US20070178559A1 - Platelet promoting protein and the usage thereof - Google Patents

Platelet promoting protein and the usage thereof Download PDF

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Publication number
US20070178559A1
US20070178559A1 US11/729,727 US72972707A US2007178559A1 US 20070178559 A1 US20070178559 A1 US 20070178559A1 US 72972707 A US72972707 A US 72972707A US 2007178559 A1 US2007178559 A1 US 2007178559A1
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protein
ppp
protein according
platelets
plasmid
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Peilin Xu
Yichen Ge
Yan Yang
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • 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.
  • 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.
  • platelet transfusion is a top choice for treatment for patients of thrombocytopenia.
  • 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.
  • Thrombopoietin plays its role of growth factor for thrombopoiesis by binding to its receptor MPL, which is made up of three parts, MPL-EC( 26-491aa ) (extracellular domain), transmembrane domain, and cytoplasmic domain.
  • MPL-EC( 26-491aa ) extracellular domain
  • transmembrane domain transmembrane domain
  • cytoplasmic domain cytoplasmic domain
  • TPO activated platelets stimulated its aggregation, and thus lead to the formation of blood clotting
  • [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.
  • 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.
  • the kits Matchmaker Two-Hybrid System 3, Matchmaker LexA Two-Hybrid System (Clontech) are commercially-available examples of the system.
  • the object of the current invention is to provide an isolated protein that has a function equivalent to the TPO.
  • the current invention also provides a nucleotide sequence for encoding the protein of the present invention and a plasmid that containing the DNA sequence.
  • 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.
  • 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.
  • MPL-EC extracellular domain of 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.
  • 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.
  • 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.
  • 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 2+ IMAC) column.
  • 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.
  • 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.
  • 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).
  • MCSF macrophage colony stimulating factor
  • 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.
  • concentration of the solution may range from 1 to 100 ⁇ g PPP per milliliter of the solution.
  • 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 ⁇ 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.
  • 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.
  • FIG. 1 shows the construction of plasmid pLexA-MPL-EC.
  • FIG. 2 shows the outline of plasmid pB42AD.
  • FIG. 3 shows the outline of expression vector pET-28b.
  • FIG. 4 shows the construction of the PPP expression vector pET-PPP.
  • FIG. 5 shows SDS-PAGE of His-PPP.
  • the protein samples were separated on 10% SDS-PAGE and stained with Coomassie blue. His-PPP, with a size of 45 kDa, was indicated by the arrow.
  • Lane 1 shows a protein size ladder; lanes 2 and 3 shows E. coli cell lysates before and after IPTG induction, respectively; lane 4 shows a soluble His-PPP crude extract; and lane 5 shows the His-PPP after cobalt affinity chromatography (Co 2+ IMAC) purification.
  • FIG. 6 shows the stimulation of platelet production by His-PPP in BALB/c mice.
  • Normal BALB/c mice were subcutaneously injected with PBS (control, white column), or 10 ⁇ g/kg His-PPP (grey column), or 50 ⁇ g/kg His-PPP (black column) for 7 days.
  • Twenty ⁇ L venous blood was collected from a small lateral cut in the tail vein on day 0, 4, 7, 10, 13, 16 and 19.
  • the platelets were counted using an F-820 Sysmex electronic blood cell analyzer, and expressed as the mean ⁇ SD ⁇ 10 9 /L.
  • FIG. 7 shows the reduction of bleeding time by His-PPP in BALB/c mice.
  • Normal BALB/c mice were subcutaneously injected with PBS (control, white column) or 10 ⁇ g/kg His-PPP (black column) for 7 days. The bleeding times were measured on day 0, 4, 7, 10, 13, 16 and 19.
  • MPLEC-F and MPLEC-R were synthesized based on the sequence of MPL-EC as follows: MPLEC-F: 5′-CCG GAATTC CAAGATGTCTCCTTGCTGGCATCAGA-3′; MPLEC-R: 5′-CCG CTCGAG TTATCCGACCACGAGCTCCAGGG-3′ ⁇
  • MPL-EC was PCR amplified with using the total human DNA as the template as indicated in FIG. 1 .
  • the PCR reaction mixture of a total 50 ⁇ l contained 1 ⁇ PCR reaction buffer, 5 ⁇ M MPLEC-F, 0.5 ⁇ M MPLEC-R, 1 ⁇ g human total DNA, 2U Taq DNA polymerase (Fermentas), 50 ⁇ M dATP, 50 ⁇ M dTTP, 50 ⁇ M dCTP, 50 ⁇ M dGTP, 1.5 mM MgCl 2 .
  • the PCR program used was: 94° C., 5 min; then 30 cycles of 94° C., 1.5 min, 55° C., 1 min, 72° C., 2 min; with an additional of 72° C., 10 min at the end of the program.
  • the resultant PCR product of approximately 1,450 bps long was separated by and purified from 1% agarose gel, digested using EcoRI and XhoI, and ligated by using T4 ligase into pLexA (Clontech) to generate pLexA-MPL-EC ( FIG. 1 ).
  • the screening system MATCHMAKER LexA Two-hybrid System (Clontech) is based on LexA and used for the detection of protein-protein interaction in the yeast (Gyuris et al., 1993). The detailed procedure was carried out as described by the protocol of the manufacturer.
  • the human fetal liver cDNA library in pB42AD (Clontech) was diluted and spreaded on LB plates and incubated overnight at 37° C. The cell colonies were collected by using sterile cotton tips and transferred into a LB broth and incubated overnight at 37° C. The plasmids were isolated by using E.Z.N.A.® Fastfilter Plasmid Miniprep Kit (Omega Bio-Tek).
  • pLexA-MPL-EC DNA from Example 1.1 and 100 ⁇ g human fetal liver cDNA library DNA were co-transformed into Saccharomyces cerevisiae EGY48 containing p80p-lacZ (Ura + , Lac + , Leu + ); and the transformants were selected on SD/Gal/Raf/-His/-Trp/-Ura/-Leu auxotrophic medium containing X-Gal, prepared as suggested by the manufacturer.
  • One positive clone of blue colony, named pB42AD-PPP, carrying an insert of approximate 1,300 bps was identified. The insert was sequenced and analyzed.
  • the insert carried a complete coding region of 993 bps encoding a 331 amino acid peptide, as shown in Sequence 1 and Sequence 2.
  • Blast analysis revealed that the sequence shares no homology with TPO but is identical to that of Human Nuclear Distribution Gene C (Matsumoto, N. and Ledbetter, D. H, Hum. Genet. 104, 498-504, 1999; Genbank database gi: 12052969).
  • the protein was named as PPP, as it stimulates the formation of platelets (Example 3) and enhances the blood clotting function of platelets (Example 4).
  • PPP was cloned into His-tag containing expression vector pET-28B (Novagen) ( FIG. 3 ).
  • the expressed protein His-PPP carried six continuous histidine residues at the N-terminus and can be purified by affinity chromatography.
  • pET-28b contains multiple cloning sites.
  • the primes PPP-F and PPP-R were designed based on the restriction sites on the vector and the cDNA sequence of PPP: PPP-F: 5′-CG GGATCC GATGGGCGGAGAGCAGGAGGAGGA-3′, containing BamHI (underlined); PPP-R: 5′-CCG CTCGAG CTAGTTGAATTTAGCCTTGGAAA-3′, containing XhoI (underlined).
  • PPP DNA was amplified with using pB42AD-PPP DNA as the template.
  • the PCR reaction mixture of a total 50 ⁇ l contained 1 ⁇ PCR reaction buffer, 0.5 ⁇ M PPP-F, 0.5 ⁇ M PPP-R, 1 ⁇ g pB42AD-PPP DNA, 2U Taq DNA polymerase (Fermentas), 50 ⁇ M dATP, 50 ⁇ M dTTP, 50 ⁇ M dCTP, 50 ⁇ M dGTP, 1.5 mM MgCl 2 .
  • the PCR program used was: 94° C., 5 min; then 30 cycles of 94° C., 1 min, 55° C., 1 min, 72° C., 1.5 min; and with an additional of 72° C., 10 min at the end of the program.
  • the resultant PCR product of approximately 1,000 bps long was separated by and purified from 1% agarose gel, and digested by BamH1 and Xho1, and ligated into pET28b downstream and in frame with the His-tag to generate the construct pET-PPP ( FIG. 4 ).
  • the pET-PPP was used to transform into E. coli strain BL21 (DE3), from Novagen.
  • the transformant expressing His-PPP was grown in a LB medium containing 0.05 mg/ml kanamycin overnight at 37° C., and diluted 100 fold into the same medium and incubated at 37° C. to an A600 of 0.5-0.6, and induced with 0.5 mM IPTG for 4 h at 30 C.
  • the bacterial cells were collected by centrifugation, suspended with a phosphate buffer of pH 7.4 containing 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, and 1 mM PMSF, lysed by sonication and centrifuged at 10,000 g for 30 minutes to remove cell debris.
  • the His-PPP containing supernatants was purified by using cobalt-based immobilized metal affinity chromatography (Co 2+ IMAC) column (Clontech) as described by the protocol of the manufacturer to yield a soluble His-PPP of 45 kDa in size.
  • the protein was more than 95% pure on 10% SDS-PAGE ( FIG. 5 ).
  • the His-PPP purified as described at Example 2 was diluted into a stock solution of 10 ⁇ g/ml with PBS containing 0.1% BSA and used for the injection.
  • Thirty normal male BALB/c mice of 6-7 week old were divided randomly into three groups of ten mice each. The first group was subcutaneously injected with 10 ⁇ g/kg His-PPP once per day for seven consecutive days; the second group was subcutaneously injected with 50 ⁇ g/kg His-PPP once per day for seven consecutive day; and the third group (control) was subcutaneously injected with PBS containing 100 ⁇ g/ml of BSA once per day for seven consecutive days.
  • the bleeding time test measures the time taken for the blood flow, caused by incision of the mouse tail veins, to stop.
  • the hemostasis test evaluates the blood clotting function of the platelets. The measurement was conducted as described by Alves-Rosa et al., Blood, Vol. 96, 2000, 2834-2840 with modifications.
  • the His-PPP purified as described at Example 2 was diluted into a stock solution of 10 ⁇ g/ml with PBS containing 0.1% BSA and used for the injection. Twenty BALB/c mice were divided randomly into two groups of 10 mice each. The first group was subcutaneously injected with 10 ⁇ g/kg His-PPP once per day for seven consecutive days; the second group (control) was subcutaneously injected with PBS containing 100 ⁇ g/ml of BSA once per day for seven consecutive days. The bleeding times were recorded on day 0, 4, 7, 10, 13, 16 and 19. The measurement was as follows: a wound of 20 mm wide was made using scissors at the tails of the mice and the blood was removed by gently contacting the cut site with paper filters every 30 seconds until there was no blood stain on the filter. The duration between the initiation and stoppage of the bleeding was recorded as the bleeding time.
  • FIG. 7 shows.
  • the bleeding time of His-PPP group started to bounce back gradually and returned to the pre-injection level on day 19, i.e., 12 days after the last injection.

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CN200410080297.3 2004-09-30
CNB2004100802973A CN100432102C (zh) 2004-09-30 2004-09-30 血小板增进蛋白及其应用
PCT/CN2004/001358 WO2006034610A1 (en) 2004-09-30 2004-11-26 A platelet promoting protein and the usage thereof

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130344047A1 (en) * 2011-12-21 2013-12-26 Abbott Cardiovascular Systems, Inc. Methods And Composition For Treating Heart Failure And Ischemia

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Publication number Priority date Publication date Assignee Title
WO2008009634A2 (en) * 2006-07-17 2008-01-24 Novo Nordisk Health Care Ag Factor viia analogues with increased activity for treating thrombocytopenia

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5986049A (en) * 1994-12-30 1999-11-16 Zymogenetics, Inc. Purified thrombopoietin and method of making it

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US5989538A (en) 1995-02-15 1999-11-23 Amgen Inc. Mpl ligand analogs
EP1961760A3 (en) * 1995-06-07 2008-09-03 Glaxo Group Limited Peptides and compounds that bind to a thrombopoietin receptor
CN1129805A (zh) * 1995-09-08 1996-08-28 济南金鲁生物工程有限公司 人促血小板生成素变异体、其cDNA克隆表达及检测方法
WO1998033911A1 (en) 1997-01-31 1998-08-06 Incyte Pharmaceuticals, Inc. Human signal transduction protein regulating the nuclear movement
AU3071399A (en) * 1998-03-05 1999-09-20 Penn State Research Foundation Human homolog of a nuclear migration and its use

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Publication number Priority date Publication date Assignee Title
US5986049A (en) * 1994-12-30 1999-11-16 Zymogenetics, Inc. Purified thrombopoietin and method of making it

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130344047A1 (en) * 2011-12-21 2013-12-26 Abbott Cardiovascular Systems, Inc. Methods And Composition For Treating Heart Failure And Ischemia

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CN1754886A (zh) 2006-04-05
WO2006034610A1 (en) 2006-04-06
US8012927B2 (en) 2011-09-06
US20100279938A1 (en) 2010-11-04

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