Aim: To prepare a soluble hemangiopoietin(HAPO) protein and to construct pET22b(+) expression vector, to obtain pure recombinant HAPO protein and to measure its bioactivity.
Methods: HAPO cDNA was amplified using RT-PCR method from a commercial human fetal liver cDNA library. The resulting product was cloned into pET22b(+) vector and transformed into E.coli BL21(DE3). The recombinant protein was isolated and purified by Ni(2+)-NTA chelating resin and the chromatographies of SP Sepharose FF. The adhesion of human umbilical vein endothelial cells (HUVEC) were measured by adhesion assay.
Results: HAPO gene with a reading frame of 897 bp was successfully cloned from human fetal liver cDNA library, the expressed pET22b(+)-HAPO fused protein existed in a soluble form, with the yield above 10% total bacterial protein and its purity achieved above 80%. The activity assay showed that the treatment of HAPO enhanced total adherence of HUVEC in a concentration-dependent manner.
Conclusion: HAPO protein can be expressed in a soluble form. HAPO may facilitate the homing of hematopoietic stem/progenitor cells in vitro.