J Korean Cancer Assoc.  1999 Dec;31(6):1236-1245.

cDNA Cloning and Expression of Angiostatin, an Angiogenesis Inhibitor , from Human Liver Tissue mRNA

• Park MJ
• Hwang BG
• Son YS
• Yi DH
• Lee SH
• Hong SI

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.

Keyword

Angiogenesis inhibitor; Angiostatin; Refolding; Plasminogen

MeSH Terms

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
Angiostatins
DNA, Complementary
Isopropyl Thiogalactoside
Lysine
Pancreatic Elastase
Plasminogen
RNA, Messenger
Sepharose
Urea