Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-11-14
2003-12-16
Fredman, Jeffrey (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S006120, C435S091100, C435S094000
Reexamination Certificate
active
06664383
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel polypeptide, a cDNA encoding it and a pharmaceutical use of it.
TECHNICAL BACKGROUND
Until now, when a man skilled in the art intends to obtain a particular polypeptide or a cDNA encoding it, he generally utilizes methods by confirming an aimed biological activity in a tissue or in a cell medium, isolating and purifying the polypeptide and then cloning a gene or methods by “expression-cloning” with the guidance of the said biological activity. However, physiologically active polypeptides in living body have often many kinds of activities. Therefore, it happens increasingly that after cloning a gene, the isolated gene is found to be identical to that encoding a polypeptide already known. In addition, some factors could be generated in only a very slight amount and/or under specific conditions and it makes difficult to isolate and to purify the factor and to confirm its biological activity.
Recent rapid developments in techniques for constructing cDNAs and sequencing techniques have made it possible to quickly sequence a large amount of cDNAs. By utilizing these techniques, a process, which comprises constructing cDNAs library using various cells or tissues, cloning the cDNA at random, identifying the nucleotide sequences thereof, expressing novel polypeptides encoded by them, is now in progress. Although this process is advantageous in that a gene can be cloned and information regarding its nucleotide sequence can be obtained without any biochemical or genetic analysis, the target gene can be discovered thereby only accidentally in many cases.
DISCLOSURE OF THE PRESENT INVENTION
The present inventors have studied cloning method to isolate genes encoding proliferation and/or differentiation factors functioning in hematopoietic systems and immune systems. Focusing their attention on the fact that most of the secretory proteins such as proliferation and/or differentiation factors (for example various cytokines) and membrane proteins such as receptors thereof (hereafter these proteins will be referred to generally as secretory proteins and the like) have sequences called signal peptides in the N-termini, the inventors have conducted extensive studies on a process for efficiently and selectively cloning a gene encoding for a signal peptide. Finally, we have successfully developed a screening method for the signal peptides (signal sequence trap (SST)) by using mammalian cells (See Japanese Patent Application Kokai Hei 6-315380). We also developed yeast SST method on the same concept. By the method based on the same conception using yeast, (yeast SST method), genes including sequence encoding signal peptide can be identified more easily and efficiently (See U.S. Pat. No. 5,536,637).
The present inventors et al. have diligently performed certain investigation using the present invention in order to isolate novel factors (polypeptides) useful for treatment, diagnosis and/or study, particularly, secretory proteins containing secretory signal and membrane protein. From the result, the present inventors achieved to find novel secretory proteins and membrane proteins produced from cell lines and tissue, for example, human adult brain tissue, cell lines derived from human brain tissue and cell line derived from human bone marrow, and cDNAs encoding them, and then completed the present invention.
The present invention provides the cDNA sequences identified as clones OC001, OM237, OA004b which were isolated by the said yeast SST method using cDNA libraries prepared from human adult brain tissue and cell lines derived from human brain tissue (T98G, ATCC No. CRL-1690). Clones OC001, OM237, OA004b were full-length cDNA including full cDNA sequences encoding membrain proteins (Each protein is represented as OC001, OM237, OA004b protein, respectively).
It was indicated from the results of homology search for the public database of the nucleic acid sequences by using BLASTN and FASTA, and for the public database of the amino acid sequences by using BLASTX, BLASTP and FASTA, that there was no sequence identical to the polypeptide sequence and the nucleotide sequences of OC001, OM237, OA004b of the present invention. In addition, the polypeptides of the present invention were expected to possess the transmembrane region by hydrophobisity analysis of the obtained amino acid sequences. From these results, it was proved that polypeptides OC001, OM237, OA004b of the present invention were new membrane proteins.
The present invention provides the cDNA sequence identified as clone OAF075b which was isolated by the said yeast SST method using cDNA libraries prepared from human bone marrow cell line HAS303 (human bone marrow cell line: provided from Prof. Keisuke Sotoyama, Dr. Makoto Aizawa, First Medicine, Tokyo Medical College. see J. Cell. Physiol. 148, 245-251, 1991 and Experimental Hematol. 22, 482-487, 1994). Clone OAF075b was a full-length cDNA including a full cDNA sequence encoding secretory protein (this protein is represented as OAF075b protein).
It was indicated from the results of homology search for the public database of the nucleic acid sequences by using BLASTN and FASTA, and for the public database of the amino acid sequences by using BLASTX, BLASTP and FASTA, that there was no sequence identical to the polypeptide sequence and the nucleotide sequence of OAF075b of the present invention. In addition, the polypeptide of the present invention was expected to possess no transmembrane region by hydrophobisity analysis of the obtained amino acid sequence. From these results, it was proved that polypeptide of the present invention was a new secretory protein.
The present invention relates to
(1) a polypeptide comprising an amino acid sequence of SEQ ID NOS. 1, 4, 6, 9 or 12,
(2) a cDNA encoding the polypeptide described in (1),
(3) a cDNA comprising a nucleotide sequence of SEQ ID NOS. 2, 5, 7, 10 or 13, and
(4) a cDNA comprising a nucleotide sequence of SEQ ID NOS. 3, 8, 11 or 14.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The present invention relates to a substantially purified form of the polypeptide comprising the amino acid sequence shown in SEQ ID NOS. 1, 4, 6, 9 or 12, homologue thereof, fragment thereof or homologue of the fragment.
Further, the present invention relates to cDNAs encoding the above peptides. More particularly the invention is provided cDNAs comprising nucleotide sequence shown in SEQ ID NOS. 2, 5, 7, 10 or 13, and cDNA containing a fragment which is selectively hybridizing to the cDNA comprising nucleotide sequence shown in SEQ ID NOS. 2, 5, 7, 10, 13, 3, 8, 11 or 14. A said cDNA capable for hybridizing to the cDNA includes the contemporary sequence of the above sequence.
A polypeptide comprising amino acid sequence shown in SEQ ID NOS. 1, 4, 6, 9 or 12 in substantially purified form will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is that of the SEQ ID NOS. 1, 4, 6, 9 or 12.
A homologue of polypeptide comprising amino acid sequence shown in SEQ ID NOS. 1, 4, 6, 9 or 12 will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the polypeptide comprising the said amino acid sequence over a region of at least 20, preferably at least 30, for instance 40, 60 or 100 more contiguous amino acids. Such a polypeptide homologue will be referred to a polypeptide of the present invention.
Further, a fragment of polypeptide comprising amino acid sequence shown in SEQ ID NOS. 1, 4, 6, 9 or 12 or its homologues will be at least 10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 amino acids in length.
A cDNA capable of selectively hybridizing to the cDNA comprising nucleotide sequence shown in SEQ ID NOS. 2, 5, 7, 10, 13, 3, 8, 11 or 14 will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the cDNA comprising the said nucleotide sequence over a region of at least 20, preferably at least 30, for instance 40, 60 or 100 or
Fukushima Daikichi
Shibayama Shiro
Tada Hideaki
Chakrabarti Arun Kr.
Fredman Jeffrey
Ono Pharmaceutical Co. Ltd.
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