Cloning, Expression, Purification And Bioassay Of Recombinant Human Platelet Factor 4

Platelet factor 4 (PF4) is a 7.8-kD protein 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 disulfide bridges to the protein core, which consists of three antiparallel P -strands and a carboxyl-terminal cc-helix. The mechanism of action of PF4 remains poorly understood, because no cell surface receptor has been yet identified. The basic nature of the C-terminus of PF4, which confers to the molecule a high affinity for heparin and other sulfated glycans, has been considered to be responsible for most of the activities of PF4.Platelet factor 4 (PF4), a member of the C-X-C chemokine family, has been recognized as an inhibitor of megakaryocytopoiesis, neovascularization, and endothelial cell proliferation. Recombinant humanplatelet factor 4 can therapy solid tumor by inhibition of angiogenesis. The migration of human endothelial cells was also inhibited by PF4, which can be used for the development of novel therapeutic approaches to angiogenic diseases.Native PF4 is purified from human platelet.lt is expensive and little production,so it does not enough for scientific research and clinic therapy so far. The aim of our experiment is to obtain human platelet factor 4(hPF4) with biological activity by means of genetic engineering.In our study, we firstly designed and synthesized specific primers for PF4, recombinant plasmid pUC19-PF4 as the template, then the PF4 cDNA gene was amplified by PCR and cloned into pDH2. The recombinant plasimid pDH2-PF4 was constructed successfully and identified by the sequence analysis.Then it was inserted into the prokaryotic fusion expression vector pRSET to construct recombinant expression plasmid pRSET-PF4. pRSET-PF4 was transformed into E.coli BL-21, then induced by IPTG in 37°C. The SDS-PAGE showed E.coli BL-21 containing pRSET-PF4 could express a fusion protein , which existed as inclusion bodies. The molecular weight of fusion protein was 18 KD, which more than theory estimated. The expression level was about 11% of the total bacterial proteins.In order to induce a soluble fusion protein, we changed the condition of inducement. The pRSET-PF4 was induced at 28 to 30°C. The SDS-PAGE showed E.coli BL-21 containing pRSET-PF4 could express a fusion protein in which about 50% exsisted in a soluble form. The soluble protein was purified by Ni-NTA affinity chromatography. The result of...