Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-06-28
2004-11-23
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S325000
Reexamination Certificate
active
06822082
ABSTRACT:
BACKGROUND OF THE INVENTION
Within the field of genetic engineering, polynucleotides encoding proteins of interest have been identified and cloned by methods that require a detailed knowledge of the structure and/or function of the polynucleotide or the encoded protein. These methods include hybridization screening, polymerase chain reaction (PCR), and expression cloning.
With the more recent advent of large DNA sequence databases and the accompanying data analysis tools, identification of genes of interest is possible through the analysis of raw sequence data. Databases can be “mined” to locate sequences that resemble (are “homologous to”) sequences of known function. Alignment of similar sequences can be used to place novel sequences within families of structurally similar sequences. These analytical tools can be combined with structural information obtained from, for example, X-ray crystallography to predict the higher order structure of a novel polypeptide. These analyses also facilitate prediction of polypeptide function. These recent technological advances have greatly increased the pace of gene discovery.
Genetic engineering has made available a number of genes and proteins of pharmaceutical or other economic importance. Such proteins include, for example, tissue plasminogen activator (t-PA) (U.S. Pat. No. 4,766,075), coagulation factor VII (U.S. Pat. No. 4,784,950), erythropoietin (U.S. Pat. No. 4,703,008), platelet derived growth factor (U.S. Pat. No. 4,889,919), and various industrial enzymes (e.g., U.S. Pat. Nos. 5,965,384; 5,942,431; and 5,922,586).
Although estimates vary as to the amount of the human genome that has been identified to date, there remains a need in the art for further characterization of the human genome and the proteins encoded thereby. Previously unknown genes and proteins will be useful in the treatment and/or prevention of many human diseases, included diseases that have heretofore been refractory to treatment.
SUMMARY OF THE INVENTION
Within one aspect of the invention there is provided an isolated polypeptide comprising fifteen contiguous amino acid residues of a polypeptide as shown in SEQ ID NO:M, wherein M is an even integer from 2 to 328. Within one embodiment, the isolated polypeptide is from 15 to 723 amino acid residues in length. Within another embodiment, the at least fifteen contiguous amino acid residues of SEQ ID NO:M are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:123. Within another embodiment, the polypeptide comprises at least 30 contiguous residues of SEQ ID NO:M. Within a further embodiment, the polypeptide comprises at least 47 contiguous residues of SEQ ID NO:M.
Within a second aspect of the invention there is provided an isolated, mature protein encoded by a sequence selected from the group consisting of SEQ ID NO:N, wherein N is an odd integer from 1 to 327.
A third aspect of the invention provides isolated polynucleotides encoding the polypeptides disclosed above. Within certain embodiments of the invention the polynucleotides comprise a sequence of nucleotides as shown in SEQ ID NO:N, wherein N is an odd integer from 1 to 327.
Within a fourth aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as shown in SEQ ID NO:M, wherein M is an even integer from 2 to 328; and a transcription terminator.
A fifth aspect of the invention provides a cultured cell comprising the expression vector disclosed above. The cultured cell can be used, inter alia, within a method of producing a polypeptide, the method comprising (a) culturing the cell under conditions whereby the sequence of nucleotides is expressed, and (b) recovering the polypeptide. The invention also provides a polypeptide produced by this method.
Within a sixth aspect of the ivention there is provided an isolated polynucleotide encoding a fusion protein, wherein the fusion protein comprises a secretory peptide selected from the group consisting of secretory peptides shown in SEQ ID NO:M, wherein M is an even integer from 2 to 328, operably linked to a second polypeptide.
Within a seventh aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a fusion protein as disclosed above; and a transcription terminator. The invention further provides a cultured cell comprising this expression vector, wherein the cell expresses the DNA segment and produces the encoded fusion protein. Also provided is a method of producing a protein comprising culturing the cell under conditions whereby the DNA segment is expressed, and recovering the second polypeptide. Within one embodiment the recovered second polypeptide is joined to a portion of a protein of SEQ ID NO: M, wherein M is an even integer from 2 to 328.
Within a further aspect of the invention there is provided a computer-readable medium encoded with a data structure comprising SEQ ID NO:X, wherein X is an integer from 1 to 328.
Within an additional aspect of the invention there is provided an antibody that specifically binds to a protein selected from of the group consisting of SEQ ID NO:M, wherein Ml is an even integer from 2 to 328.
Within an additional aspect, the invention provides an isolated polypeptide comprising fourteen contiguous amino acid residues of a polypeptide as shown in SEQ If) NO:M, wherein M is an even integer from 2 to 328. Within an embodiment, said isolated polypeptide comprising said isolated fourteen contiguous amino acid residues is selected from the polypeptides as shown in Table 1. Within another embodiment, said fourteen contiguous amino acid residues can be used in a fusion protein to facilitate the secretion of a second polypeptide of interest outside a cell.
Within an additional aspect the invention provides a method of detecting protein secretion from a cell or tissue comprising detecting a mature MSP selected from the group consisting of SEQ ID NO:M, wherein M is an even integer from 2 to 328 in conditioned media or membrane extracts.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:
The term “affinity tag” is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al.,
EMBO J
. 4:1075, 1985; Nilsson et al.,
Methods Enzymol
. 198:3, 1991), glutathione S transferase (Smith and Johnson,
Gene
67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al.,
Proc. Natl. Acad. Sci. USA
82:7952-7954, 1985; see SEQ ID NO:123), substance P, Flag™ peptide (Hopp et al.,
Biotechnology
6:1204-1210, 1988), maltose binding protein (Kellerman and Ferenci,
Methods Enzymol
. 90:459-463, 1982; Guan et al.,
Gene
67:21-30, 1987), streptavidin binding peptide, thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, immunoglobulin constant domain, or other antigenic epitope or binding domain. See, in general, Ford et al.,
Protein Expression and Purification
2: 95-107, 1991. Affinity tags can be used individually or in combination. DNAs encoding affinity tags and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.; Eastman Kodak, New Haven, Conn.; New England Biolabs, Beverly, Mass.).
The term “allelic variant” is used herein to denote any of two
Presnell Scott R.
Sheppard Paul O.
Adams Robyn
Marschel Ardin H.
Smith Carolyn L.
ZymoGenetics Inc.
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