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  Platelet promoting protein and the usage thereofUSPTO Application #: 20070178559Title: Platelet promoting protein and the usage thereof Abstract: The present invention discloses a protein that has strong affinity to thrombopoietin receptor (C-MPL) and the nucleotide sequences of the protein. The protein is capable of increasing the numbers of platelets and enhancing the blood clotting in vivo and is named as platelet promoting protein (PPP). The protein and its nucleotide sequences can be used for the treatment of blood diseases including thrombocytopenia. (end of abstract) Agent: The Webb Law Firm, P.C. - Pittsburgh, PA, US USPTO Applicaton #: 20070178559 - Class: 435069100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Recombinant Dna Technique Included In Method Of Making A Protein Or Polypeptide The Patent Description & Claims data below is from USPTO Patent Application 20070178559. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to a bio-medicament, and more specifically relates to a protein for increasing the numbers of platelets and its applications in the treatment of blood diseases. BACKGROUND OF THE INVENTION [0002] As an important component of blood, platelets are responsible for hemostasis in response to vascular injury and involved in the repairment of injured blood vessels. Low level of blood platelets can be life-threatening as it is prone to a mass loss of blood. At the present, platelet transfusion is a top choice for treatment for patients of thrombocytopenia. However, like other blood products, the platelets are short in shelf life, and are easy to be contaminated with blood pathogens such as hepatitis B virus and AIDS virus, and often elicit allergenic reactions among recipients. [0003] Thrombopoietin (TPO) plays its role of growth factor for thrombopoiesis by binding to its receptor MPL, which is made up of three parts, MPL-EC(.sub.26-491aa) (extracellular domain), transmembrane domain, and cytoplasmic domain. In 1994, five groups of scientists simultaneously cloned TPO. The successful cloning of TPO had injected new hopes and approaches for the treatment of thrombocytopenia. However, the clinical data indicated that the efficacy of TPO towards thrombocytopenia varied among patients. At the same time, the side effects of TPO were also observed. Two of the major side effects were: [1] TPO activated platelets, stimulated its aggregation, and thus lead to the formation of blood clotting; and [2] antibodies to TPO being generated after TPO administration (Archimbaud E, et al. Blood. 1999; 94:3694-3701; Schiffer C A, et al, Blood. 2000; 95:2530-2535; Nash R, et al. Blood. 1997; 90 suppl. 1:262a). The search for alternative therapeutic proteins or cytokines continues in the art. [0004] The yeast two-hybrid system is a suitable system for the study of protein-protein interactions. The system, which contains two expression plasmids (plasmid A and plasmid B) as well as a yeast host, takes advantage of the fact that yeast transcription factors, such as LEXA or Gal4, comprises two separate domains: DNA-binding domain (DNA-BD) and transcription activating domain (AD). Plasmid A expresses a fusion protein of a bait protein and the DNA-BD; and Plasmid B expresses a fusion protein of a protein of interest and AD. After co-transformation of the two plasmids into the yeast host, the interaction of the bait protein and the protein of interest brings the DNA-BD and AD into close contact, which activates the transcription of the reporter gene. Therefore the system can be used to isolate ligands of bait proteins. The kits Matchmaker Two-Hybrid System 3, Matchmaker LexA Two-Hybrid System (Clontech) are commercially-available examples of the system. SUMMARY OF THE INVENTION [0005] The object of the current invention is to provide an isolated protein that has a function equivalent to the TPO. [0006] The current invention also provides a nucleotide sequence for encoding the protein of the present invention and a plasmid that containing the DNA sequence. [0007] Another object of the current invention is to provide the application of the protein, including using the protein as a medicament for treatment of a blood disease. [0008] The current invention is carried out by using a yeast two-hybrid system, in particular, by using the extracellular domain of MPL (MPL-EC) as a bait protein to screen proteins in a human fetal liver cDNA library that interact with the MPL. The screening identified a protein that binds specifically to the extracellular domain of MPL. The protein of the present invention has 331 amino acids in length and has no homology to TPO in BLAST analysis. It is capable of stimulating the maturation of megakaryocytes and the formation of platelets and is consequently named as Platelet Promoting Protein, or PPP. The amino acid sequence of the PPP is shown as Sequence 2 in the Sequence Listing and its nucleic acid sequence is shown as Sequence 1 in Sequence Listing. The Platelet Promoting Protein or PPP of the present invention refers to the protein having the amino sequence as shown by Sequence 2 in the Sequence Listing. [0009] Also provided by the invention are derivatives of the PPP. The derivatives of the "PPP" include: [1] mutants of the PPP, provided that the mutants retain the ability of increasing the numbers of platelets and enhancing the blood clotting in vivo; [2] variants of the PPP, which, as compared with the Sequence 2, comprise one or more conservative substitutions of amino acids; one or more deletions of amino acids; or one or more additions of amino acids; [3] a carboxyl terminal-truncated form or amino terminal-truncated form of the PPP having the Sequence 2; [4] a tandem repetition of partial or complete Sequence 2; and [5] a fusion protein of the PPP having the Sequence 2 and another protein or cytokine. One of such derivatives, for example, carries additional 2-6 histidines at the N-terminus of the Sequence 2. [0010] The IUPAC nomenclature and symbolism for amino acid abbreviations was used in the present invention (European Journal of Biochemistry, 138:9-37, 1984). [0011] To complete the invention, the inventors first amplified and isolated a 1.3 kb MPL-EC cDNA from the total human DNA using PCR primers MPLEC-F and MPLEC-R, which are complementary to the ends of the MPL-EC and contain appropriate restriction sites. The MPL-EC fragment was restricted and ligated into the polyclonal site of pLexA to generate a plasmid, named as pLexA-MPL-EC. The pLexA-MPL-EC and human fetal liver cDNA library were then co-transformed into Saccharomyces cerevisiae EGY48 and a positive clone was identified on an auxotrophic media and then DNA sequencing was conducted. The sequencing analysis of the positive clone revealed the clone had an insert having a nucleotide sequence shown as Sequence 1 in Sequence Listing. Its deduced amino acid sequence is given as Sequence 2 in Sequence Listing. [0012] Insertion of PPP gene into expression vector pET-28b formed a expression vector, named as pET-PPP, which was subsequently transformed into E. coli BL21(DE3). The transformants were induced to produce His-PPP containing six continuous histidine residues at the N-terminus of the protein. The His-tag served as an affinity tag for the purification of PPP by using a cobalt-based immobilized metal affinity chromatography (Co.sup.2+IMAC) column. [0013] The purified PPP was injected into normal mice and the amount of the circulating platelets was measured and the bleeding times were monitored. The results indicated that the His-PPP stimulated significantly the formation of platelets and increased the amount of platelets in the circulating blood. [0014] The PPP of the invention is a potential medicament for the treatment of thrombocytopenia or/and hemorrhage. The protein of the present invention may be formulated into injections, powders, tablets, capsules, solutions, suspensions, or emulsions. The medicament may be administrated by oral administration or may be administered via subcutaneous injection, intravenous injection or intramuscular injection. [0015] The present invention also provides a pharmaceutical composition comprising the PPP of the invention. The pharmaceutical composition may be prepared by mixing the PPP or the derivatives of the PPP that have the function of increasing the numbers of platelets, with pharmaceutically acceptable excipients. The excipients may be a liquid such as water, salines, phosphate buffers or albumin solutions; or a solid such as antioxidant agents, starches or dextrins. The pharmaceutical compositions are preferably to contain other hematopoietic growth factors such as interleukins, erythropoietins, macrophage colony stimulating factor (MCSF). [0016] The PPP of present invention may be readily prepared in to various solutions by applying any known methods in the pharmaceutical field, such as by using a sterile saline, phosphate buffer and albumin solution. The concentration of the solution may range from 1 to 100 .mu.g PPP per milliliter of the solution. [0017] The PPP of the present invention may be administrated to patients in a dosage with the dosage of TPO as a reference, e.g. in the range of 1 to 1000 .mu.g per kilogram of body weight per day. The dosage will be determined by a medically qualified physician, based on a variety of factors of the patients, including age, weight, severity of sickness, the cause and history of the disease. [0018] The vectors and host cells described in the invention were obtained commercially. For example, pET-28b and E. coli BL21(DE3) were from Novagen, the yeast two hybrid system Matchmaker LexA Two-Hybrid System and Talon Metal Affinity Resin were from Clontech. [0019] The current invention is further described with the figures and Examples. The invention is not limited by the detailed description provided in the Examples. Various modifications can be made by those skilled in the field and these modifications should be construed to fall within the scope of the invention defined by the Claims. BRIEF DESCRIPTION OF THE DRAWINGS [0020] FIG. 1 shows the construction of plasmid pLexA-MPL-EC. Continue reading... Full patent description for Platelet promoting protein and the usage thereof Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Platelet promoting protein and the usage thereof patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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