Cloning, Expression, Purification And Characterization Of Hemangiopoietin(HAPO), A Factor Stimulating Both Hematopoietic Stem Cells And Endothelial Cells

A protein was purified from the urine of patients with aplastic anemia. It was shown to support the proliferation and survival of hematopioetic cells and vascular endothelial cells in vitro. So, we named the protein hemangiopoietin (HAPO). We have cloned the N-terminal cDNA functional region of HAPO from human fetal liver mRNA. When this sequence was subjected to a BLAST search for sequence homologies, a high scoring match was obtained against PRG4 mRNA (GenBank Accession No.U70136). PRG4 gene was located in lq24-25. By analysis of the structure, HAPO includes two somatomedin B homology domains and one heparin-binding domain. The somatomedin B homology domain plays an important role in the proliferation of cells. Sequence comparisons and analyses indicated that the cDNA of HAPO might be an alternatively spliced variant lacking exon 5 of PRG4.In order to investigate further the function of HAPO, the cDNA of HAPO was subcloned into the pET32c (+) expression vector downstream to the his-tag site and transformated into Escherichia Coli (BL21). The HAPO protein was expressed efficiently as soluble protein, with the yield of 5mg per liter of Escherichia coli cells. Purification of HAPO protein involved a combination of DEAE sepharose Fast Flow column, his-tag affinity column and SP sepharose Fast Flow column. After enterokinase digestion, the recombinant HAPO was further isolated and tested in vitro and in vivo for its biological activities.Recombinant HAPO markedly increased the proliferation of human bone marrow cells in liquid culture and semisolid culture. To investigate further the target cells on which HAPO acted, the human fetal hematopoetic cells cultured for three weeks were analyzed by flow cytometry. FACS analysis showed that...