Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-12-10
2001-10-16
Spector, Lorraine (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C530S350000
Reexamination Certificate
active
06303770
ABSTRACT:
BACKGROUND OF THE INVENTION
Proliferation, maintenance, survival and differentiation of cells of multicellular organisms are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
Hormones and growth factors influence cellular metabolism by binding to proteins. Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.
Of particular interest are cytokines, molecules that promote the proliferation, maintenance, survival or differentiation of cells. Examples of cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF), which stimulates development of neutrophils. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia or receiving chemotherapy for cancer. The demonstrated in vivo activities of these cytokines illustrates the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists.
SUMMARY OF THE INVENTION
The present invention addresses this need by providing a novel polypeptide and related compositions and methods. Within one aspect, the present invention provides an isolated polynucleotide encoding a mammalian protein termed “Alpha helical protein-1” or Zalpha1. The human Zalpha1 polypeptide is comprised of a sequence of amino acids 146 amino acids long with the initial Met as shown in SEQ ID NO:1 and SEQ ID NO:2. It is believed that amino residues 1-20 are signal sequence, and the mature Zalpha1 polypeptide is represented by the amino acid sequence comprised residues 21, an isoleucine, through amino acid residue 146, a tyrosine. The mature Zalpha1 polypeptide is also defined by SEQ ID NO:45. Within an additional embodiment, the polypeptide further comprises an affinity tag. Within a further embodiment, the polynucleotide is DNA.
Also claimed are polypeptides which are at least 90% identical to SEQ ID NO:2 or SEQ ID NO:45 and polynucleotides which encode the polypeptides.
Within a second aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a Zalpha1 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.
Within a third aspect of the invention there is provided a cultured eukaryotic or bacterial cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a Zalpha1 polypeptide encoded by the DNA segment.
Within a further aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond. The first portion of the chimeric polypeptide is either (a) a Zalpha1 polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO:45 or (b) protein polypeptides that are at least 90% identical to SEQ ID NO:2 or SEQ ID NO:45. The second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag. Within one embodiment the affinity tag is an immunoglobulin F
c
polypeptide. The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zalpha1 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalpha1 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule. Antibodies produced from these epitope-bearing portions of Zalpha1 can be used in purifying Zalpha1 from cell culture medium. Examples of such epitope-bearing polypeptides are the polypeptides of SEQ ID NOs: 46-56. Also claimed are proteins or polypeptide which contain a sequence which is at least 90% identical to an epitope-bearing polypeptide described above.
Within an additional aspect of the invention there is provided an antibody that specifically binds to a Zalpha1 polypeptide as disclosed above, and also an anti-idiotypic antibody which neutralizes the antibody to a Zalpha1 polypeptide.
These and other aspects of the invention will become evident upon reference to the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The term “allelic variant” is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The term “expression vector” is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
The term “isolated”, when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
“Operably linked”, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.
A “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
The term “promoter” is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.
A “soluble protein” is a protein polypeptide that is not bound to a cell membrane.
Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selec
Conklin Darrell C.
Lok Si
Parrish Julia E.
Lunn, Esq. Paul G.
O'Hara Eileen B.
Spector Lorraine
ZymoGenetics Inc.
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