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Title   사람 간 조직으로 부터 혈관신생 저해제인 Angiostatin 유전자의 클로닝 및 발현 ( cDNA Cloning and Expression of Angiostatin , an Angiogenesis Inhibitor , from Human Liver Tissue mRNA )
Publicationinfo   1999 Jan; 031(06): 1236-1246.
Key_word   Angiogenesis inhibitor, Angiostatin, Refolding, Plasminogen
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Abstract   Purpose: Angiostatin, a 38 kDa internal fragment of plasminogen, is a potent inhibitor of angiogenesis. It blocks neovascularization and growth of primary and metastatic tumors in mice. To produce recombinant angiostatin protem comprising kringle 1-4 of plas- minogen, we cloned the angiostatin cDNA from human liver tissue mRNA and expressed it in E. coli. Materials and Methods: We cloned angiostatin cDNA from human liver tissue mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Cloned cDNA was ligated to pET22b (+) expression vector, transformed into E. coli stram BL21 (DE3) and expressed by IPTG induction. Recombinant human angiostatin protein was purified from the inclusion bodies of lysated bacterial pellet with 8 M urea solubilization, refolding, single step Lysine-Sepharose 4B affinity chromatography and 0.2 M E-aminocarproic acid elution. The anti-angiogenic activity of purified recombinant angiostatin was assayed with endothelial cell proliferation assay and chorioallantoic membrane assay (CAM). Results: The identification of cloned angiostatin cDNA was confirmed by Southern hy- bridization and Pst I restriction enzyme digestion pattern. Angiostatin cDNA was expressed in E. coli, refolded in vitro and purified by Lysine Sepharose 4B affinity chromatography. The molecular weight of purified recombinant angiostatin was about 55 kDa on the SDS-PAGE. It inhibited the proliferation of bovine capillary endothelial (BCE) cells in vitro with a half-maximal inhibition concentration (ED50) of approximately 500 ng/mL. It also suppressed neovasculrization on the CAM assay. Conclusion: These results demonstrated that recombinant human angiostatin has similar function and biological activity compared with human angiostatin which is purified from porcine elastase digested human plasminogen fragment.
저 자   박명진(Myung Jin Park),황병갑(Byung Gap Hwang),손영숙(Young Sook Son),이동희(Dong Hee Yi),이승훈(Seong Hoon Lee),홍석일(Seok II Hong)