Identification and characterization of a novel angiostatin-binding protein by the display cloning method.
Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of 3.4 x 10(-7) M. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.
Kang
Ha-Tan
HT
Division of Life Sciences, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Korea.
Bang
Won-Ki
WK
Yu
Yeon Gyu
YG
D000595:Amino Acid Sequence; D043168:Angiostatins / Q000737:chemistry / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D018082:Kringles; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019151:Peptide Library; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D019151:Peptide Library; D012333:RNA, Messenger; D043168:Angiostatins