Clone, Expression, Purification And Quantitative Bioassay Of Recombinant Human Platelet Factor 4

Platelet factor 4 (PF4) is a 7.8-kD protein that is synthesized by megakaryocytes, stored in a-granules as a noncovalent tetramer and released from activated platelets. Each monomer has a conformational flexible N-terminal region that is anchored by two disulflde bridges to the protein core, which consists of three antiparallel P-strands and a-6-carboxyl-terminal a-helix. PF4 is a protein belonging to the family of CXC chemokines for the two cysteins from nearest the N-termini of the protein are separated by a single amino acid. The gene encoding PF4 clusters at human chromosome 4q.PF4 have many biologic activities including modulating inflammation, hemostasis, hematopoiesis, cell proliferation, angiogenesis. and glycosaminoglycan activity. PF4 has previously been demonstrated to be an important negative regulator of hematopoiesis, particularly megakaryocytopoiesis. Data indicate that PF4 accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment, it suggests the molecule may have a haemoprotective effect against chemotherapeutic agents. The addition of PF4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis which exerts its inhibitory effect on cell growth in a reversible and S phase-specific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity. PF4 also inhibits tumor growth by mechanisms involving antiangiogenesis. PF4 preferentially binds to regions of active angiogenesis in vivo, supporting the concept of-7-using rhPF4 conjugates to target tumors in cancer patients.The present study was designed to clone and express recombinant human platelet factor 4, then purified it and analysed its activity. The work will lay steady foundation for further study and clinical amplication of platelet factor 4.In our study, we first designed and synthesized specific primers for PF4, then the PF4 cDNA gene was amplified by PCR and cloned into pUC19 The recombinant plasimid pUC19-PF4 was constructed successfully and identified by the sequence analysis. The nucleotide sequence of the rhPF4 cDNA was the same as the origin reported in GeneBank. Then it was inserted into the prokaryotic fusion expression vector pGEX-4T-3 to construct recombinant expression plasm id pGEX-4T-3-PF4. PGEX-4T-3-PF4 was tranformated into E.coli DH5ot. then induced by IPTG in 37癈. The SDS-PAGE showed E.coli DH5oc containing pGEX-4T-3-PF4 could express a fusion protein, which existed as inclusion bodies. The molecular weight of fusion protein was 34KD The expression level was about 20% of the total bacterial proteins.In order to induce a soluble fusion protein, we changed the condition-8-for inducement. The pGEX-4T-3-PF4 was transformed into E.coli BL-21 and induced at 28 to 30癈. The SDS-PAGE showed E.coli BL-21 containing pGEX-4T-3-PF4 could express a fusion protein in which about 50% exsisted in a soluble form. The soluble protein was purified by Glutathione Sepharose 4B chromatograph and then isolated by thrombin The result of activity analysis showed rhPF4 could inhibit the formation of the new vessel on CAM.In conclusion, we cloned the rhPF4 and made it expressed in a soluble form, furthermore purified it to pursue its primary bioassay. Our experiments lay solid foundation for further study and clinlical application to PF4.