dulbecco's modified Eagle liquid culture medium
• HTC cells were suspended in DMEM liquid culture medium containing 5% FCS and transferred into a 175 cm 2 plastic tissue culture flask at 2.5 x 10 5 cells/flask. After 3 days of culturing at 37 ° c in a 5% CO2 incubator, the medium was exchanged with
• IMDM culture medium containing 5% FCS IMDM culture medium containing 5% FCS and the culturing was continued for additional 3 days to recover the resulting culture supernatant.
• the resulting eluate was applied to a Q-Sepharose FF column, the column was washed with 20 mM Tris-HCI (pH 8.0) and then the adsorbed fraction was eluted with 20 mM Tris-HCI (pH 8.0) containing 175 mM NaCl.
• the thus eluted fraction was applied to a WGA-Agarose column, the column was washed with PBS and the adsorbed fraction was eluted with 20 mM sodium phosphate (pH 7.2) containing 0.2 M GlcNAc and 0.15 M NaCl.
• the resulting eluate was applied to an TSK-gel AF-BLUE 650MH column, the column was washed with 20 mM sodium phosphate (pH 7.2) containing 1 M NaCl and then the adsorbed fraction was eluted with 2 M NaSCN.
• the resulting eluate was applied to a Phenyl- Sepharose 6 FF/LS column, the column was washed with 50 mM sodium phosphate (pH 7.2) containing 1.5 M ammonium sulfate followed by 0.8 M ammonium sulfate containing 36 mM sodium phosphate, and then the adsorbed fraction was eluted with 20 mM sodium phosphate (pH 7.2).
• the thus obtained adsorption fraction was concentrated and then fractionated using a reverse phase Vydac Protein C4 column (manufactured by The Separations Group, catalog No. 214TP51015; 1 cm in diameter and 15 cm in bed height). That is, the sample was applied to the C4 column which has been equilibrated in advance with 20% B and then elution was effected by 90 minutes of linear gradient of from 20% B to 40% B at a flow rate of 1 ml/min, using 0.1% TFA in the developing solvent A and 1-propanol containing 0.05% TFA in the developing solvent B. As the result, each of the TPO-active proteins derived from these cell lines was eluted at a 1-propanol concentration of 30 to 43%.
• TPO activities in samples of each purification step were measured by the rat CFU-MK assay system, the results showed that TPO activities of the 3 cell lines behaved with the similar patterns to those of the XRP- derived TPO (cf. Example 1-2).
• Relative activity, activity yield and the like at each purification step measured by the rat CFU- MK assay system are shown in Table 2.
• TPO activities in the eluates from the final reverse phase column were measured by the rat CFU-MK assay system, TPO activities of the 3 cell lines and the XRP-derived TPO were found in the same peak area.
• Example 1-2 Each of the pooled, active fractions from the reverse phase column was subjected to SDS-PAGE in accordance with the procedure described in Example 1-2.
• TPO activity was measured by the rat CFU-MK assay system, apparent molecular weights of the XRP-derived TPO, McA-RH8994 cell- derived TPO and H4-II-E cell-derived TPO were found to be 17,000 to 22,000, 33,000 to 39,000 and 31 ,000 to 38,000, respectively, and the HTC cell-derived TPO showed apparent molecular weights of 17,000 to 22,000 and 28,000 to 35,000.
• TPO proteins produced by McA-RH8994 cells, H4-II-E cells and HTC cells are equivalent to the XRP-derived TPO in terms of their biological properties, though their biochemical properties are slightly different from one another in terms of apparent molecular weight.
• a remarkable finding in the present invention is a possibility that a TPO-active protein molecule contained in blood may be a product of partial digestion at its specific or irregular site in its production cells or after its secretion from the production cells. It also suggests possible existence of TPO genes (mRNA and cDNA) having various lengths.
• a vector into which a cDNA fragment can be integrated easily and which can exert a high expression efficiency of the cloned cDNA was constructed for use in the cloning and expression of TPO cDNA. That is, an expression vector pEF18S was constructed from an expression vector pME18S by replacing the SR ⁇ promoter with an elongation factor 1 ⁇ (EF1 ⁇ ) promoter which is known as a high expression promoter (cf. Fig. 6).
• EF1 ⁇ elongation factor 1 ⁇
• the promoter of elongation factor 1 ⁇ was obtained by partially digesting 1 ⁇ g of an expression vector pEF-BOS (Mizushima ef al., Nucleic Acids Res., vol.18, p.5322, 1990) with restriction enzymes H ⁇ dlll and EcoR I , subjecting the digest to 2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a DNA fragment of about 1 ,200 bp and purifying the DNA fragment using a Prep-A- Gene DNA purification kit (manufactured by Bio-Rad Laboratories, Inc.; a kit for use in the selective purification of DNA which is effected by porous silica matrix-aided DNA adsorption, developed on the basis of the procedure reported by Willis et al.
• pEF-BOS Mazushima ef al., Nucleic Acids Res., vol.18, p.5322, 1990
• the clone pEF18S obtained above was cultured overnight at 37 * c in 50 ml of an LB medium (1 % Bacto-tryptone, 0.5% Bacto-yeast extract, 0.5% NaCl) containing 50 ⁇ g/ml of ampicillin, and the resulting cells collected by centrifugation were suspended in 4 ml of TEG- lysozyme solution (25 mM Tris-HCI, pH 8, 10 mM EDTA, 50 mM glucose, 0.5% lysozyme).
• the resulting pellet was again dissolved in TE solution to which were subsequently added NaCl and polyethylene glycol 3,000 to their final concentrations of 0.63 M and 7.5%, respectively. After centrifugation, the pellet was dissolved in TE solution and precipitated with ethanol. In this way, about 300 ⁇ g of the plasmid DNA was obtained.
• McA-RH8994 cells which showed relatively high activity in the colony assay of Example 4 were selected as the material for use in the rat TPO cDNA cloning and subjected to the following experiments.
• RNA isolation of total RNA was carried out basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). That is, McA-RH8994 cells were grown to confluence in 15 culture dishes of 90 mm in diameter. After removing the liquid medium, cells in each dish were thoroughly suspended in 0.8 ml of a 5 M guanidine solution (5 M guanidine thiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M ⁇ -mercaptoethanol, 0.5% sodium sarcosylsulfate), and the resulting suspensions in all dishes were collected in a single tube and the total volume was adjusted to 20 ml with the guanidine solution.
• a 5 M guanidine solution 5 M guanidine thiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M ⁇ -mercaptoethanol, 0.5% sodium sarcosylsulfate
• the resulting mixture which became viscous due to cell disruption, was subjected to about 20 repetitions of suction/discharge with a 20 ml syringe equipped with a 18G needle and then 21 G needle until the mixture became almost non-viscous.
• a 18 ml portion of 5.7 M CsCI-0.1 M EDTA (pH 7.5) was used as a cushion in a polyallomer centrifugation tube for use in Beckman SW28 rotor, and the aforementioned mixture of about 20 ml was gently layered over the cushion in such a manner that the layers were not disturbed and the tube was almost filled.
• the thus prepared tube was subjected to 20 hours of centrifugation at 25,000 rpm and at 20 - c .
• RNA was obtained from about 10 8 cells.
• Double-stranded cDNA having an EcoRI recognition site at its 5'-end and a Notl recognition site at the 3'-end was synthesized from 5 ⁇ g of the poly (A) + RNA obtained in
• Two anti-sense nucleotide primers AP8-1 R and AP8-2R which correspond to the amino acid sequence of the peptide fragment AP8 described in Example 2, and two sense nucleotide primers EF1 ⁇ -1 and EF1 ⁇ -2, which correspond to the first intron of the human elongation factor 1 ⁇ of the plasmid vector pEF-18S (cf. Fig. 6) used for the preparation of cDNA library, were synthesized.
• the primers AP8-1 R and AP8-2R are mixed primers consisting of 17 continued nucleotides in which deoxyinosine is incorporated, as reported by Takahashi et al. (Proc. Natl. Acad. Sci. USA, vol.82, pp.1931 - 1935, 1985).
• the primers EF1 ⁇ -1 and EF1 ⁇ -2 consisting of 21 and 20 nucleotides, respectively, were synthesized based on the nucleotide sequences of positions 1491 to 1512 and 1513 to 1532 of the genomic sequence reported by Uetsuki et al. (J. Biol. Chem., vol.264, pp.5791 - 5798, 1989).
• AP8-1R 3'-ACG CTG GGG GCI GAI GA-5' (SEQ ID NO:21)
• AP8-2R 3 ' -GGG GGG CGG ACG CTG GG-5 ' (SEQ ID NO: 25)
• EFla-1 5' -GGA TCT TGG TTC ATT CTC AAG-3 ' (SEQ ID NO: 29)
• EFla-2 5"-CCT CAG ACA GTG GTT CAA AG-3 ' (SEQ ID NO:30)
• PCR was carried out by GeneAmpTM PCR system 9600 (manufactured by Perkin-Elmer; a thermal cycker for PCR) (100 ⁇ l volume; heating at 95 * c for 2 minutes, a total of 35 cycles, each cycle consisting of denaturation at 95 * c for 1 minute, annealing at 40 * c for 1 minute and synthesis at 72 * c for 1 minute, and final incubation at 72 * c for 7 minutes).
• reaction solution was subjected to 2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a DNA fragment of about 330 bp as the main product of the PCR which was subsequently purified making use of the aforementioned Prep-A-Gene DNA purification kit (manufactured by Bio-Rad Laboratories, Inc.).
• Prep-A-Gene DNA purification kit manufactured by Bio-Rad Laboratories, Inc.
• T4 DNA ligase manufactured by Life Technologies
• the thus purified DNA fragment was subcloned into pCR ⁇ II vector (a vector for the use of TA cloning of PCR products, manufactured by Invitrogen).
• thermostable polymerase to be used in PCR has a terminal transferase activity
• the amplified DNA fragment is directly subcloned into the pCR ⁇ II vector which has a 5'-dT cohesive end, making use of the enzyme's property to add one deoxyadenylic acid to the 3'-end of the PCR-amplified DNA.
• Plasmid DNA molecules were purified from 28 clones selected at random from the resulting clones using QIAGEN-tip100 (manufactured by DIAGEN) and their nucleotide sequences were determined by a 373A DNA sequencer (a fluorescence sequencer, manufactured by Applied Biosystems), making use of a Taq Dye Terminater Cycle Sequening Kit (manufactured by Applied Biosystems; a kit for use in PCR-aided nucleotide sequence determination using fluorescent dyes, based on the dideoxy method of Sanger et al., Proc. Natl. Acad. Sci. USA, vol.74, pp.5463 - 5467, 1977).
• the aforementioned cDNA library was divided into pools of about 10,000 clones, cultured overnight in 1 ml of aforementioned LB medium containing 50 ⁇ g/ml of ampicillin and then , subjected to plasmid DNA extraction using an automatic plasmid isolation apparatus PI-100 (VER-3.0, manufactured by Kurabo Industries, Ltd.; an automatic plasmid DNA extraction apparatus based on a modified method of the alkali SDS method disclosed in Molecular Cloning, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
• PI-100 VER-3.0, manufactured by Kurabo Industries, Ltd.
• an automatic plasmid DNA extraction apparatus based on a modified method of the alkali SDS method disclosed in Molecular Cloning, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989.
• the following two oligonucleotides were synthesized based on the cDNA fragment A1 and purified. 5' CGAGGGTGTACCTGGGTCCTG 3' (S
• PCR was carried out by GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) (a total of 30 cycles, each cycle consisting of denaturation at 94 * c for 30 seconds, annealing at 66 ° c for 30 seconds and synthesis at 72 * c for 1 minute). As the result, a specific band of 236 bp was detected in 3 of the 100 pools examined.
• Example 9 Purification of plasmid DNA was carried out basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Each of the two clones obtained in Example 9 were cultured overnight in 50 ml of LB medium containing 50 ⁇ g/ml of ampicillin, and about 300 ⁇ g of plasmid DNA was obtained after purification in the same manner as described in Example 6.
• the thus obtained plasmid DNA was subjected to sequencing in the same manner as described in Example 8 using the Taq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems) to determine complete nucleotide sequence containing the A1 fragment.
• nucleotide sequences of the two clones completely coincided with each other, showing that they are the same clone.
• One of these clones was selected, and the plasmid carried by this clone was named pEF18S-A2 ⁇ . Its nucleotide sequence and an amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 2).
• sequence shown in the Sequence Listing (SEQ ID NO: 2) has distinct characteristics.
• the sequence downstream of the 172 bp 5' non-translation region encodes an amino acid sequence rich in hydrophobic amino acids which is presumed to be a protein secretion signal sequence consisting of 21 amino acids starting with methionine.
• This protein contains 126 amino acid residues, and a 3' non-translation sequence of 1 ,025 nucleotides and a poly A tail following the termination codon (TAA).
• TAA termination codon
• This protein contains a sequence which corresponds to the amino acid sequence AP8 analyzed in Example .2 (amino acid numbers 1 to 12 in SEQ ID NO: 2), but does not contain a consensus sequence for N-glycosylation.
• the 1624-1629 nucleotide sequence located closely to the terminal of the 3' non-translation sequence is different from a consensus sequence but seems to be a potential polyadenylation sequence.
• the vector pEF18S-A2 ⁇ carried by an E. coli strain DH5 has been deposited by the present inventors on February 14, 1994, under the deposit No. FERM BP-4565, in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.
• COS 1 cells (ATCC CRL1650) were suspended in the aforementioned DMEM medium containing 10% FCS, put into a 100-mm plastic tissue culture dish and cultured at 37 ° c in a 5% CO2 incubator until the cells reached a stage of about 40% confluence.
• plasmid pEF18S-A2 ⁇ (10 ⁇ g) dissolved in 30 ⁇ l of HBS (21 mM HEPES,
• DEAE-dextran manufactured by Pharmacia
• 80 ⁇ M of chloroquine manufactured by Sigma Chemical Co.
• DMEM culture medium DMEM culture medium, and the culturing was continued for 5 hours at 37 ° c in the 5% CO2 incubator. Thereafter, culture supernatant in the dish was removed by suction, and the remaining cells in the dish were washed twice with the DMEM culture medium, supplemented with 15 ml of the DMEM culture medium containing 10% FCS and cultured at 37 ' c for 3 to 5 days in the 5% CO2 incubator to recover the resulting culture supernatant.
• TPO activity was found in a dose-dependent fashion in the culture supernatant of COS 1 cells in which the plasmid pEF18S-A2 ⁇ was expressed (Fig. 7). Similar to the case of XRP-derived TPO, many megakaryocytes formed elongated cytoplasmic processes on 4 days of culture. By contrast, TPO activity was not found in the culture supernatant of COS 1 cells to which the plasmid pEF18S alone was transfected (Fig. 7).
• M-07e cell proliferation enhancing activity was also found in a dose dependent fashion in the culture supernatant of COS 1 cells in which the plasmid pEF18S-A2 ⁇ was expressed, but not in the culture supernatant of COS 1 cells in which the plasmid pEF18S alone was expressed.
• a partially purified TPO sample was prepared in order to examine for the ability of this TPO activity to promote platelet production in vivo.
• COS 1 cells transfected with the plasmid pEF18S-A2 ⁇ were cultured for 3 days in a serum free culture medium which has been supplemented with 0.2 mg of BSA, thereby obtaining about 5.8 liters of serum free culture supernatant.
• Protease inhibitor p- APMSF was added to the serum-free culture supernatant to the final concentration of 1 mM, and the mixture was filtered using a 0.22 ⁇ m filter.
• TSK- gel AF-BLUE 650MH-adsorbed TPO-active fraction F2 (2840 ml) was concentrated to 6.81 ml using an ultrafiltration unit with YM 3 membrane (76 mm in diameter, manufactured by Amicon Corp.). Total protein in this TPO-active fraction F2 was 12.5 mg, and protein yield of F2 at this step was 0.62%. Relative activity of TPO was calculated to be 240.
• the F2 was applied to a HiLoad 26/60 Superdex 200 pg (manufactured by Pharmacia Biotech, catalog No.
• TPO activity was found in eluates in the range of from 194 to 260 ml after application. These eluates were pooled to obtain a Superdex 200 pg TPO-active fraction F2 (66 ml; protein concentration, 0.112 mg/ml; total protein, 7.41 mg; relative activity, 142,860; total activity, 1 ,058,600).
• buffer A (20 mM sodium acetate, pH 5.5) and buffer B (20 mM sodium phosphate, pH 7.2, containing 500 mM NaCl) were prepared, and the TPO-active fraction was applied at a flow rate of 1 ml/min to a strong cation exchange column RESOURCE S (manufactured by Pharmacia Biotech, catalog No. 17-1178- 01 ; 0.64 cm in diameter and 3 cm in bed height) which has been equilibrated in advance with 100% A. Thereafter, elution was carried out at a flow rate of 0.3 ml/min with 40 minutes of a linear gradient of from 100% A to 100% B.
• RESOURCE S strong cation exchange column
• TPO activity was detected in a broad range of eluates (from 5% B to 32% B).
• TPO-active eluates were pooled and concentrated using an ultrafiltration unit with YM 3 membrane (25 mm in diameter, manufactured by Amicon Corp.) to obtain a RESOURCE S TPO-active fraction F2 (1.65 ml; protein concentration, 4.74 mg/ml; total protein, 7.82 mg; relative activity, 71 ,400; total activity, 558,600).
• the partially purified TPO sample was subcutaneously administered for consecutive 5 days (474 ⁇ g (100 ⁇ l)/mouse/day) to ICR male mice (9 weeks old) whose platelet numbers had been measured on the day before the administration.
• BSA 200 ⁇ g (100 ⁇ l)/mouse/day
• a buffer used as the solvent of the partially purified TPO 100 ⁇ l/mouse/day
• blood was collected from the heart of each mouse to measure platelet counts using a hemocytometer (F800, manufactured by Toa lyo Denshi).
• RNA was extracted from various rat tissues.
• a total of 6 rats were subjected to X-ray irradiation in the same manner as described in Example 1 , and various tissues (brain, thymus, lung, liver, heart, spleen, small intestine, kidney, testis and bone marrow cells) were excised from the rats on the 11 th to 14th day after the X-ray irradiation and immediately frozen in liquid nitrogen. Extraction of total RNA was effected by the use of an RNA isolation reagent ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.).
• Amount of ISOGEN to be used was determined according the weight of each frozen tissue sample, and the reagent-added tissue sample was treated with a hom ⁇ genizer (Physcotron ⁇ NS-60, manufactured by NITI-ON Medical & Physical Instruments MFG. Co., LTD) until complete disintegration of the tissue was attained (approximately 45 to 60 seconds at 10,000 rmp).
• the tissue homogenate was then subjected to total RNA extraction making use of a procedure based on the acid guanidium phenol chloroform method of Chomczynski et al. (Anal. Biochem., vol.162, pp.156 - 159, 1987). As the result, 1.1 to 5.6 mg of total RNA was obtained from respective tissues.
• first strand of cDNA was synthesized from 1 ⁇ g of the poly (A) + RNA obtained from each tissue. That is, 1 ⁇ g of the poly (A) + RNA was dissolved in 10 ⁇ l of sterile water, incubated at 70 " c for 15 minutes and then rapidly cooled down. To this were added 75 pmoles of random primer (Takara Shuzo Co., Ltd.), 10 U of RNase inhibitor (Boehringer-Mannheim Corp.), 50 mM of Tris-HCI (pH 8.3), 75 mM of KCl, 3 mM of MgCI 2 and 200 U of Super ScriptTM II (a reverse transcriptase manufactured by Life Technologies).
• rTPO-l 5'-CCT GTC CTG CTG CCT GCT GTG-3 * (SEQ ID NO:33) (positions 347 to 367 in SEQ ID NO: 2)
• rTPO-N 5'-TGA AGT TCG TCT CCA ACA ATC-3' (SEQ ID NO:34) (anti-sense primer corresponding to positions 1005 to 1025 in SEQ ID NO: 2)
• PCR was carried out in a volume of 100 ⁇ l, making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and heating at 95 * c for 2 minutes, repeating a total of 30 cycles, each cycle consisting of denaturation at 95 * c for 1 minute, annealing at 57 - c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 * c for 7 minutes.
• the thus synthesized cDNA was ligated with 1 .2 ⁇ g of the aforementioned expression vector pEF18S which had been digested in advance with EcoRI and Notl and transformed into 8.4 ml of the aforementioned Competent High E. coli DH5 (manufactured by Toyobo Co., Ltd.) As the result, 1.2 x 10 ⁇ transformants were obtained.
• first strand of cDNA was synthesized from 1 ⁇ g of the commercial normal human liver- derived poly (A) + RNA (manufactured by Clontech). That is, 1 ⁇ g of the poly (A) + RNA was dissolved in 10 ⁇ l of sterile water, incubated at 70 ° c for 15 minutes and then rapidly cooled down. To this were added 75 pmoles of random primers (Takara Shuzo Co., Ltd.), 10 U of RNase inhibitor (Boehringer-
• Primers for PCR use were synthesized based on the rat TPO cDNA sequence (SEQ ID NO: 2). Sequences of the thus synthesized primers are as follows. rTPO-AIN: 5'-ATG GAG CTG ACT GAT TTG CTC-3' (SEQ ID NO:35) (positions 173 to 193 in SEQ ID NO: 2) rTPO-N: 5'-TGA AGT TCG TCT CCA ACA ATC-3' (SEQ ID NO:36) (anti-sense primer corresponding to positions 1005 to 1025 in SEQ ID NO: 2)
• PCR was carried out with a volume of 100 ⁇ l, making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and heating at 95 * c for 2 minutes, repeating a total of 35 cycles, each cycle consisting of denaturation at 95 ° c for 1 minute, annealing at 40 ⁇ c for 1 minute and synthesis at 72 " c for 1 minute, followed by the final incubation at 72 * c for 7 minutes.
• reaction solution was subjected to 2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a DNA fragment of about 620 bp as the main product of the PCR which was subsequently purified making use of the aforementioned Prep-A-Gene DNA purification kit (manufactured by Bio-Rad Laboratories, Inc.).
• Nucleotide sequence of the thus purified DNA fragment was directly determined by a 373A DNA sequencer (manufactured by Applied Biosystems) making use of the aforementioned Taq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems).
• the thus determined nucleotide sequence excluding the primer moiety and an amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 3).
• This DNA fragment excluding the primer sequences, has a length of 580 bp.
• the human cDNA showed a homology of 86% with the rat cDNA nucleotide sequence, indicating that this DNA fragment encodes a portion of human TPO cDNA.
• hTPO-l 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO:37) (positions 60 to 80 in SEQ ID NO: 3)
• hTPO-J 5'-TGA CGC AGA GGG TGG ACC CTC-3' (SEQ ID NO:38) (anti-sense primer corresponding to positions 479 to 499 in SEQ ID NO: 3)
• the human cDNA library constructed in Example 13 was amplified and divided into pools (each pool containing about 100,000 clones), cultured overnight in 1 ml of the aforementioned LB medium containing 50 ⁇ g/ml of ampicillin and then subjected to plasmid DNA extraction using an automatic plasmid isolation apparatus PI-100 (VER-3.0, manufactured by Kurabo Industries, Ltd.). The thus extracted DNA was dissolved in TE solution.
• PCR was carried out in 20 ⁇ l volume by GeneAmpTM PCR System 9600 (manufactured by PERKIN- ELMER) (a total of 35 cycles, each cycle consisting of denaturation at 95 " c for 1 minute, annealing at 59 * c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 - c for 7 minutes).
• a specific band was detected in 3 of the 90 pools used.
• One of these 3 pools was divided into sub-pools, each containing about 5,000 clones, and plasmid DNA was purified from 90 sub-pools to carry out PCR in the same manner.
• specific band was detected in 5 sub-pools.
• One of these 5 pools was divided into sub-pools, each containing 250 clones, and plasmid DNA was extracted from 90 sub-pools. When these extracted samples were subjected to PCR in the same manner, a specific band was detected in 3 sub-pools.
• One of these 3 pools was further divided into sub-pools, each containing 30 clones, and plasmid DNA was purified from 90 sub-pools to carry out PCR in the same manner. As the result, a specific band was detected in 3 sub-pools.
• One of the candidate pools was cultured on the aforementioned LB plate containing 50 ⁇ g/ml of ampicillin and each of the thus formed 90 colonies was subjected to plasmid DNA extraction and PCR in the same manner. .As the result, a clone HL34 was finally obtained.
• plasmid DNA Purification of plasmid DNA was carried out basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
• the clone HL34 was cultured overnight in 50 ml of the LB medium containing 50 ⁇ g/ml of ampicillin, and the resulting cells collected by centrifugation were suspended in 4 ml of the aforementioned TEG-lysozyme solution. To this were added 8 ml of 0.2 N NaOH/1% SDS solution and then 6 ml of 3 M potassium/5 M acetate solution to suspend the cells thoroughly.
• the resulting supernatant fluid was treated with phenol/chloroform (1 :1 ), mixed with the same volume of isopropanol and then centrifuged.
• the resulting pellet was dissolved in the TE solution and treated with RNase and then with phenol/chloroform (1 :1 ) , followed by ethanol precipitation.
• the resulting pellet was again dissolved in the TE solution to which were subsequently added NaCl and polyethylene glycol 3,000 to their final concentrations of 0.63 M and 7.5%, respectively.
• the pellet was dissolved in the TE solution and precipitated with ethanol. In this way, about 300 ⁇ g of the pEF18S-HL34 plasmid DNA was obtained.
• the thus purified plasmid DNA was applied to the aforementioned 373A DNA sequencer (manufactured by Applied Biosystems) to determine its complete nucleotide sequence using the aforementioned Taq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems).
• the thus determined nucleotide sequence and an amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 4).
• oligonucleotides synthesized based on the nucleotide sequence of SEQ ID NO: 3 and synthetic oligonucleotides designed based on the internal sequence obtained by the sequence analysis were used in the nucleotide sequence determination as primers.
• the plasmid clone pEF18S-HL34 comprises a cDNA fragment of 861 bp and contains highly homologous sequences with the amino acid sequences AP8 (amino acid numbers 1 to 12 in SEQ ID NO: 4) and TP2/TP3 (amino acid numbers 157 to 162 in SEQ ID NO: 4) analyzed in Example 2.
• This DNA fragment seems to encode an open reading frame starting at its position 25, but has no termination codon and contains a poly A tail-like sequence of 76 bases on its 3'-end.
• Transfection of the thus obtained plasmid clone pEF18S-HL34 to COS 1 cells was carried out according to the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of the plasmid DNA by the DEAE-dextran method which includes chloroquine treatment. The COs-1 cells were cultured for 3 to 5 days at 37 * c, and then the supernatant was collected. "
• the thus obtained culture supernatant was extensively dialyzed against the IMDM culture medium and evaluated by the rat CFU-MK assay system.
• TPO activity was detected in a dose-dependent fashion in the culture supernatant of COS 1 cells in which a plasmid pEF18S-HL34 was expressed (Fig. 8). After 4 days of culturing, many megakaryocytes formed elongated cytoplasmic processes. In contrast, TPO activity was not found in the culture supernatant of COS 1 cells in which the plasmid pEF18S alone was expressed (Fig. 8).
• M-07e cell proliferation enhancing activity was found only in the culture supernatant of COS 1 cells in which the plasmid pEF18S-HL34 was expressed through its transfection.
• pEF18S-HL34 contains a gene which encodes a protein having a TPO activity.
• human TPO acts on rat megakaryocytic progenitor cells (no species specificity).
• the clone HL34 obtained in Example 15 contained the cDNA comprising a poly A tail-like continuous sequence on its 3'-side, which did not exist in the rat TPO cDNA and therefore seemed to be an artificial product of the experiment.
• a cDNA molecule from which the poly A tail ⁇ like sequence was deleted was prepared in order to see if a protein expressed by the deletion cDNA had a TPO activity.
• the deletion cDNA was prepared making use of PCR. Sequences of primers used for PCR are as follows in which a restriction enzyme recognition site is added to the 5'-end of each primer (EcoRI to hTPO ⁇ and Notl and two stop codons TAA and TGA to hTP03).
• hTP05 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO:170) (positions 1 to 21 in SEQ ID NO: 4)
• hTP03 5'-TTT GCG.
• PCR was carried out with a volume of 100 ⁇ l, making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and repeating a total of 15 cycles, each cycle consisting of denaturation at 95 ' c for 1 minute, annealing at 65 " c for 1 minute and synthesis at 72 • c for 1 minute, followed by the final incubation at 72 * c for 7 minutes.
• a band of about 800 bp thus obtained was digested with restriction enzymes EcoRI and Notl, purified and then sub-cloned into the expression vector pEF18S which has been treated in advance with the same restriction enzymes. From the resulting transformants, 5 clones which contained a DNA fragment of about 800 bp were selected to prepare a large quantity of plasmid DNA in accordance with the procedure described in Example 5. The whole length of the amplified region of about 800 bp of each plasmid was subjected to nucleotide sequence analysis to find its complete identity with the nucleotide sequence analyzed in Example 16 (positions 1 to 780 in SEQ ID NO: 4).
• the plasmid clone thus obtained was named pHT1- 231.
• the vector pHT1-231 carried by an E. coli strain DH5 has been deposited by the present inventors on February 14, 1994 under the deposit No. FERM BP-4564, in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.
• Transfection of the thus obtained plasmid to COS 1 cells was carried out according to the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of the plasmid DNA by the DEAE-dextran method which includes chloroquine treatment.
• the COS1 cells were cultured for 3 to 5 days at 37 ° c, and then the supernatant was collected.
• the clone HL34 prepared in Example 15 contained the cDNA comprising poly (A) tail-like sequence so that it was suggested that its 3'-end region was incomplete. It was therefore tried to obtain a full-length 3'-end region by PCR.
• the following four kinds of 5'-side primer for PCR were synthesized based on the sequences determined in Example 16 hTPO-H: 5'-AGC AGA ACC TCT CTA GTC CTC-3' (SEQ ID NO:39) (positions 574 to 594 in SEQ ID NO: 4)
• hTPO-K 5'-ACA CTG AAC GAG CTC CCA AAC-3' (SEQ ID NO:40) (positions .595 to 615 in SEQ ID NO: 4)
• hTPO-N 5'-AAC TAC TGG CTC TGG GCT TCT-3' (SEQ ID NO:41) (positions 660 to 680 in SEQ ID NO: 4)
• hTPO-O 5'
• 3'-side primers containing mixed nucleotides at the four bases of 3'-end were synthesized in order to amplify the cDNA from the beginning of the poly (A) portion.
• anchor primer without the mixed nucleotide, was also synthesized.
• hTP03mix 5'-TAG CGG CCG C(T) ⁇ 7 G GGG-3' (SEQ ID NO:43)
• AAA-3' (SEQ ID NO:44)
• the first strand of cDNA was synthesized from 1 ⁇ g of the commercial normal human liver-derived poly (A) + RNA (manufactured by Clontech), such as in Example 14, but using 0.5 ⁇ g of oligo dT primer (it is included in TimeSaverTMcDNA Synthesis Kit manufactured by Pharmacia).
• PCR was carried out with volume of 50 ⁇ l, making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN- ELMER) and heating at 96 * c for 2 minutes, repeating a total of 10 cycles, each cycle consisting of heat denaturation at 96 * c for 1 minute, annealing at 48 * c for 1 minute and synthesis at 72 " c for 1 minute, followed by the final incubation at 72 ° c for 7 minutes.
• the second PCR was carried out by using 1/10 volume of the first PCR resulting solution as a template, 20 ⁇ M of the hTPO-K primer and 10 ⁇ M of the hTP03mix primer with volume of 50 ⁇ l, heating at 96 - c for 2 minutes, repeating a total of 10 cycles, each cycle consisting of heat denaturation at 96 ° c for 1 minute, annealing at 63 * c for 1 minute and synthesis at 72 ° c for 1 minute, followed by the final incubation at 72 " C for 7 minutes.
• the third PCR was carried out by using 1/10 volume of the second PCR resulting solution as a template, 20 ⁇ M of the hTPO-N primer and 10 ⁇ M of the hTP03mix primer with volume of 50 ⁇ l, heating at 96 * c for 2 minutes, repeating a total of 10 cycles, each cycle consisting of heat denaturation at 96 • c for 1 minute, annealing at 63 ° c for 1 minute and synthesis at 72 ° c for 1 minute, followed by the final incubation at 72 * c for 7 minutes.
• the fourth PCR was carried out by using 1/10 volume of the third PCR resulting solution as a template, 20 ⁇ M of the hTPO-0 primer and 10 ⁇ M of the hTP03mix primer with volume of 50 ⁇ l, heat at 96 - c for 2 minutes, repeating a total of 10 cycles, each cycle consisting of heat denaturation at 96 * c for 1 minute, annealing at 63 * c for 1 minute and synthesis at 72 # c for 1 minute, followed by a final incubation at 72 ' c for 7 minutes.
• the fifth PCR was carried out by using 1/10 volume of the fourth PCR resulting solution as a template, 20 ⁇ M of the hTPO-0 primer and 20 ⁇ M of the hTP03anchor primer with volume of 50 ⁇ l, heating at 96 * c for 2 minutes, repeating a total of 10 cycles, each cycle consisting of heating denaturation at 96 ° c for 1 minute, annealing at 58 * c for 1 minute and synthesis at 72 ° c for 1 minute, followed by the final incubation at 72 * c for 7 minutes.
• reaction solution was subjected to 2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a DNA fragment of about 600 bp as the main product of the PCR which was subsequently purified making use of the aforementioned Prep-A-Gene DNA purification kit (manufactured by Bio-Rad Laboratories, Inc.).
• Nucleotide sequence of the thus purified DNA fragment was directly determined by a 373A DNA sequencer (manufactured by Applied Biosystems) making use of the aforementioned Taq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems).
• SEQ ID NO: 5 The thus determined nucleotide sequence and an amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 5).
• This DNA fragment has a nucleotide sequence coding for 130 amino acids starting with the primer hTPO-O, followed by sequence of more than 180 nucleotides at 3'-end (the nucleotides after 577 position could not determined).
• the 30 amino acid sequence starting with glycine coincides with the amino acid sequence of the positions 203 to 232 in SEQ ID NO: 4.
• the nucleotide sequence of positions 1 to 94 also coincides with the nucleotide sequence of the positions 692 to 785 in SEQ ID NO: 4.
• human TPO protein consists of 353 amino acids containing 21 amino-acid signal sequence, as shown in SEQ ID NO: 6.
• Example 15 Since the clone HL34 obtained in Example 15 contained the poly A tail-like sequence directly on its 3'-end of its open reading frame without termination codons and seemed therefore to be an artificial product of the cDNA synthesis, a cDNA library was reconstructed using 5 ⁇ g of a commercial normal human liver-derived poly (A) + RNA preparation (manufactured by Clontech). Synthesis of cDNA was carried out using SuperscriptTM Lambda System for cDNA Synthesis . and ⁇ Cloning Kit and SuperscriptTM II RNase H " (both manufactured by LIFE TECHNOLOGIES).
• the poly (A)+ RNA was subjected to heat denaturation and then added to 20 ⁇ l of a reaction solution containing a Notl sequence-included oligo dT as a primer attached to the kit (50 mM Tris-HCI, pH 8.3, 75 mM KCl, 3 mM MgCI 2 , 1 mM DTT, 1 mM dNTP mix, 200 U
• the reaction solution was heated at 65 * c for 10 minutes and extracted with the same volume of phenol/chloroform, and cDNA molecules having a length of less than 400 bp were removed using SizeSepTM 400 spun column (a spun column for the removal of low molecular weight DNA, attached to TimeSaverTM cDNA Synthesis Kit manufactured by Pharmacia).
• SizeSepTM 400 spun column a spun column for the removal of low molecular weight DNA, attached to TimeSaverTM cDNA Synthesis Kit manufactured by Pharmacia.
• EcoRI Adaptor attached to Directional Cloning Toolbox manufactured by Pharmacia
• the thus synthesized 1.3 ⁇ g of the double-stranded cDNA having an EcoRI recognition sequence on its 5'-end and a Notl recognition sequence on its 3'-end was ligated with the expression vector pEF18S which had been previously digested with EcoRI and Notl, and then transformed into 9.2 ml of Competent High E. coli DH5 (manufactured by Toyobo Co., Ltd.)
• the thus obtained human liver cDNA library (hTPO-F1 ) contained 1.0 x 10 6 transformants.
• hTPO-l 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO:48) (positions 60 to 80 in SEQ ID NO: 3)
• hTPO-KU 5'-AGG ATG GGT TGG GGA AGG AGA-3' (SEQ ID NO:49) (anti-sense primer corresponding to the sequence of positions 901 to 921 in SEQ ID NO: 6)
• the human liver cDNA library hTPO-F1 (1.0 x 10 6 transformants) constructed in Example 20 was divided into 3 pools (pool # 1 - 3) and the pools were frozen. PCR was carried out using 2 ⁇ g of the plasmid DNA prepared from each pool as a template and 1 ⁇ M each of the synthesized oligonucleotides (hTPO-l and hTPO-KU) as primers. Using GeneAmpTM PCR Reagent Kit with AmpliTaqTM DNA Polymerase (manufactured by Takara Shuzo.
• PCR was performed in 100 ⁇ l volume by GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) (a total of 35 cycles, each cycle consisting of denaturation at 95 " c for 1 minute, annealing at 59 # c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 * c for 7 minutes).
• GeneAmpTM PCR System 9600 manufactured by PERKIN-ELMER
• a DNA fragment having the expected size was amplified when the plasmid DNA prepared from the pool # 3 was used.
• the # 3 pool was then divided into subpools, each containing 15,000 transformants, and these subpools were cultured overnight in 1 ml of LB medium containing 50 ⁇ g/ml of ampicillin, followed by extraction of plasmid DNA using an automatic plasmid isolation apparatus PI-100.
• the thus extracted DNA was dissolved in TE solution, and 5% of the resulting solutions were used as templates to carry out PCR using the same primers and under the same conditions described above. As the result, amplification of DNA having the expected size was found in 6 of the 90 pools.
• amplification of DNA having the expected size was found in 6 of the 90 pools.
• the # 3 pool was spread on 100 LB agar plates of 15 cm in diameter in such an inoculum size that 4,100 colonies grew on each LB agar plate.
• one of the duplicate plates was cultured at 37 * c for 6 hours to recover colonies grown on the plate and to extract the plasmid DNA samples.
• PCR of these DNA samples was carried out in the same manner as described above, amplification of a band equivalent to the expected size was observed in one of 100 subpools.
• Two replica filters were prepared from the plate of this subpool using BIODYNETM A TRANSFER MEMBRANE (manufactured by PALL).
• Denaturation of the filters were performed by soaking them in 10% SDS for 10 minutes, in 0.5 N NaOH/1.5 M NaCl for 10 minutes and in 0.5 M Tris-HCI (pH 8.0)/1 .5 M NaCl for 10 minutes in that order, followed by 30 minutes of air-drying and subsequent 1 hour of baking at 80 " c in a vacuum oven.
• the thus baked filters were washed with 6 x SSC (prepared by diluting 20 x SSC stock solution consisting of 175.3 g of NaCl and 88.2 g of Na citrate dissolved in 1 liter water, pH 7.0) supplemented with 1 % SDS.
• Prehybridization of the thus washed filters were carried out by incubating it at 42 * c for 30 minutes with shaking in 30 ml of a reaction solution consisting of 50% formamide, 5 x SSC, 5 x Denhardt's solution (prepared from 50 x Denhardt's solution containing 5 g of Ficoll, 5 g of polyvinylpyrrolidone and 5 g of bovine serum albumin fraction V in 500 ml water), 1% SDS and 20 ⁇ g/ml of salmon sperm DNA.
• a reaction solution consisting of 50% formamide, 5 x SSC, 5 x Denhardt's solution (prepared from 50 x Denhardt's solution containing 5 g of Ficoll, 5 g of polyvinylpyrrolidone and 5 g of bovine serum albumin fraction V in 500 ml water), 1% SDS and 20 ⁇ g/ml of salmon sperm DNA.
• the reaction solution was exchanged with 30 ml of a hybridization solution having the same composition, mixed with a probe which had been labeled with [ ⁇ - 32 P] dCTP (manufactured by Amersham) and then incubated with shaking at 42 * c for 20 hours.
• the labeled probe used in this experiment was the EcoRI/SamH I fragment of the plasmid pEF18S-HL34, which was obtained by purifying a portion of SEQ ID NO: 4 ranging from its 5'-end to the 458 position base and by labeling the purified portion by random primer technique with Megaprime DNA Labelling System (a kit manufactured by Amersham based on the method disclosed in Anal.
• the resulting supernatant was treated with phenol/chloroform (1 :1 ), mixed with the same volume of isopropanol and then centrifuged.
• the resulting pellet was dissolved in TE solution and treated with RNase and then with phenol/chloroform (1 :1 ), followed by ethanol precipitation.
• the resulting pellet was again dissolved in TE solution to which were subsequently added NaCl and polyethylene glycol 3,000 to their final concentrations of 0.63 M and 7.5%, respectively.
• the pellet was dissolved in TE solution and precipitated with ethanol. In this way, about 300 ⁇ g of the plasmid DNA pHTF1 was obtained.
• the thus purified plasmid DNA was applied to the aforementioned 373A DNA sequencer (manufactured by Applied Biosystems) to determine its complete nucleotide sequence using the aforementioned Taq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems).
• the thus determined nucleotide sequence and the deduced amino acid sequence are shown in the Sequence Listing (SEQ ID NO: 7).
• Oligonucleotides synthesized based on the nucleotide sequence of SEQ ID NO: 6 and on the internal sequence obtained by their sequence, reaction analysis were used in the nucleotide sequence determination as primers.
• the clone pHTF1 contains a cDNA fragment of 1 ,721 bp and has high homology with the amino acid sequences AP8 (amino acid numbers 1 to 12 in SEQ ID NO: 7) and TP2/TP3 (amino acid numbers 157 to 162 in SEQ ID NO: 7) analyzed in Example 2.
• This DNA fragment seems to have a 5' noncoding region of 101 bases, an open reading frame consisting of 353 amino acid residues, starting at a methionine residue encoded at the 102 to 104 nucleotide positions and ending at a glycine residue encoded at the 1 ,158 to 1 ,160 nucleotide positions, the subsequent termination codon (TAA), a 3' noncoding region of 531 bases and a poly A tail sequence of 30 bases.
• TAA termination codon
• the amino acid sequence of a protein considered to be encoded by the open reading frame completely coincided with the predicted amino acid sequence of human TPO shown in SEQ ID NO: 6.
• the cDNA sequence of pHTFI has a larger size than the putative cDNA sequence shown in SEQ ID NO: 6, and contains 77 more additional bases at the 5' side and 347 more additional bases at the 3' side in front of its poly A tail sequence.
• the nucleotide sequence was different from SEQ ID NO: 6 at 3 positions. That is, A (position 84), A (position 740) and G (position 1 ,198) in SEQ ID NO: 7 were C, T and A in SEQ ID NO: 6, respectively. Only the mutation at position 740 was included in the protein-encoding region, but this mutation did not cause amino acid exchange because both bases A and T were the third base of threonine codons.
• the vector pHTFI carried by an E. coli strain DH5 has been .deposited by the present inventors on March 24, 1994 under the accession No. FERM BP-4617, at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.
• Transfection to COS 1 cells of the thus obtained plasmid clone pHTFI was carried out according to the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of the plasmid DNA by the DEAE-dextran method which includes chloroquine treatment. The transfected COS1 cells were cultured for 3 days at 37 ° c , and the culture supernatant was collected.
• Cloning of human TPO chromosomal DNA was carried out using human TPO cDNA as a probe.
• a genomic library used in the cloning was a gift from Prof. T. Yamamoto at the Gene Research Center, Tohoku University (the library reported by Sakai et al. in J. Biol. Chem., 269, 2173 - 2182,
• NZYM 10 g of NZ amine, 5 g of NaCl, 5 g of Bacto Yeast Extract, 2 g of MgS04-7H 2 0 and 15 g of agar in
• Prehybridization was carried out by incubating the thus treated filters at 42 - c for 1 hour in 500 ml of a reaction solution consisting of 50% formamide, 5 x SSC, 5 x Denhardt's solution, 1% SDS and 20 ⁇ g/ml of salmon sperm DNA.
• a human TPO cDNA fragment (base position numbers 178 to 1 ,025 in SEQ ID NO: 7) was amplified by PCR and purified, and the purified fragment was labeled with 32 P making use of Random Primer DNA Labelling Kit (a DNA labeling kit manufactured by Takara Shuzo based on the random primer method disclosed in Anal. Biochem., 132, 6 - 13, 1983).
• hTPO-l 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO:50) (positions 178 to 198 in SEQ ID NO: 7)
• hTPO-N 5'-AGG GAA GAG CGT ATA CTG TCC-3' (SEQ ID NO:51) (anti-sense primer corresponding to the sequence of positions 1 ,005 to 1 ,025 in SEQ ID NO: 7)
• hybridization was carried out at 42 ° c for 20 hours in 500 ml of a reaction solution having the same composition of the prehybridization solution.
• the resulting filters were washed 3 times in 2 x SSC/0.1% SDS solution at room temperature for 5 minutes, and then once in 0.1 x SSC/0.1% SDS solution at 68 ° c for 1 hour.
• the filters were then subjected to 16 hours of autoradiography at -70 ° c using an intensifying screen and an X-OMATTM AR5 film (manufactured by Eastman Kodak). As the result, 13 positive signals were obtained.
• Plaques approximately corresponding to each of the positive signals were collected from the original plates and inoculated again onto 15-cm NZYM plates in such an inoculum size that 1 ,000 plaques were formed on each plate.
• Two replica filters were prepared from each of the resulting plates to carry out hybridization under the same conditions described above. As the result, positive signals were detected on all filters of the 13 groups.
• a single plaque was recovered from each of the resulting plates to prepare phage DNA by the plate lysate method described in Molecular Cloning. Phage DNA samples thus prepared from the 13 clones were checked for the presence of the cDNA coding region, by PCR using primers having the following sequences.
• hTPO-L 5'-GGC CAG CCA GAC ACC CCG GCC-3 * (SEQ ID NO:52) (positions 1 to 21 in SEQ ID NO: 6)
• hTPO-F 5'-ATG GGA GTC ACG AAG CAG TTT-3' (SEQ ID NO:53) (anti-sense primer corresponding to the sequence of positions 127 to 147 in SEQ ID NO: 6)
• hTPO-P 5'-TGC GTT TCC TGA TGC TTG TAG-3' (SEQ ID NO:54) (positions 503 to 523 in SEQ ID NO: 6)
• hTPO-V 5'-AAC CTT ACC CTT CCT GAG ACA-3' (SEQ ID NO:55) (anti-sense primer corresponding to the sequence of positions 1 ,070 to 1 ,090 in SEQ ID NO: 6)
• the resulting filter was air-dried for 30 minutes and subjected to 2 hours of baking at 80 * c in a vacuum oven.
• Prehybridization was performed by incubating the thus treated filter at 42 ° c for 1 hour in 50 ml of a reaction solution consisting of 50% formamide, 5 x SSC, 5 x Denhardt's solution, 1% SDS and 20 ⁇ g/ml of salmon sperm DNA.
• a human TPO cDNA fragment base position numbers 178 to 1 ,025 in SEQ ID NO: 7 was amplified by PCR and purified, and the purified fragment was labeled with 3 2 P using Random Primer DNA Labelling Kit (manufactured by Takara Shuzo).
• hybridization was carried out at 42 ° c for 20 hours in 50 ml of a reaction solution having the same composition of the prehybridization solution.
• the resulting filter was washed 3 times in 2 x SSC/0.1% SDS solution at room temperature for 5 minutes, and then once in 0.1 x SSC/0.1% SDS solution at 68 * c for 1 hour.
• the filter was then subjected to 16 hours of autoradiography at -70 ° c using an intensifying screen and an X-OMATTM AR5 film (manufactured by Eastman Kodak). As the result, a single band of about 10 kb was observed in the case of the Hindlll digestion.
• FIG.11 A restriction map of phage clone ⁇ HGT1 is shown in Fig.11.
• Culturing of the clone pHGTI and purification of plasmid DNA were carried out basically according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
• the clone pHGTI was cultured overnight in 50 ml of the LB medium containing 50 ⁇ g/ml of ampicillin.
• the resulting cells were collected by centrifugation and suspended in 4 ml of the aforementioned TEG-lysozyme solution. To this were added 8 ml of 0.2 N NaOH/1% SDS solution and then 6 ml of 3 M potassium/5 M acetate solution to suspend the cells thoroughly.
• the resulting supernatant was treated with phenol/chloroform (1 :1 ), mixed with the same volume of isopropanol and then centrifuged.
• the resulting pellet was dissolved in the TE solution and treated with RNase and then with phenol/chloroform (1 :1), followed by ethanol precipitation.
• the resulting pellet was again dissolved in the TE solution to which were subsequently added NaCl and polyethylene glycol 3,000 to their final concentrations of 0.63 M and 7.5%, respectively.
• the pellet was dissolved in the TE solution and precipitated with ethanol. In this way, about 300 ⁇ g of the plasmid DNA pHGTI was obtained.
• the thus purified plasmid DNA was applied to the aforementioned 373A DNA sequencer (manufactured by Applied Biosystems) to determine its nucleotide sequence around the protein-encoding region predicted from the cDNA nucleotide sequence.
• the thus determined nucleotide sequence and the deduced amino acid sequence are shown in the Sequence Listing (SEQ ID NO: 8).
• oligonucleotides used in Example 22 for the nucleotide sequence analysis of cDNA and synthetic oligonucleotides designed based on the internal sequence obtained by their sequence reaction analysis were used as primers in the nucleotide sequence determination.
• chromosomal DNA carried by the plasmid clone pHGTI was found to contain an entire coding region of the amino acid sequence deduced from SEQ ID NO: 6, and nucleotide sequence of the coding region coincided completely with that of SEQ ID NO: 6.
• a region corresponding to the amino acid-encoding exon contained 4 introns having lengths of 231 bp, 286 bp, 1 ,932 bp and 236 bp in that order counting from the 5' side (Fig. 11 ).
• the nucleotide sequence was different from the cDNA nucleotide sequence of SEQ ID NO: 7 at 3 positions which were the same as the positions described in Example 22 (different positions between Sequence ID Nos.
• nucleotide sequence of the human TPO cDNA clone pHTFI obtained in Example 21 is different from the nucleotide sequence of the human chromosomal DNA clone pHGTI at 3 positions. Therefore, in order to analyze if these mutations in the cDNA clone pHTFI sequence reflect the chromosomal DNA sequence, nucleotide sequences of other 4 clones among the 5 chromosomal DNA clones independently selected by the screening were determined.
• sequence analysis was carried out by a direct nucleotide sequence determination method using a phage DNA sample prepared according to the plate lysate method described in Molecular Cloning. Sequencing of each clone was performed by applying it to the aforementioned 373A DNA sequencer (manufactured by Applied Biosystems), using the sequence primers used in Example 22, which have been synthesized based on such sequence portions that the aforementioned 3 mutation positions in the nucleotide sequence could be analyzed, and the aforementioned Taq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems). It was found that nucleotide sequences of all 4 clones were identical to that of SEQ ID NO: 6.
• the positions 84 and 740 corresponding to SEQ ID NO: 7 were substituted by C and T, respectively, in these 4 clones. But the position 1 ,198 was G in 2 clones and A in other 2 clones. In other words, it was revealed that there are two types of nucleotide sequences inherent to the Original chromosomal DNA. At present, it is not clear if such differences in the nucleotide sequence are derived from homologous chromosome or from plural genes. In addition, it is suggested that the differences in the nucleotide sequence may be due to a racial difference, because the poly (A) + RNA purchased from Clontech is a Caucasian origin while the chromosomal DNA is a Japanese origin.
• the vector pHGTI carried by an E. coli strain DH5 has been deposited by the present inventors on March 24, 1994 under the accession No. FERM BP-4616, at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.
• the plasmid clone pHGTI obtained by the subcloning contained a total of 4 EcoRI recognition sequences, 3 in the insert moiety and 1 in the vector.
• the nucleotide sequence analysis revealed that the entire human TPO protein-encoding region was included in a DNA fragment of about 4.3 kbp which was interposed between the EcoRI recognition sequence closest to the 5' side in the insert and the EcoRI recognition sequence in the vector. Therefore, this fragment was ligated with an EcoRI-treated expression vector pEF18S, and obtained 4 human TPO expression plasmid pEFHGTE#1-4 (see Fig. 11 ).
• an expression experiment was carried out. Purification of plasmid DNA was carried out basically according to the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), thereby obtaining about 250 ⁇ g of the plasmid DNA.
• Transfection to COS 1 cells of the thus obtained clones pEFHGTE#1 -4 was carried out according to the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of the plasmid DNA by the DEAE-dextran method which includes chloroquine treatment. The transfected COS1 cells were incubated for 3 days at 37 ° c and then the culture supernatants were collected.
• the culture supernatants were extensively dialyzed against the IMDM culture medium and evaluated by the rat CFU- MK assay system. TPO activity was detected in a dose- dependent fashion in the supernatant of COS1 cells in which each of four clones (pEFHGTE#1 -4) was expressed. By contrast, TPO activiy was not found in the supernatant of COS1 cells in which the plasmid pEF1 8S alone was expressed. Representative data with pEFHGTE#1 is shown in Fig. 12a. Similar results were obtained in the M-07e assay system.
• Example 18 Preparation of human TPO deletion type DNA and its expression in COS 1 cells and confirmation of TPO activity The results in Example 18 indicated that human TPO could exhibit its biological activity even after the removal of its carboxyl third. Thus, in order to further evaluate the biologically active portions, deletion derivative experiments were performed. In this Example, TPO deletion derivatives lacking 20, 40, 60 or 68 amino acids from the carboxyl-terminal end of the TPO protein (amino acids 1 -231 ) encoded by pHT1-231 were examined for their ability to exert TPO in vitro biological activity. The shortest derivative (amino acids 1 -163) still included amino acid sequences corresponding to TP2/3 of rat plasma TPO described in Example 2.
• the deletion plasmids were prepared by PCR using the DNA of the plasmid clone pHT1 -231 obtained in Example 18 as template and synthesized oligonucleotides as primers. Sequences of primers used for PCR were as follows: hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO:56) (prepared by adding an EcoRI recognition sequence to the sequence of positions 1 to 21 in SEQ ID NO:4; this is the identical sequence described in Fig.9); hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG ACA GCT GTG GTG GGT-3' (SEQ ID NO:57) (an antisense primer corresponding to the sequence of positions 555 to 576 in SEQ ID NO:4, prepared by adding two termination codons TAA and TGA and a Notl recognition sequence for use in the preparation of a deletion derivative coding for positions 1 to 163 amino acid
• PCR was carried out with a volume of 100 ⁇ l, using GeneAmpTM PCR System 9600 (manufactured by PERKIN- ELMER) and repeating a total of 20 cycles, each cycle consisting of denaturation at 95 * c for 1 minute, annealing at 65 ° c for 1 minute and synthesis at 72 ° c for 1 minute, followed by the final incubation at 72 * c for 7 minutes.
• a band of interest thus obtained by each PCR was digested with restriction enzymes EcoRI and Notl and the resulting digest was subjected to 1 % agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a main DNA fragment having an expected size amplified by each PCR.
• the thus isolated DNA fragment was purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad) and then sub ⁇ cloned into the expression vector pEF18S which had been previously treated with the same restriction enzymes.
• Competent-High E. coli DH5 manufactured by TOYOBO was used as the host strain.
• plasmid DNA samples were obtained from a deletion derivative coding for positions 1 to 163 amino acid residues (pHT1 -163 #1 -5), a deletion derivative coding for positions 1 to 171 amino acid residues (pHT1 -171 #1 -4), a deletion derivative coding for positions 1 to 191 amino acid residues (pHT1-191 #1-4) and a deletion derivative coding for positions 1 to 211 amino acid residues (pHT1-211 #1 -4).
• the whole length of the amplified regions of each plasmid DNA was subjected to nucleotide sequence analysis to find its complete identity with the nucleotide sequence shown in SEQ ID NO:4.
• Transfection to COS 1 cells of the thus obtained clones was carried out according to the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of each of the plasmid DNA samples by the DEAE-dextran method which includes chloroquine treatment. The transfected COS1 cells were cultured for 3 days and then the culture supernatants were recovered.
• the culture supernatants were extensively dialyzed against the IMDM culture medium and evaluated by the rat CFU- MK assay system. TPO activity was detected in a dose- dependent fashion in the supernatant of COS1 cells in which pHT1 -21 1 , pHT1 -191 , pHT1 -171 or pHT1 -163 each was expressed. Representative data with pHT1 -21 1 #1 , pHT1 - 191 #1 and pHT1-171#2 is shown in Fig. 13a and with pHT1 - 163#2 in Fig. 13b. Similar results were obtained in the M-07e assay system.
• the C-terminal amino acids were further deleted from the deletion derivatives obtained in Example 27 and the TPO activity expressed in COS 1 cells was examined.
• expression plasmids were prepared by deleting nucleotide sequences coding for C-terminal amino acid residues starting from the 163 position serine. Construction of these deletion plasmids was effected making use of PCR.
• hTPO-5 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO:61 ) (prepared by adding an EcoR I recognition sequence to the sequence of positions 1 to 21 in SEQ ID NO: 4; the same sequence shown in Example 18);
• hTPO-150 5 * -TTT GCG GCC GCT CAT TAG AGG GTG GAC CCT CCT ACA AGC AT-3' (SEQ ID NO:62) (an antisense primer corresponding to the sequence of positions 514 to 537 in SEQ ID NO:4; prepared by adding two termination codons TAA and TGA and a Notl recognition sequence for use in the preparation of a deletion mutant coding for positions 1 to 150 amino acid residues);
• hTPO-151 5'-TTT GCG GCC GCT CAT TAG CAG AGG GTG GAC CCT CCT ACA A-3' (SEQ ID NO:63) (SEQ ID NO:63) (SEQ ID NO:63)
• PCR was carried out using 1 ⁇ g of plasmid DNA of the clone pEF18S-HL34 obtained in Example 16 as a template and 10 ⁇ M each of the thus synthesized oligonucleotides (hTPO-5 for 5' side and hTPO-150, -151 , -153, -154, -155, - 156 and -157 for 3' side) as primers.
• PCR reaction was performed in 100 ⁇ l volume making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and repeating a total of 20 cycles, each cycle consisting of denaturation at 95 * c for 1 minute, annealing at 66 * c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 ° c for 7 minutes.
• a band of interest thus obtained by each PCR was digested with restriction enzymes EcoRI and Notl and the resulting digest was subjected to 1 % agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a main DNA fragment having a size expected by each PCR.
• the thus isolated DNA fragment was purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad) and then sub ⁇ cloned into the expression vector pEF18S which has been treated in advance with the same restriction enzymes.
• Competent-High E. coli DH5 manufactured by TOYOBO was used as the host strain.
• plasmid DNA samples were obtained from a deletion derivative coding for positions 1 to 150 amino acid residues (pHT1 -150 # 21 , 22 and 25), a deletion derivative coding for positions 1 to 151 amino acid residues (pHT1 -151 # 16, 17 and 18), a deletion derivative coding for positions 1 to 153 amino acid residues (pHT1 -153 # 1 to 5), a deletion derivative coding for positions 1 to 154 amino acid residues (pHT1-154 # 1 to 5), a deletion derivative coding for positions 1 to 155 amino acid residues (pHT1 -155 # 1 to 5), a deletion derivative coding for positions 1 to 156 amino acid residues (pHT1 -156 # 1 to 5) and a deletion derivative coding for positions 1 to 157 amino acid residues (pHT1-157 # 1 to 5).
• Transfection of COS 1 cells with each of the thus obtained clones was carried out in accordance with the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of each of the plasmid DNA samples by the DEAE- dextran method which includes chloroquine treatment, and the culture supernatant was recovered after 3 days of the culture. The thus obtained culture supernatants were dialyzed against the IMDM culture medium described in the foregoing and evaluated by the M-07e assay system.
• TPO activities were detected in the culture supernatant of COS 1 cells transfected with respective clones coding for C-terminal deletion derivatives, consisting of 1 to 151 position amino acids, 1 to 153 position amino acids, 1 to 154 position amino acids, 1 to 155 position amino acids, 1 to 156 position amino acids or 1 to 157 position amino acids. All of these derivatives contain a cysteine residue at position 151. However, no TPO activities were detected in the culture supernatant of COS 1 cells transfected with the clone coding for a C-terminal side deletion derivative consisting of 1 to 150 position amino acids whose cysteine residue at the 151 position was also deleted.
• the N- terminal side amino acids of the deletion derivatives obtained in Example 28 were further deleted and expression of TPO activity of the thus prepared deletion derivativess was examined.
• expression plasmids were prepared by deleting nucleotide sequences coding for N-terminal amino acid residues after the signal sequence. Construction of these deletion plasmids were effected making use of PCR.
• hTPO-5 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO:69) (prepared by adding an EcoR I recognition sequence to the sequence of positions 1 to 21 in SEQ ID NO:4; the same sequence shown in Example 18);
• hTP03 5'-TTT GCG GCC GCT CAT TAT TCG TGT ATC CTG TTC AGG TAT CC-3' (SEQ ID NO:70) (an antisense primer corresponding to the sequence of positions 757 to 780 in SEQ ID NO:4; prepared by adding two termination codons TAA and TGA and a Notl recognition sequence; the same sequence synthesized for use in the preparation of a deletion derivative coding for positions 1 to 231 amino acid residues);
• hTPO-S 5'-TTT GCG GCC GCT CAT TAG CTG GGG ACA GCT GTG GTG GGT-3' (SEQ ID NO:
• PCR was carried out using 1.4 ⁇ g of plasmid DNA of the clone pEF18S-HL34 obtained in Example 18 as a template and 5 ⁇ M each of the thus synthesized oligonucleotides (hTPO- 13 and hTP03 in one combination and hTPO-5 and hTPO-13R in another combination) as primers.
• PCR products were subjected to 1.2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a main DNA fragment having an expected size which was subsequently purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad) and dissolved in 15 ⁇ l of TE buffer. Thereafter, second PCR was carried out using 1 ⁇ l portion of each of the thus prepared solution as a template.
• the second PCR was carried out using 5 ⁇ M each of the synthesized primers (hTPO-5 and hTP03).
• the PCR reaction was performed in 100 ⁇ l volume making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and, after 5 minutes of denaturation at 95 ° c , repeating a total of 30 cycles, each cycle consisting of denaturation at 95° c for 1 minute, annealing at 60 ° c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 ° c for 7 minutes.
• PCR products were subjected to 1.2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a main DNA fragment having an expected size which was subsequently purified using Prep-A- Gene DNA Purification Kit (manufactured by Bio-Rad) and dissolved in 15 ⁇ l of TE buffer. After digestion with restriction enzymes EcoRI and Notl, the resulting solution was subjected to extraction with the same volume of phenol/chloroform and subsequent ethanol precipitation. After centrifugation, the resulting precipitate was dissolved in 15 ⁇ l of TE buffer and then sub-cloned into the expression vector pEF18S which has been treated in advance with the same restriction . enzymes.
• Competent-High E. coli DH5 manufactured by TOYOBO was used as the host strain.
• 45 clones containing the insert of expected size were selected to prepare plasmid DNA samples basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, it was able to obtain plasmid DNA of a deletion derivative (pHT13-231 ) coding for a protein molecule in which amino acids residues of positions 1 to 12 have been deleted from the original amino acid residues of positions 1 to 231.
• pHT13-231 deletion derivative coding for a protein molecule in which amino acids residues of positions 1 to 12 have been deleted from the original amino acid residues of positions 1 to 231.
• sequence of 3 clones were checked by 373A DNA Sequencer manufactured by Applied Biosystems making use of Taq Dye DeoxyTM Terminater Cycle Sequening Kit (Applied Biosystems), thereby obtaining a clone pHT13-231 #3 having a TPO cDNA sequence as designed and a deletion as expected with no substitutions and the like over the entire nucleotide sequence.
• the 163 position amino acid was used as the C-terminal for the preparation of deletion derivativess because, though the 231 position amino acid was designed as the C-terminal of TPO protein for the preparation of deletion derivativess in the above step (1 ), it was found that TPO activity could be expressed even when the C-terminal amino acids were further deleted. On the basis of the same reason, the 163 position amino acid was used as the C-terminal also in the subsequent step (3).
• PCR was carried out using 1.4 ⁇ g of plasmid DNA of the clone pHT1-163 obtained in Example 27 as a template and 5 ⁇ M each of the synthesized oligonucleotides (hTPO-7 and hTPO-S in one combination and hTPO-5 and hTPO- 7R in another combination) as primers.
• PCR products were subjected to 1.2% agarose gel (manufactured by FMC BioProducts) electrophoresis to isolate a main DNA fragment having an expected size which was subsequently purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad) and dissolved in 15 ⁇ l of TE buffer. Thereafter, second PCR was carried out using 1 ⁇ l portion of each of the thus prepared solution as a template.
• the second PCR was carried out using 5 ⁇ M each of the synthesized primers (hTPO-5 and hTPO-S).
• the PCR reaction was performed in 100 ⁇ l volume making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and, after 5 minutes of denaturation at 95 * c , repeating a total of 30 cycles, each cycle consisting of denaturation at 95 * c for 1 minute, annealing at 60 ° c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 ° c for 7 minutes.
• PCR products were subjected to 1.2% agarose gel (man ufactu red by FMC BioProducts) electrophoresis to isolate a main DNA fragment having an expected size which was subsequently purified using Prep-A- Gene DNA Purification Kit (manufactured by Bio-Rad) and dissolved in 15 ⁇ l of TE buffer. After digestion with restriction enzymes EcoRI and Notl, the resulting solution was subjected to extraction with the same volume of phenol/chloroform and subsequent ethanol precipitation. After centrifugation, the resulting precipitate was dissolved in 15 ⁇ l of TE buffer and then sub-cloned into the expression vector pEF18S which has been treated in advance with the same restriction enzymes.
• Prep-A- Gene DNA Purification Kit manufactured by Bio-Rad
• Competent-High E. coli DH5 manufactured by TOYOBO was used as the host strain. From the resulting transformants, 30 clones containing the insert of expected size were selected to prepare plasmid DNA samples basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, it was able to obtain plasmid DNA of a deletion derivative (pHT7-163) coding for a protein molecule in which amino acids residues of positions 1 to 6 have been deleted from the original amino acid residues of positions 1 to 163 in SEQ ID NO: 4.
• a derivative (pHT8-163) having deletion of amino acid residues of positions 1 to 7 was prepared basically in the same manner as the above case for the preparation of a derivative (pHT7-163) having deletion of amino acid residues of positions 1 to 6.
• PCR was carried out using 1.4 ⁇ g of plasmid DNA of the clone pHT1-163 obtained in Example 27 as a template and 5 ⁇ M each of the synthesized oligonucleotides
• the second PCR was carried out using 5 ⁇ M each of the synthesized primers (hTPO-5 and hTPO-S).
• the PCR reaction was performed in 100 ⁇ l volume making use of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) and, after 5 minutes of denaturation at 95 ° c , repeating a total of 30 cycles, each cycle consisting of denaturation at 95 * c for 1 minute, annealing at 60 * c for 1 minute and synthesis at 72 * c for 1 minute, followed by the final incubation at 72 ° c for 7 minutes.
• PCR products were subjected to 1.2% agarose gel (manufactu red by FMC Bio Products) electrophoresis to isolate a main DNA fragment having an expected size which was subsequently purified using Prep-A- Gene DNA Purification Kit (manufactured by Bio-Rad) and dissolved , in 15 ⁇ l of TE buffer. After digestion with restriction enzymes EcoRI and Notl, the resulting solution was subjected to extraction with the same volume of phenol/chloroform and subsequent ethanol precipitation. After centrifugation, the resulting precipitate was dissolved in 15 ⁇ l of TE buffer and then sub-cloned into the expression vector pEF18S which has been treated in advance with the same restriction enzymes.
• Prep-A- Gene DNA Purification Kit manufactured by Bio-Rad
• Competent-High E. coli DH5 manufactured by TOYOBO was used as the host strain. From the resulting transformants, 30 clones containing the insert of expected size were selected to prepare plasmid DNA samples basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, it was able to obtain plasmid DNA of a deletion derivative (pHT8-163) coding for a protein molecule in which amino acids residues of positions 1 to 7 have been deleted from the original amino acid residues of positions 1 to 163 in SEQ ID NO:4.
• Transfection of COS 1 cells with each of the thus obtained deletion clones was carried out according to the procedure of Example 11. That is, transfection was performed with 10 ⁇ g of each of the plasmid DNA samples by the DEAE- dextran method which includes chloroquine treatment, and the culture supernatants were recovered after 3 days of culture. The thus obtained culture supernatant was thoroughly dialyzed against the IMDM culture medium described in the foregoing and evaluated by the M-07e assay system.
• TPO activity was detected in the culture supernatant of COS 1 cells transfected with a clone coding for the deletion derivative consisting of 7 to 163 position amino acids.
• no TPO activity was detected in the culture supernatant of COS 1 cells transfected with a clone coding for the deletion derivative consisting of 8 to 163 position amino acids or of 13 to 231 position amino acids.
• An expression vector having all amino acid coding regions of human TPO cDNA presumed in SEQ ID NO:6 was constructed for the expression in mammalian cells.
• PCR was carried out in the following manner in order to prepare a DNA fragment which covers all human TPO cDNA coding regions.
• Nucleotide sequences of the used primers are as follows: hTPO-l: 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO:78) (positions 105 to 125 in SEQ ID NO:6);
• SA 5'-CAG GTA TCC GGG GAT TTG GTC-3' (SEQ ID NO:79) (an antisense primer corresponding to the sequence of positions 745 to 765 in SEQ ID NO:6)
• hTPO-P 5'-TGC GTT TCC TGA TGC TTG TAG-3' (SEQ ID NO:80) (positions 503 to 523 in SEQ ID NO:6)
• hTPO-KO 5'-GAG AGA GCG GCC GCT TAC CCT TCC TGA GAC.
• AGA TT-3' (SEQ ID NO:81) (a sequence prepared by adding a restriction enzyme Notl recognition sequence and a GAGAGA sequence (SEQ ID NO:82) to an antisense sequence corresponding to the sequence of positions 1066 to 1086 in SEQ ID NO:6).
• a first PCR was carried out using 300 ng of the clone pEF18S-HL34 obtained in Example 16 as a template.
• composition of the reaction solution in final concentration is as follows: 10 mM KCl, 10 mM (NH 4 ) 2 S0 4 , 20 mM Tris-HCI (pH
• second PCR (repetition of a total of 30 cycles, each cycle consisting of incubations at 96 ° c for 1 minute, at 58° c for 1 minute and at 72 * c for 1 minute) was carried out using 1/20 volume of the reaction solution of synthesized cDNA as a template and 2.5 ⁇ M each of the primers hTPO-P and hTPO-KO and 2.5 units of AmpliTaqTM DNA polymerase (manufactured by Takara Shuzo).
• Each of the resulting solutions of the first and second PCR was subjected to 1 % agarose gel electrophoresis to isolate, respective main DNA fragments of expected size which was subsequently purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad). Thereafter, third PCR was carried out using 1/20 volume of each of the thus prepared solutions as a template.
• This reaction (heating at 96 * c for 2 minutes, followed by the repetition of a total of 3 cycles, each cycle consisting of incubations at 96 * c for 2 minute and at 72 ° c for 2 minute, and subsequent incubation at 72 * c for 7 minutes) was carried out using 1 unit of Vent R ⁇ M DNA polymerase (manufactured by New England BioLabs).
• the resulting reaction solution was mixed with 1 ⁇ M each of hTPO- I and hTPO-KO, heated at 96° c for 2 minutes, subjected to 25 cycles of reaction, each cycle consisting of incubations at 96 ° c for 1 minute, at 62 * c for 1 minute and at 72 ° c for 1 minute, and then incubated at 72 ° c for 7 minutes.
• the resulting reaction solution was extracted with the same volume of water saturated phenol-chloroform and then with the same volume of chloroform, and the extract was subjected to ethanol precipitation (2.5 volumes of ethanol in the presence of 0.3 M sodium acetate and 0.5 ⁇ l of glycogen manufactured by Boehringer-Mannheim) to recover DNA.
• the thus recovered DNA was digested with restriction enzymes B a m HI and No tl and subjected to 1 % agarose gel electrophoresis, and a main band having an expected size thus isolated was purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), ligated with pBluescript II SK + vector (manufactured by Stratagene) which had been digested in advance with the restriction enzymes EamHI and Notl and then transformed into Competent-High E. coli DH5 (manufactured by TOYOBO). From the resulting colonies, 4 clones were selected to prepare plasmid DNA samples.
• the sequence of purified plasmid DNA samples were checked by 373A DNA Sequencer manufactured by Applied Biosystems making use of Taq Dye DeoxyTM Terminater Cycle Sequening Kit (Applied Biosystems), thereby obtaining a clone, pBLTP, having a TPO cDNA sequence as designed with no substitution in nucleotide sequence within the region between BamHI and Notl.
• the clone pBLTP was digested with restriction enzymes EcoRI and BamHI and subjected to 1 % agarose gel electrophoresis, and a band of high molecular weight thus isolated was purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad).
• pEF18S-HL34 was treated with the restriction enzymes to purify a DNA fragment of about 450 bp.
• Each of the thus obtained DNA samples was subjected to ligation and transformed into Competent-High E. coli DH5 (manufactured by TOYOBO). Plasmid DNA samples were prepared from the resulting colonies to obtain a clone, pBLTEN, containing human TPO cDNA insert.
• pBLTEN was digested with restriction enzymes EcoRI and Notl and subjected to 1% agarose gel electrophoresis, and a DNA fragment of about 1 ,200 bp thus isolated was purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), ligated with the expression vector pEF18S which has been digested in advance with the same restriction enzymes and then transformed into Competent-High E. coli DH5 (manufactured by TOYOBO). Plasmid DNA samples were prepared from the resulting colonies to obtain the clone of interest, pHTPI , which contains entire human TPO cDNA coding region.
• Plasmid DNA was prepared from this clone in a large quantity and used in the following experiments. Preparation of plasmid DNA was carried out basically in accordance with the procedure described in Molecular Cloning. (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
• DHFR mouse dihydrofolate reductase
• the mammalian expression vector pEF18S was digested with a restriction enzyme Smal, dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo) and then ligated with a DNA fragment containing mouse DHFR minigene by using T4 DNA ligase (manufactured by Takara Shuzo) to obtain an expression vector pDEF202.
• the constructed expression vector pDEF202 was digested with restriction enzymes, EcoRI and Spel, and then subjected to agarose gele electrophoresis to isolate a larger DNA fragment.
• Human TPO cDNA which has been obtained by digesting the plasmid pHTPI containing human TPO cDNA (clone p1) with restriction enzymes, EcoRI and Spel, was ligated with this linearized pDEF202 vector by using T4 DNA ligase (manufactured by Takara Shuzo) to obtain a plasmid pDEF202-hTPO-P1 for expression of human TPO cDNA.
• This constructed plasmid contains SV40 replication origin, human elongation factor 1 - ⁇ -promoter, and SV40 early polyadenylation signal for transcription of human TPO cDNA, mouse DHFR minigene, and pUC18-replication origin and ⁇ - lactamase gene (Amp r ).
• CHO cells ( a DHFR- strain; Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, vol.77, p4216, 1980) were maintained in ⁇ -minimum essential medium ( ⁇ -MEM(-), supplemented with hypoxanthine and thymidine) containing 10% fetal calf serum (FCS) by using a 6 cm diameter tissue culture plate (by Falcon).
• ⁇ -MEM(-) ⁇ -minimum essential medium
• FCS fetal calf serum
• the transformation of CHO cells with a pDEF202-hTPO-P1 plasmid was carried out by a calcium phosphate method (CellPhect, manufactured by Pharmacia) as described below.
• Example 31 Ten micrograms of the plasmid pDEF202-hTPO-P1 prepared in Example 31 was mixed with 120 ml of buffer A and 120 ml of H 2O, and the resulting mixture was incubatedat room temperature for 10 minutes. This solution was further mixed with 120 ml of buffer B and allowed to stand at room temperature for additional 30 minutes. This DNA mixture solution was then added into the culture and incubated for 6 hours in a C0 2 incubator.
• the culture was washed twice with serum-free ⁇ -MEM(-), treated with ⁇ - MEM(-) containing 10% dimethyl sulfoxide for 2 minutes, and then incubated for 2 days in non-selection medium ( ⁇ -M EM (-) supplememted with hypoxanthine and thymidine) containing 10 % dialyzed FCS.
• non-selection medium ⁇ -M EM (-) supplememted with hypoxanthine and thymidine
• ⁇ -MEM(-) supplememted with hypoxanthine and thymidine
• the cells in 6 cm tissue culture plate were split into five 10 cm tissue culture plates or twelve 24-well tissue culture plates, and then cultured in the selection medium.
• the culture medium was exchanged at the intervals of two days.
• the human TPO activity in the cultured medium of CHO cells with DHFR- positive phenotype was measured by CFU-MK assay, M-07e assay or. Ba F3 assay.
• the cells secreting human TPO into culture medium were further selected in the selection medium supplemented with 25 nM methotrexate in order to isolate the cell clone producing a higher amount of human TPO.
• transfection of CHO cells may also be effected by carrying out co-transfection of CHO cells with pHTPI and pMG1 plasmids.
• CHO strain (CHO-DUKXB11) transfected with the plasmid pDEF202-hTPO-P1 has been deposited by the present applicant on January 31 , 1995, under the accession No. FERM BP-4988, at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.
• a mammalian expression vector pBMCGSneo was digested with restriction enzymes, Xhol and Notl, and the subjected to agarose gel electrophoresis to isolate the vector DNA portion.
• This DNA fragment and human TPO cDNA (P1 clone) which had been obtained by digesting the plasmid pBLTEN containing human TPO cDNA with the restriction enzymes Xhol and Notl were ligated using T4 DNA ligase to obtain the expression vector of interest, pBMCGSneo- hTPO-P1.
• This expression plasmid contains cytomegalovirus early promoter, parts of rabbit ⁇ globin gene-derived intron and polyadenylated region for transcription of human TPO cDNA, human ⁇ globin gene, 69% of bovine papilloma virus 1 gene, thymidine kinase promoter and polyadenyl region, phosphotransferase I gene (neomycin resistant gene) and pBR 322 replication origin and ⁇ -lactamase gene (Amp r ), and human TPO cDNA was ligated at a downstream site of the cytomegalovirus promoter.
• X 63.6.5.3 cells were cultured in Dulbecco's minimal essential (DME) medium containing 10% fetal bovine serum. The transfection of X63.6.5.3 cells with BMCGSneo hTPO plasmid was carried out by electroporation method as described below.
• DME Dulbecco's minimal essential
• the transfected cells were suspended in 50 ml of DEM containing 10% fetal bovine serum, dispensed into wells of five 96 well tissue culture plate and then cultured for 2 days in a C0 2 incubator. After the 2 days culture, the culture medium was exchanged with DEM containing 10% fetal bovine serum and 1 mg/ml of G418 (manufactured by GIBSCO), and the medium exchange was carried out every 3 days thereafter.
• the human TPO activity in the culture medium of transformants was measured by CFU-MK, M-07e or Ba/F3 assay. The transformants secreting human TPO into culture medium were cloned twice by a limiting dilution to establish a human TPO producing cell lines.
• COS 1 cells Transfection of COS 1 cells was carried out according to the DEAE-dextran method which includes chloroquine treatment as described in Example 11.
• COS 1 cells (ATCC CRL1650) were cultured in IMDM containing 10% (v/v) FCS using a collagen-coated 175 cm 2 culture flask at 37° c in a 5% carbon dioxide ' incubator until the cells became about 100% confluent.
• Transfection was effected by mixing 20 ml of IMDM solution containing 250 ⁇ g/ml of DEAE-dextran (manufactured by Pharmacia), 60 ⁇ M of chloroquine (manufactured by Sigma) and 10% (v/v) of Nu- Serum (manufactured by Collaborative) with the plasmid pHTPI (40 ⁇ g) which has been dissolved in 500 ⁇ l of HBS, adding the resulting mixture to the COS 1 cells described above contained in one 175 cm 2 culture flask, which have been washed once with IMDM just before the transfection, and then culturing the cells for 3 hours at 37 ° c in a 5% carbon dioxide incubator.
• the culture supernatant was removed by suction, and the cells were washed once with IMDM, mixed with 50 ml of a serum-free medium arid then cultured for 5 days at 37 ° c in the 5% carbon dioxide incubator to recover the culture supernatant.
• 100 to 260 culture flasks of 175 cm 2 were used and 5 to 13 liters of the culture supernatant was recovered.
• the serum-free medium was prepared by supplementing IMDM with 5 ⁇ g/ml of insulin (manufactured by Sigma) , 5 ⁇ g/ml of transferrin (manufactured by Sigma), 10 ⁇ M of monoethanolamine (manufactured by Wako Pure Chemical Industries), 25 nM of sodium selenite (manufactured by Sigma) and 200 ⁇ g/ml of BSA (fatty acid-free high purity bovine albumin, manufactured by Nichirei).
• TPO activity was carried out to obtain a high purity product.
• the M-07e assay system was mainly used for the measurement of TPO activity in the following preparation steps. TPO activity was similarly found in the rat CFU-MK assay system. In carrying out these assays, human serum albumin (HSA) was added to each sample to a final concentration of 0.02 to 0.05%.
• HSA human serum albumin
• COS 1 cells transfected with the plasmid pHTPI according to the method of Ohashi and Sudo (Hideya).
• proteolytic enzyme inhibitors p-APMSF and Pefabloc SC 4-(2- ami noethyl)-benzenesulfo nyl fl uo ride hydroch lo ride , manufactured by Merk, catalog No. 24839) to a final concentration of about 1 mM, followed by filtration using a 0.2 ⁇ m filter to recover the supernatant.
• fraction F2 (1 ,092 ml; protein concentration, 0.776 mg/ml; total protein, 847 mg; and relative activity, 150,000).
• the eluting solution was changed to 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl to collect fraction F2 (856 ml; protein concentration, 0.189 mg/ml; total protein, 162 mg; and relative activity, 300,000).
• the eluting solution was changed to 20 mM sodium citrate buffer (pH 5.2) containing 1 ,000 mM NaCl to collect eluted fraction F3 (370 ml; protein concentration, 0.034 mg/ml; total protein, 12.6 mg; and relative activity, 150,000).
• the main TPO active fraction F2 (845 ml) of the SP Sepharose Fast Flow column step was mixed with about 10% in final concentration of 1 -propanol and applied at a flow rate of 3 ml/min to LA-WGA column (2 cm in diameter and 14 cm in bed height, manufactured by Honen, catalog No. WG-007) which has been equilibrated in advance with 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl.
• fraction F1 (64.4 ml; protein concentration, 0.0178 mg/ml; total protein, 1 .15 mg; and relative activity, 17,220).
• the eluting solution was changed to 20 mM sodium citrate buffer (pH 6.1) containing 0.4 M GlcNAc and 10% 1 -propanol to collect eluted fraction F2 (45 ml; protein concentration, 0.0104 mg/ml; total protein, 0.470 mg; and relative activity, 675,000).
• the main TPO active fraction F2 (340 ml) of the LA-WGA column operation was mixed with about 0.005% in final concentration of TFA and subjected to YMC-Pack CN-AP (6 mm in diameter and 250 mm in bed height, manufactured by YMC, (catalog No. AP-513) column chromatography. That is, using 0.1 % TFA as a developing solvent A and 1 -propanol containing 0.05% TFA as a developing solvent B, the sample was applied at a flow rate of 0.6 ml/min to the YMC-Pack CN- AP column which has been equilibrated in advance with 15% B.
• the propanol concentration was increased from 15% B to 25% B, and the elution was carried out with a linear gradient of from 25% B to 50% B for 65 minutes while collecting the eluates in 1.5 ml (2.5 min) portions in polypropylene tubes.
• TPO active fraction FA 13.5 ml
• the fraction FA obtained by the YMC-Pack CN-AP column step was subjected to Capcell Pak Cl 300A (4.6 mm in diameter and 150 mm in bed height, manufactured by Shiseido, catalog No. Cl TYPE:SG300A) column chromatography using 0.1% TFA as a developing solvent A and 1-propanol containing 0.05% TFA as a developing solvent B.
• a portion (8.9 ml) of the fraction FA obtained by the YMC-Pack CN-AP column operation was mixed with 0.3 ml of glycerol, concentrated by centrifugation evaporation and then mixed with about 2.5 ml of 10% B, and the resulting solution was subsequently applied at a flow rate of 0.4 ml/min to the Capcell Pak Cl 300A column which has been equilibrated in advance with 20% B.
• the elution was carried out firstly with 20% B for 5 minutes and then with a linear gradient of from 20% B to 40% B for 50 minutes while collecting the eluates in 1 ml (2.5 min) portions in polypropylene tubes.
• a 1 ⁇ l portion (1/1 ,000 fraction) of the eluate in each tube was mixed with HSA and concentrated by ultrafiltration to obtain 0.25 ml of IMDM assay culture solution containing 0.05% HSA for use in the identification of TPO active fractions by the assay.
• strong TPO activity (relative activity of about 3,000,000 to 22,500,000) was found in tube Nos. 20 to 23 (within the range of 28.5 and 32.5% as. propanol concentration) which were pooled as a high activity fraction.
• a partially purified TPO fraction was prepared in the following manner from a culture supernatant containing the human complete length TPO (derived from the expression plasmid pHTPI , F1 clone) expressed in COS 1 cells.
• TPO activity was examined by the M-07e assay system after extracting protein from a gel of SDS-PAGE electrophoresis carried out under a non-reducing condition as described in Example 1 , or molecular weight was measured based on reduction-treated DPCIII markers by means of a western analysis which will be described later in Example 45.
• TPO activity was found within a broad apparent molecular weight range of approximately from 69,000 to 94,000, thus confirming variation in the molecular weight.
• apparent molecular weight of TPO based on reduction- treated DPCIII markers after its N-glycosidic linkage type sugar chain digestion with N-glycanase was measured to be 36,000 to 40,000 which was larger than the molecular weight of about 35,000 presumable from the size of the peptide chain, thus strongly indicating the presence of an O-glycosidic linkage type sugar chain too.
• human TPO stimulated the formation of significant number of megakaryocyte colonies. For example, 11.5 megakaryocyte colonies were formed by 6,000 CD34+DR+ cells in the presence of 10% (v/v) of culture supernatant from COS1 cells transfected with pHT1 -231.
• CD34 + cells were prepared from human peripheral blood as follows. A leukocyte fraction obtained from human peripheral blood by a Ficoll-Paque density gradient was depleted of cells adherent to plastic dishes.
• the nonadherent cells subsequently, were depleted of SBA (soybean agglutinin) positive cells by a panning using AIS Microselecter SBA(Asahi Medical).
• the resultant cells were incubated on AIS Microselecter CD 34, and the adsorbed cells, CD 34 + cells, were collected when CD3 + cells were cultured for 10 days in the presence of culture supernatant from transfected COS 1 cells in a liquid medium, selective proliferation of large size Gpllb/llla + cells was observed, and increase in the ploidy was found in these Gpllb/llla + cells.
• the plastic nonadherent cell fraction contains various hematopoietic progenitor cells other than CFU-MK, only megakaryocyte colonies were formed in the presence of human TPO, strongly suggesting that human TPO exerts its selective action on progenitor cells of the megakaryocyte lineage.
• the Gpllb/llla + cells obtained from rat bone marrow was cultured for 3 to 5 days in the presence of 20% (v/v) of culture supernatant from transfected COS 1 cells in a liquid culture, increase in the ploidy was observed clearly on 5 days of the culture.
• an artificial gene which encodes human TPO and contains an E. coli preference codon was prepared.
• the nucleotide sequence of this DNA contains an amino-terminal methionine codon (ATG) at the -1 position for use in the translation initiation in E. coli.
• GGCTCCGCCAGCTTGTGACCTTCGTGTTCTGTCT-3' (SEQ ID NO.83); 2:5-CAGTTTAGACAGM( ⁇ CGAAGGTCACAAGCTGGCGG
• CTTCCAGAAGCAGAGTTACTGCACCCAGGATGTC-3' (SEQ ID NO.-90); 9: S-CCGACCTGCX ⁇ TGTCTRCXX ⁇ GC ⁇ GGC AGCTG ' TO
• Single-stranded cDNA was synthesized from 1 ⁇ g of human normal liver-derived poly (A) + RNA using oligo dT primer, and PCR was carried out using 0.1 volume of the cDNA synthesis reaction solution as a template.
• the PCR reaction (incubation at 96 ⁇ c for 2 minutes, repetition of a total of 30 cycles of reaction, each cycle consisting of incubations at 95 ° c for 1 minute, at 58 ° c for 1 minute and at 72 ° c for 1 minute, and final incubation at 72 ° c for 7 minutes) was carried out with a volume of 100 ⁇ l using hTPO-C and hTPO- Z(EcoRI) as primers.
• the human TPO cDNA fragment obtained in this way was digested with BamHI and EcoRI and subjected to 2% agarose gel electrophoresis to recover a fragment of about 600 bp in. size which was subsequently purified using Prep-A- Gene DNA Purification Kit and sub-cloned into pUC18 which has been digested in advance with EcoRI and BamHI (E. coli DH5 ⁇ was used as the host).
• a clone encoding correct human TPO nucleotide sequence was selected by nucleotide sequence analysis and named pUC18(BE)(124-332).
• Oligonucleotide sequences of the primers used in the PCR reaction are as follows: hTPO-C: 5'-GGA GGA GAC CAA GGC ACA GGA-3' (SEQ ID NO:95) (positions 329 to 349 of pHTFI clone); and hTPO-Z(EcoRI): 5'-CCG GAA TTC TTA CCC TTC CTG AGA CAG ATT-3' (SEQ ID NO:96) (prepared by adding an EcoR I recognition sequence to an antisense primer corresponding to positions 1 ,143 to 1 ,163 of pHTFI clone).
• the clone pUC18(BE)(124-332) was digested with B a m HI and Eco RI and subjected to 2% agarose gel electrophoresis to recover a human TPO cDNA C-terminal side fragment of about 600 bp in size which was subsequently purified using Prep-A-Gene DNA Purification Kit and sub ⁇ cloned into pUC18(XB)(1 -123) which has been digested in advance with EcoRI and BamHI (E. coli DH5 ⁇ was used as the host).
• a clone obtained in this manner was named pUC18(XE)(1-332).
• SSE1 and SSE2 were annealed in a solution composed of 10 mM Tris/HCI (pH 7.5), 10 mM MgCI 2 and 50 mM NaCl, and, making use of DNA Ligation Kit (manufactured by Takara Shuzo), the thus obtained double-stranded DNA fragment was introduced into pUC18(XE)(1-332) from which the human TPO cDNA C- terminal fragment of about 480 bp has been removed by its digestion with Sacl and EcoRI, thereby obtaining a clone pUC18(XS)(1 -174) (E. coli DH5 ⁇ was used as the host). Sequences of the synthetic oligonucleotides used herein are as follows:
• SSE2 AATTCTCATTAGAGCT (SEQ ID NO:98); S d SSE1 EccEI ⁇ ' -CTAATGAG-S ' ( SEQ ID NO : 99 ) 3 * -TCGAGATTACTCTTAA-5 ' ( SEQ ID NO : 100 ) SSE2
• the clone pUC18(XS)(1 -174) obtained above was digested with Xbal and EcoRI and subjected to 2% agarose gel electrophoresis to recover a fragment of about 600 bp in size which encodes amino acid residues 1 to 174 of human TPO, and the fragment was subsequently purified using Prep-A-Gene DNA Purification Kit and sub-cloned into pBluescript IISK+ (manufactured by Stratagene) which had been digested in advance with Xbal and EcoRI (E. coli DH5 ⁇ was used as the host).
• a clone obtained in this way was named pBL(XS)(1-174).
• the clone pBL(XS)(1 -174) was digested with Xbal and Hindlll to recover a fragment of about 550 bp in size which encodes amino acid residues 1 to 174 of human TPO, and the fragment was subsequently purified using Prep- A-Gene DNA Purification Kit and sub-cloned into pCFM536 (EP- A-136490) which had been digested in advance with Xbal and Hindlll (E. coli DH5 ⁇ was used as the host).
• pCFM536/hT(1-174) A clone obtained in this way was named pCFM536/hT(1-174).
• the human TPO encoding fragment obtained in this way was digested with Nael and EcoRI and subjected to 2% agarose gel electrophoresis to recover a fragment of about 550 bp in size, the fragment was subsequently purified using Prep-A-Gene DNA Purification Kit and cloned into pGEX-2T which has been digested in advance with EcoRI and Smal (E. coli DH5 was used as the host) and then a clone, named pGEX-2T/hT(1 -174) , which encodes correct human TPO nucleotide sequence was selected by nucleotide sequence analysis to prepare a transformant for use in the expression of GST-TPO(1 -174). Oligonucleotide sequences of the primers used in the PCR reaction are as follows:
• GEX1 5'-ATC GCC GGC TCC GCC AGC TTG TGA C-3' (SEQ ID NO:101) (prepared by adding a ⁇ /ael recognition sequence to the sequence of positions 21 to 39 in SEQ ID NO:10); and
• GEX3 5'-GCC GAA TTC TCA TTA GAG CTC GTT CAG TGT-3' (SEQ ID NO:102) (an antisense primer corresponding to the sequence of positions 523 to 549 in SEQ ID NO:10).
• This expression plasmid contains a DNA sequence which encodes GST protein followed by a thrombin recognition peptide and a human TPO (amino acid residues 1 to 174).
• the DNA sequence which encodes thrombin recognition peptide and human TPO are shown in the Sequence Listing (SEQ ID NO:10).
• the transformant prepared in Example 39 was cultured overnight at 37 ° c on a shaker using 60 ml of LB medium containing 50 ⁇ g/ml of ampicillin, and 25 ml portion of the resulting culture broth was added to 1 ,000 ml of LB medium containing 50 ⁇ g/ml of ampicillin and cultured at 37 ° c on the shaker until OD at A 6 Q 0 reached 0.7 to 0.8.
• IPTG was added to the culture broth to a final concentration of 0.1 mM and the shaking culture was continued for additional 3 hours in order to induce expression of GST- TPO(1 -174).
• a 5.9 g portion of frozen cells of the recombinant strain which produces GST-TPO(1 -174) derived from human TPO nucleotide sequence-encoding clone pGEX-2T/hT(1 -174) were suspended in 10 ml of water and disruptured using a high pressure disintegrator. By subjecting the suspension to centrifugation, GST-TPO(1 -174) was recovered in the precipitated fraction and most parts of contaminated proteins, cell components and the like were removed.
• the thus recovered precipitated fraction containing GST-TPO(1 -174) was suspended in 5 ml of water to which were subsequently added, with stirring, 6 ml of 1 M Tris buffer (pH 8.5), 120 ml of 10 M urea and 16 ml of water. After 5 minutes of stirring to solubilize the contents, the resulting solution was evenly divided into 4 portions to carry out the following steps (1) to (4).
• step (1 ) All operations of the step (1 ) were carried out in the presence of 0.1% polysorbate 80.
• GST-TPO(1 -174) in the solution was applied to SP Sepharose Fast Flow cation exchange resin which has been equilibrated in advance with 20 mM sodium citrate buffer pH 5.5. After washing the resin with 20 mM sodium citrate buffer pH 5.5, elution of GST-TPO(1-174) was effected using 20 mM sodium citrate buffer pH 5.5 containing 500 mM sodium chloride.
• the eluate was adjusted to pH 8 with 1 M Tris buffer pH 8.5, mixed with 320 NIH units of thrombin to remove GST polypeptide sequences via cleavage at a thrombin recognition peptide linker, allowed to stand for 4 hours at room temperature, diluted by a factor of 5 with 20 mM sodium citrate buffer pH 5.5 and then applied to SP Sepharose Fast Flow cation exchange column which has been equilibrated in advance with the same buffer, and the compound of interest was subsequently eluted with a linear density gradient of 0 to 500 mM sodium chloride sissolved in the same buffer.
• step (3) All operations of the step (3) were carried out in the presence of 0.1% polysorbate 80.
• each of the fractions eluted at sodium chloride densities of about 200 to 400 mM by the SP Sepharose Fast Flow cation exchange column chromatography was thoroughly dialyzed against IMDM culture medium and then evaluated by the rat CFU-MK assay system.
• the fraction was analyzed by SDS-PAGE in the presence of a reducing agent, a band corresponding to a molecular weight of 19 kd was detected (purity, 1 to 20%) as one of the main protein bands of the fraction.
• the clone pUC18(XE)(1-332) prepared in Example 39 was digested with Xbal and EcoRI and subjected to 2% agarose gel electrophoresis to recover a fragment of about 1 ,000 bp in size which encodes amino acid residues 1 to 332 of human TPO, and the fragment was subsequently purified using Prep- A-Gene DNA Purification Kit and sub-cloned into pBluescript II SK + (manufactured by Stratagene) which has been digested in advance with Xbal and EcoRI (E. coli DH5 ⁇ was used as the host).
• a clone obtained in this way was named pBL(XE)(1 -332).
• Synthetic oligonucleotides 13 to 20 shown below were prepared, and each pair- of synthetic oligonucleotides 13 and 14, 15 and 16 and 17 and 18 were phosphorylated in the same tube containing a solution composed of 0.1 mM ATP, 10 mM Tris-acetate, 10 mM Mg-acetate and 50 mM K-acetate. After further addition of
• PCR product was digested with BamHI and H dlll and subjected to 2% agarose gel electrophoresis to recover a fragment of about 130 bp in size using Prep-A-Gene DNA Purification Kit.
• This was sub-cloned into pBL(XE)(1 -332) which has been digested in advance with BamHI and Hindlll (E. coli DH5 was used as the host).
• a clone having the following nucleotide sequences was selected from the thus obtained clones by sequence analysis and named pBL(XH)(1-163): 13: 5 , -GATCCGAACGCTATCTTCCTGTCT_TCCAGCACCTGCTGCGT-3'

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