[Soluble expression, purification and bioactivity of hemangiopoietin protein].
OBJECTIVE
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.


CONCLUSIONS
HAPO protein can be expressed in a soluble form. HAPO may facilitate the homing of hematopoietic stem/progenitor cells in vitro.
Li
Bin
B
State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, CAMS and PUMC, Tianjin 300020, China. libin_fdm@yahoo.com.cn
Wang
Xiao-jing
XJ
Liu
Yong-jun
YJ
Han
Zhong-chao
ZC
Pang
Tian-xiang
TX
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D000099158:Proteoglycan 4
D011509:Proteoglycans; C419197:PRG4 protein, human; D000099158:Proteoglycan 4