Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-08-25
2002-07-30
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S412000, C530S402000, C435S069100, C435S070100, C435S325000, C204S166000
Reexamination Certificate
active
06426404
ABSTRACT:
BACKGROUND OF THE INVENTION
ITF is abundantly expressed by specialized mucus-producing cells of the gastrointestinal tract and is secreted onto the mucosal surface, where it appears to act extracellularly on epithelial cells through undefined mechanisms.
ITF plays an important role in restitution, a rapid process during which epithelial cells spread and migrate across the basement membrane to cover shallow defects of the mucosal surfaces of the gastrointestinal tract. Colonic restitution is absent in mice made ITF deficient by homologous recombination; the otherwise trivial colonic injury induced by 2.5% dextran sodium sulfate is lethal in these animals. Moreover, ITF has been shown to prevent acute gastrointestinal injury caused by alcohol or indomethacin and to rapidly reseal erosions caused by these agents.
SUMMARY OF THE INVENTION
The invention is based on the discovery of ITF receptor (ITFR). ITFR can be purified and/or isolated from intestinal cells using an ITF fusion protein, e.g., ITF-thioredoxin. An ITF fusion protein such as ITF-thioredoxin is capable of binding to ITFR expressed on intestinal cells. The specificity of such binding activity can be confirmed by adding excess of ITF which competes with the ITF fusion protein such as ITF-thioredoxin for binding to ITFR. Subsequently, the ITF fusion protein/ITFR complex can be purified using an affinity column, e.g., a thioredoxin affinity resin. The ITFR present in the ITF fusion protein/ITFR complex can be isolated using routine protein purification methods.
The present invention features a substantially pure intestinal trefoil factor receptor (ITFR), characterized in that it specifically binds to human ITF, has a molecular weight of about 50 to 60 kD or about 75 to 80 kD, and is expressed by intestinal or colonic cells.
Also within the invention is a preparation of antibodies which specifically bind to ITFR and do not specifically bind to other proteins.
Another aspect of the invention provides a method of obtaining a substantially pure intestinal trefoil factor receptor (ITFR) which comprises
incubating intestinal trefoil factor-thioredoxin (ITF-thioredoxin) with an intestinal or colonic cell lysate in the presence and absence of ITF,
obtaining an ITF-thioredoxin complex, wherein the complex binds to a thioredoxin affinity resin, and
isolating the substantially pure ITFR from the ITF-thioredoxin complex, wherein the polypeptide is associated with the ITF-thioredoxin complex in the absence of ITF, but not in the presence of ITF.
As used herein, the term “substantially pure polypeptide” is meant a ITFR polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of ITFR polypeptide.
A substantially pure ITFR polypeptide may be obtained, for example, by extraction from a natural source (e.g., intestinal epithelial cells), by expression of a recombinant nucleic acid encoding a ITFR polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in
E. coli
or other prokaryotes.
The present invention provides substantially pure ITFR, e.g., human ITFR, and enables molecular characterization and modification of ITFR. For example, the substantially pure ITFR of the present invention can be used to generate anti-ITFR antibodies and to isolate gene(s) encoding ITFR. The cDNA encoding ITFR can be used to make recombinant ITFR, mutants of ITFR, and fragments of ITFR. Human ITFR can be used to develop treatments for gastrointestinal disease conditions associated with the activities of ITF.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
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Basi Nirmal S.
Clark & Elbing LLP
Eyler Yvonne
The General Hospital Corporation
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