Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-10-24
2002-04-30
Jones, W. Gary (Department: 1655)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C536S023500, C435S006120, C435S320100
Reexamination Certificate
active
06380361
ABSTRACT:
BACKGROUND OF THE INVENTION
To teach basic molecular biology full-length cDNAs are need to be provided to the students in educational kits for use in laboratory practica. Students can then use these full-length cDNAs to learn and practice to make expression vector, learn to transfect the cells and produce full-length protein. The DNA that encodes the protein can also be used to teach Northern blot analysis to learn how to determine in which tissues the mRNA is produced. Having identified where the mRNA is expressed, the mRNA in the tissue can then be isolated and cDNA produced. The student can then learn how to make a cDNA library. The novel polynucleotide can then be used to teach at student how to probe a cDNA library to determine which clone or clones contain the newly discoverd polynucleotide. The novel polynucleotide can also be used to teach chromosome localization.
To teach protein chemistry, a novel full-length protein can be used so the student can have practical learning experiences in protein purification procedures, protein re-folding when the protein is produced in prokaryotic host cells, and how to make both monoclonal and polyclonal antibodies.
Antibodies that bind to mature protein can also be used to learn how to make affinity purification columns, to do enzyme-linked immunoabsorbent assay (ELISA).
Especially useful novel polynucleotides for educational lab kits are those that encode protein that are hormones or growth factors. 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.
Even though many polynucleotide sequences that encode full-length, naturally occurring proteins are known, there is a need for the discovery of new full-length cDNAs or genomic DNA that encode for naturally-occurring proteins because each new sequence has unique features, such as unique enzyme-restriction sites, which are useful in increasing the analytical and technical skill of the student. There is also a need to discover new full-length and mature, naturally occurring polypeptides and proteins to teach protein chemistry and purification.
DESCRIPTION OF THE INVENTION
The present invention addresses this need by providing a novel education kit. The educational kit is comprised of one or more containers that contain at least one of the following, a polynucleotide that encodes a mature Zalpha34 polypeptide, full-length or mature Zalpha34 polypeptide, antibodies that specifically bind to a Zalpha34 polypeptide, a cloning or expression vector containing a polynucleotide that encodes a Zalpha34 polypeptide, or host cells, either prokaryotic or eukaryotic that have been transformed or transfected with an expression or cloning vector containing a polynucleotide that encodes a mature Zalpha34 polypeptide.
The present invention also fills this need by providing for the novel polypeptides, isolated polynucleotides that encode the polypeptides and related compositions and methods that can be used in educational kits. Within one aspect, the present invention provides an isolated polynucleotide encoding a mammalian alpha helical polypeptide termed ‘Secreted alpha protein-34’, hereinafter referred to as “Zalpha34”. The mouse Zalpha34 polypeptide (SEQ ID NO: 2) is encoded by SEQ ID NO: 1. The molecular weight of SEQ ID NO: 2 is about 32,328 Daltons (D). The polypeptide of SEQ ID NO: 2 has a signal sequence extending from amino acid residue 1 to and including amino acid residue 27. Thus, the mature sequence extends from amino acid residue 28 to and including amino acid residue 331 of SEQ ID NO: 2. The mature sequence is also represented by SEQ ID NO: 3. The human Zalpha34 polypeptide (SEQ ID NO: 22) is encoded by the polynucleotide of SEQ ID NO: 21. The polypeptide of SEQ ID NO: 22 has a signal sequence extending from amino acid residue 1 to and including amino acid residue 25. Thus, the mature sequence extends from amino acid residue 26 to and including amino acid residue 331 and is also represented by SEQ ID NO:23.
Within another aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a Zalpha34 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked. Within another aspect of the invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein 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 consists essentially of (a) a Zalpha34 polypeptide as shown in SEQ ID NO: 2 (b) allelic variants of SEQ ID NOs: 2, 3, 22 or 23; and (c) protein polypeptides that are at least 90% identical to (a) or (b). 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.
Within an additional aspect of the invention there is provided an antibody that specifically binds to a Zalpha34 polypeptide as disclosed above, and also an anti-idiotypic antibody that neutralizes the antibody to a Zalpha34 polypeptide.
An additional embodiment of the present invention relates to a peptide or polypeptide that has the amino acid sequence of an epitope-bearing portion of a Zalpha34 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalpha34 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. Examples of epitope-bearing polypeptides of the present invention are SEQ ID NOs: 4-20 for mouse Zalpha34 and SEQ ID NOs: 24-38 of human Zalpha34. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.
The teachings of all the references cited herein are incorporated in their entirety herein by reference.
1. Overview
The present invention provides novel cytokine polypeptides/proteins. The novel cytokine, termed “alpha helical protein-34” hereinafter referred to as “Zalpha34” was discovered and identified to be a cytokine by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth hormone).
2. Definitions
In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
As used herein, “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be compo
Conklin Darrell C.
Taft David W.
Jones W. Gary
Lunn, Esq. Paul G.
Taylor Janell E.
ZymoGenetics Inc.
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