Human PEC-60-like protein and DNA encoding the same

Details Human PEC-60-like protein and DNA encoding the same Human PEC-60-like protein and DNA encoding the same

1998-04-17

2001-08-28

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S252300, C435S325000, C536S023100, C530S399000

Reexamination Certificate

active

06280968

ABSTRACT:

APPLICATION FIELD
The present invention relates to a human PEC-60-like protein and a CDNA encoding this protein. The protein of the present invention can be used as pharmaceuticals for the treatment and diagnosis of the digestive system diseases, the immune system diseases, and the nervous system diseases, or as an antigen for preparing an antibody against said protein. The human cDNA of the present invention can be used as a probe for the gene diagnosis and a gene source for the gene therapy. Furthermore, the cDNA can be used as a gene source for large-scale production of the protein encoded by said cDNA.
PRIOR ART
PEC-60 (a peptide consisting of an N-terminal glutamic acid, a C-terminal cysteine, and 60 amino acid residues) was isolated from the pig small intestine as a protein that inhibits the glucose-induced insulin secretion from perfused pancreas [Agerberth, B. et al., Proc. Natl. Acad. Sci. USA 86: 8590-8594 (1989)]. In general, such gastrointestinal hormones are found also in the nervous systems in many cases, whereas recent immunohistological studies have revealed the localization of a putative PEC-60 in the central and peripheral catecholamine nerves [Fuxe, K. et al., Neuroreport 5: 1817-1821 (1994)]. Furthermore, it has been suggested that the peptide plays some roles in the immune system, from the observation of its existence in the peripheral blood monocyte in a high content as well as the excretion in the serum [Metsis, M. et al., J. Biol. Chem., 267: 19829-19832 (1992)].
Since PEC-60 is a multi-functional hormone acting in the digestive tract, the nervous system, the immune system, etc., as indicated above, the acquisition of a human PEC-60 leads to its utilization as medicines. Although a porcine PEC-60 cDNA has been cloned up to date [Metsis, M. et al., J. Biol. Chem., 267: 19829-19832 (1992)], any report has not been presented on the human cDNA.
DISCLOSURE OF THE INVENTION
As the result of intensive studies, the present inventors were successful in cloning of a human cDNA encoding a human PEC-60-like protein, thereby completing the present invention. That is to say, the present invention provides a protein containing the amino acid sequence represented by Sequence No. 1 that is a human PEC-60-like protein. The present invention, also, provides a DNA encoding said protein exemplified as a cDNA containing the base sequence represented by Sequence No. 1.
The protein of the present invention can be obtained, for example, by a method for isolation from human organs, cell lines, etc, a method for preparation of the peptide by the chemical synthesis on the basis of the amino acid sequence of the present invention, or a method for production with the recombinant DNA technology using the DNA encoding the human PEC-60-like protein of the present invention, wherein the method for obtainment by the recombinant DNA technology is employed preferably. For example, an in vitro expression can be achieved by preparation of an RNA by the in vitro transcription from a vector having the cDNA of the present invention, followed by the in vitro translation using this RNA as a template. Also, the recombination of the translation domain to a suitable expression vector by the method known in the art leads to the expression of a large amount of the encoded protein by using
Escherichia coli, Bacillus subtilis
, yeasts, animal cells, and so on.
The DNA of the present invention includes all DNA encoding said protein. Said DNA can be obtained using the method by chemical synthesis, the method by cDNA cloning, and so on.
The cDNA of the present invention can be cloned from, for example, a cDNA library of the human cell origin. The cDNA is synthesized using as a template a poly(A)
+
RNA extracted from human cells. The human cells may be cells delivered from the human body, for example, by the operation or may be the culture cells. A poly (A)
+
RNA isolated from the human stomach cancer tissue is used in Examples. The cDNA can be synthesized by using any method selected from the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell. Biol. 2: 161-170 (1982)], the Gubler-Hoffman method [Gubler, U. and Hoffman, J. Gene 25: 263-269 (1983)], and so on, but it is preferred to use the capping method [Kato, S. et al., Gene 150: 243-250 (1994)] as illustrated in Examples in order to obtain a full-length clone in an effective manner.
The cloning of the cDNA is performed by the sequencing of a partial base sequence of the CDNA clone selected at random from the cDNA library and the search of the protein data base by the amino acid sequence predicted from the base sequence. The identification of the cDNA is carried out by determination of the whole base sequence by the sequencing, the protein expression by the in vitro translation, and the expression by
Escherichia coli.
The cDNA of the present invention is characterized by containing the base sequence represented by Sequence No. 1, as exemplified by that represented by Sequence No. 2 possessing a 398-bp base sequence with a 261-bp open reading frame. This open reading frame codes for a protein consisting of 86 amino acid residues and possessed a signal sequence at the N-terminal end. The amino acid sequence of the protein encoded by bases 43 to 303 of Sequence No. 2 (SEQ ID NO:2) is shown in SEQ ID NO:3. Sequence No. 1 (SEQ ID NO: 1) is identical to residues 121 to 300 of SEQ ID NO:2,
Hereupon, the same clone as the cDNA of the present invention can be easily obtained by screening of the human cDNA library by the use of an oligonucleotide probe synthesized on the basis of the cDNA base sequence depicted in Sequence No. 1 or Sequence No. 2.
In general, the polymorphism due to the individual difference is frequently observed in human genes. Therefore, any cDNA that is subjected to insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides in Sequence No. 1 or Sequence No. 2 shall come within the scope of the present invention.
In a similar manner, any protein that is produced by these modifications comprising insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides shall come within the scope of the present invention, as far as said protein possesses the activity of the protein having the amino acid sequence represented by Sequence No. 1.
The cDNA of the present invention includes cDNA fragments (more than 10 bp) containing any partial base sequence of the base sequence represented by Sequence No. 1 or No. 2. Also, DNA fragments consisting of a sense chain and an anti-sense chain shall come within this scope. These DNA fragments can be used as the probes for the gene diagnosis.


REFERENCES:
patent: 2 199 582 A (1988-07-01), None
patent: WO 88/03171 (1988-05-01), None
patent: WO 95/17885 (1995-07-01), None
National Cancer Institute, Cancer Genome Anatomy Project, GenBank Database Accession No. AA534438, Aug. 21, 1997.
Collins et al., GeneSeq Database Accession No. P80003, Nov. 6, 1990.
Metsis et al., GenBank Database Accession No. S46866, Jan. 5, 1993.
Church et al., GenBank Database Accession No. T12635, Jan. 5, 1994.
Bandman et al., GeneSeq Database Accession No. V38073, Sep. 14, 1998.
Bandman et al., GeneSeq Database Accession No. W62074, Sep. 14, 1998.
Ahren, B. et al., “The intestinal peptide PEC-60 inhibits insulin secretion in the mouse and the rat,”Pancreas, 7:443-446 (1992).
Fuxe, K. et al. “PEC-60, a novel porcine 60-residue intestinal peptide, reduces dopamine utilization in discrete parts of the neostriatum of the male rat following an intracerebroven-tricular injection,”Acta Physiologica Scandinavica, GB, Oxford, 141(1):139-140 (1991).
Fuxe, K. et al. “Generalized presence of a PEC-60-like peptide in catecholamine neurones,”Neuroreport, GB, Rapid Communications of Oxford, Oxford 5(14):1817-1821 (1994).
Liepinsh, E. et al., “Solution structure and dynamics of PEC-60, a protein of the Kazal type inhibitor family, determined by nuclear magnetic resonance spectroscopy,”J. Mol. Biol.239(1):1

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