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Anti-angiogenic proteins and fragments and methods of use thereof

A fragment and integrin technology, applied in chemical instruments and methods, anti-animal/human immunoglobulins, immunoglobulins, etc., can solve the problem of not fully understanding the exact role of collagen in basement membrane tissue.

Inactive Publication Date: 2003-05-28
BETH ISRAEL DEACONESS MEDICAL CENT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the precise role of collagen in basement membrane organization and angiogenesis is still not fully understood

Method used

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  • Anti-angiogenic proteins and fragments and methods of use thereof
  • Anti-angiogenic proteins and fragments and methods of use thereof
  • Anti-angiogenic proteins and fragments and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0273] Embodiment 1: Separation of natural Arresten

[0274] Arresten is available in milligram quantities from human placenta and amnion tissue. Methods for the isolation of Arresten and similar proteins have been described (e.g., Langeveld, J.P. et al., 1988, J: Biol. Chem. 263: 10481-10488; Saus, J. et al., 1988, J: Biol. Chem. 263 : 13374-13380; Gunwar, S, et al., 1990, J.Biol.Chem.265: 5466-5469; Gunwar S. et al., 1991, J Biol.Chem.266: 15318-15324; Kahsai, T.Z. et al., 1997, J. Biol. Chem. 272:17023-17032). The preparation of recombinant forms of Arresten is described by Neilson et al. (1993, J Biol. Chem. 268:8402-8406). The protein can also be expressed in 293 kidney cells (eg, by the method described by Hohenester, E. et al., 1998, EMBO J 17:1656-1664). Arresten can also be isolated according to the method of Pihlajaniemi, T. et al. (1985, J. Biol. Chem. 260: 7681-7687).

[0275] The nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of the α1 chain...

Embodiment 2

[0277] Example 2: Recombinant production of Arresten in Escherichia coli

[0278] The sequence encoding Arresten was amplified by PCR from the α1NC1(IV) / pDS vector (Neilson, E.G. et al., 1993, J. Bio. Chem. 268:8402-5) using the forward primer 5'-CGG GAT CCT TCT GTT GAT CAC GGC TTC-3' (SEQ ID NO: 3) and reverse primer 5'-CCC AAG CTT TGT TCT TCT CAT ACA GAC 3' (SEQ ID NO: 4). The resulting cDNA fragment was digested with BamHI and HindIII and ligated into predigested pET22b(+) (Novagen, Madison, Wisconsin, USA). Figure 2 presents this construct. This construct is positioned downstream of Arresten in-frame and in open reading frame with the pelB leader sequence for cytoplasmic and soluble protein expression. Another vector sequence was added to the protein encoding the amino acid MDIGINSD (SEQ ID NO: 13). The 3' end of this sequence is linked in-frame with a polyhistidine tag sequence. Another vector sequence is located between the 3' end of the cDNA and the amino acid KLAAA...

Embodiment 3

[0281] Example 3: Expression of Arresten in 293 embryonic kidney cells

[0282] Amplification of Arresten using the pDS plasmid containing α1(IV)NC1 required that the leader signal sequence be added in-frame to the pcDNA 3.1 eukaryotic expression vector (InVitlogen, San Diego, California, USA). Cloning of the leader sequence from the 5' end of the full-length α1(IV) chain 5' into the NCl domain enables secretion of the protein into the medium. Arresten-containing recombinant vectors were sequenced with flanking primers. Error-free cDNA clones were further purified and used in in vitro translation studies to confirm protein expression. Arresten-containing plasmids and control plasmids were used to transfect 293 cells using the calcium chloride method. Transfected clones were selected by geneticin antibiotic treatment (LifeTechnologies / Gibco BRL, Gaithersburg, Maryland, USA). Cells were grown for 3 weeks in the presence of antibiotics until no more apoptosis was evident. Exp...

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Abstract

Proteins with anti-angiogenic properties are disclosed, and fragments thereof, and methods of using those proteins and fragments to inhibit or promote angiogenesis.

Description

[0001] related application [0002] This application is a continuation-in-part of U.S.S.N.09 / 625 191 filed July 21, 2000, U.S.S.N.09 / 543 371 filed April 4, 2000, and U.S.S.N. 09 / 479 118 filed January 7, 2000. U.S.S.N.09 / 625 191 is a continuation-in-part of U.S.S.N.09 / 543371, of which U.S.S.N.09 / 479 118 is a continuation-in-part of U.S.S.N.09 / 335 224 filed on June 17, 1999, which in turn claimed U.S. provisional application 60 / 089,689, filed on March 25, 1999, and U.S. provisional application 60 / 126,175, filed March 25, 1999. The entire contents of all these applications are incorporated herein by reference. governmental support [0003] This invention was supported, in whole or in part, by National Institutes of Health programs DK-51711 and DK55001. The state has certain rights in this invention. Background technique [0004] The basement membrane is a thin layer of specific extracellular matrix that provides a support structure on which epithelial and endothelial cells gr...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00A61K39/395A61K45/00A61P9/00A61P35/00A61P43/00C07K14/78C07K16/18C07K16/28
CPCC07K16/2839C07K14/78A61K38/00A61K2039/505A61P9/00A61P35/00A61P43/00
Inventor 拉格胡拉姆·卡路里
Owner BETH ISRAEL DEACONESS MEDICAL CENT INC
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