Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-07-11
2004-08-03
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100, C435S070300, C435S071100, C435S320100, C435S325000, C435S252300, C536S023100, C536S023500, C514S002600
Reexamination Certificate
active
06770458
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to isolated and purified proteins and nucleic acids which modulate TGF-&bgr; biological activity, including TGF-&bgr; signal transduction. More particularly, the present invention relates to an isolated and purified serine-threonine kinase receptors associated protein and an isolated and purified polynucleic acid encoding the same.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text, and respectively grouped in the appended list of references.
Table of Abbreviations
BMP
Bone morphogenetic protein
BSA
Bovine serum albumin
CDR(s)
Complementarity determining region(s)
GC-MS
Gas chromatography-Mass spectroscopy
HAT
Cell culture media comprising hypoxanthine,
aminopterin, and thymidine
HPLC
High pressure liquid chromatography
KLH
Keyhole limpet hemocyanin
PCR
Polymerase chain reaction
SMAD
Vertebrate Homologues of Sma and Mad
STRAP
Serine-Threonine Kinase Receptors Associated
Protein
TGF&bgr;
Transforming growth factor &bgr;
T&bgr;R-I
Transforming growth factor &bgr; receptor I
T&bgr;R-II
Transforming growth factor &bgr; receptor II
BACKGROUND ART
Transforming growth factor &bgr;'s (TGF&bgr;) are a family of multifunctional cell regulatory factors produced in various forms by many types of cells (for review see Spom et al.,
J. Cell Biol
. 105:1039, (1987)). Five different TGF&bgr;'s are known, and the functions of two, TGF&bgr;-1 and TGF&bgr;-2, have been characterized in detail. TGF&bgr;'s are the subject of U.S. Pat. Nos. 4,863,899; 4,816,561; and 4,742,003 which are incorporated by reference. TGF&bgr;-1 and TGF&bgr;-2 are publicly available through many commercial sources (e.g. R & D Systems, Inc. of Minneapolis, Minn.). These two proteins have similar functions and will be here collectively referred to as TGF-&bgr;.
TGF-&bgr; binds to cell surface receptors possessed by essentially all types of cells, causing profound changes in them. In some cells, TGF-&bgr; promotes cell proliferation and in others it suppresses proliferation. A marked effect of TGF-&bgr; is that it promotes the production of extracellular matrix proteins and their receptors by cells (for review see Keski-Oja et al.,
J. Cell Biochem
. 33:95 (1987); Massague,
Cell
49:437 (1987); Roberts and Sporn in “Peptides Growth Factors and Their Receptors” (Springer-Verlag, Heidelberg) (1989)).
While TGF-&bgr;, has many essential cell regulatory functions, improper TGF-&bgr; activity can be detrimental to an organism. Since the growth of mesenchyme and proliferation of mesenchymal cells is stimulated by TGF-&bgr;, some tumor cells may use TGF-&bgr; as an autocrine growth factor. Therefore, if the growth factor activity of TGF-&bgr; could be prevented, tumor growth could be controlled. In other cases the inhibition of cell proliferation by TGF-&bgr; may be detrimental, in that it may prevent healing of injured tissues. The stimulation of extracellular matrix production by TGF-&bgr; is important in situations such as wound healing. However, in some cases the body takes this response too far and an excessive accumulation of extracellular matrix ensues. An example of excessive accumulation of extracellular matrix is glomerulonephritis, a disease with a detrimental involvement of TGF-&bgr;.
Pleiotropic responses to TGF-&bgr; are mediated via ligand-induced heteromeric complex formation by type I and type II receptors. Upon ligand binding, the type if receptor (T&bgr;R-II), which is a constitutively active kinase, transphosphorylates the type I receptor (T&bgr;R-I) and activates this kinase to propagate the signals to downstream effectors, termed SMAD proteins. See Massague et al.,
Trends Cell Biol
. 7:187-192 (1997): Heldin et al.,
Nature
390:465-471 (1997). SMAD proteins can be classified according to their role in signaling by TGF-&bgr; family members. Pathway-restricted SMADs interact transiently with, and are phosphorylated by specific activated type I receptors. Smad2 and Smad3 mediate signaling by TGF-&bgr; and activin, whereas Smad1 and Smad5 are involved in BMP signaling. Smad4 is a common mediator of TGF-&bgr;, activin and BMP signals. Recently Smad6 and Smad7 have been shown to function as inhibitors of these signaling pathways by interfering with the activation of pathway-restricted SMADs. Although the nature and mechanism of activation of TGF-&bgr; receptors at the cell surface has been described and the ester of potential regulators of TGF-&bgr; signaling continues to expand, little is known at the molecular level about the signaling mechanisms immediately downstream of the TGF-&bgr; receptors.
This lack of knowledge represents a serious deficiency in the art in view of the effects of cell regulatory factors such as TGF-&bgr; as described above. Therefore, further characterization of TGF-&bgr; signaling in vertebrates, particularly in mammals, and more particularly in humans is needed. A novel isolated and purified polypeptide having a role in the modulation of TGF-&bgr; signaling would have broad utility due to the various and multiple physiological and pathophysiological roles of TGF-&bgr;, as described above.
SUMMARY OF THE INVENTION
The present invention contemplates an isolated and purified vertebrate serine-threonine kinase receptors associated protein (STRAP) which plays a role in the modulation of TGF-&bgr; biological activity. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a mammalian STRAP. Even more preferably, a polypeptide, of the present invention comprises a human STRAP. Even more preferably, a polypeptide of the present invention comprises the amino acid residue sequence of SEQ ID NO:2.
The present invention also provides an isolated and purified polynucleotide that encodes a polypeptide that plays a role in the modulation of TGF-&bgr; biological activity. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes a polypeptide designated STRAP. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequence of SEQ ID NO:1.
In another embodiment, the present invention provides an antibody immunoreactive with a STRAP polypeptide as described above. SEQ ID NO:1 and SEQ ID NO:2 set forth representative vertebrate nucleotide and amino acid sequences. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent STRAP polynucleotide and potypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the STRAP polypeptide comprises human STRAP. Even more preferably, the STRAP polypeptide comprises the amino acid residue sequence of SEQ ID NO:2.
In another aspect, the present invention contemplates a method of producing an antibody immunoreactive with STRAP as described above, the method comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes a STRAP polypeptide having a TGF-&bgr; activity-modulating function; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) purifying the polypeptide; and (d) raising the antibody to the polypeptide. Preferably, the host cell is transfected with the polynucleotide of SEQ ID NO:1. Even more preferably, the present invention provides an antibody prepared according to the method described above. Also contemplated by the present invention is the use of homologues or biologically equ
Datta Pran K.
Moses Harold L.
Jenkins & Wilson & Taylor, P.A.
Seharaseyon Jegatheesan
Spector Lorraine
Vanderbilt University
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