Li Bin, Wang Xiao-jing, Liu Yong-jun, Han Zhong-chao, Pang Tian-xiang
State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, CAMS and PUMC, Tianjin 300020, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Nov;25(11):991-3.
To prepare a soluble hemangiopoietin(HAPO) protein and to construct pET22b(+) expression vector, to obtain pure recombinant HAPO protein and to measure its bioactivity.
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.
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.
HAPO protein can be expressed in a soluble form. HAPO may facilitate the homing of hematopoietic stem/progenitor cells in vitro.
