cDNA Cloning and Expression of Angiostatin, an Angiogenesis Inhibitor , from Human Liver Tissue mRNA.
- Author:
Myung Jin PARK
1
;
Byung Gap HWANG
;
Young Sook SON
;
Dong Hee YI
;
Seong Hoon LEE
;
Seok II HONG
Author Information
1. Lab. of Cell Biology, Korea Cancer Center Hospital, Seoul, Korea.
- Publication Type:In Vitro ; Original Article
- Keywords:
Angiogenesis inhibitor;
Angiostatin;
Refolding;
Plasminogen
- MeSH:
Angiostatins*;
Animals;
Capillaries;
Chorioallantoic Membrane;
Chromatography, Affinity;
Clone Cells*;
Cloning, Organism*;
Digestion;
DNA, Complementary*;
Electrophoresis, Polyacrylamide Gel;
Endothelial Cells;
Humans*;
Inclusion Bodies;
Isopropyl Thiogalactoside;
Kringles;
Liver*;
Lysine;
Mice;
Molecular Weight;
Pancreatic Elastase;
Plasminogen;
Reverse Transcriptase Polymerase Chain Reaction;
RNA, Messenger*;
Sepharose;
Urea
- From:Journal of the Korean Cancer Association
1999;31(6):1236-1245
- CountryRepublic of Korea
- Language:Korean
-
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 plasminogen, 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 hybridization 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.
