U.S. patent application number 09/941309 was filed with the patent office on 2002-08-08 for human pec-60-like protein and dna encoding this protein.
This patent application is currently assigned to Sagami Chemical Research Center. Invention is credited to Kamata, Kouju, Kato, Seishi, Sekine, Shingo, Yamaguchi, Tomoko.
Application Number | 20020106777 09/941309 |
Document ID | / |
Family ID | 17622687 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106777 |
Kind Code |
A1 |
Kato, Seishi ; et
al. |
August 8, 2002 |
Human PEC-60-like protein and DNA encoding this protein
Abstract
The present invention provides a human PEC-60-like protein, a
gastrointestinal hormone excreted by a stomach tissue and a cDNA
encoding this protein. The protein and the gene of the present
invention can provide a protein containing the amino acid sequence
represented by Sequence No. 1 and a t DNA encoding said protein
exemplified as a cDNA containing the base sequence represented by
Sequence No. 1. as well as a human cDNA encoding a human
PEC-60-like protein and said protein by the expression of this
human cDNA recombinant.
Inventors: |
Kato, Seishi; (Kanagawa,
JP) ; Yamaguchi, Tomoko; (Tokyo, JP) ; Sekine,
Shingo; (Kanagawa, JP) ; Kamata, Kouju;
(Kanagawa, JP) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Sagami Chemical Research
Center
|
Family ID: |
17622687 |
Appl. No.: |
09/941309 |
Filed: |
August 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09941309 |
Aug 28, 2001 |
|
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09065019 |
Apr 17, 1998 |
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Current U.S.
Class: |
435/226 ;
435/200; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 2319/00 20130101; A61P 1/00 20180101; A61P 25/00 20180101;
A61P 37/00 20180101; C07K 14/575 20130101 |
Class at
Publication: |
435/226 ;
536/23.2; 435/200 |
International
Class: |
C12N 009/24; C12N
009/64; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 1995 |
JP |
7/280272/1995 |
Oct 22, 1996 |
JP |
PCT/JP96/03061 |
Claims
What is claimed is:
1. A protein containing the amino acid sequence represented by
Sequence No. 1.
2. A DNA coding for the protein as described in claim 1.
3. A cDNA containing the base sequence represented by Sequence No.
1.
4. A protein described in claim 1 which comprises the amino acid
sequence represented by Sequence No. 2.
5. A cDNA described in claim 3 which comprises the base sequence
represented by Sequence No. 2.
Description
APPLICATION FIELD
[0001] 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
[0002] 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 te 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)].
[0003] 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
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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).sup.+ 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).sup.+ 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.
[0008] 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 Ad expression by the in
vitro translation, and the expression by Escherichia coli.
[0009] The cDNA of the present invention is character zed 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a figure depicting the structure of the plasmid
pHP00839.
[0016] FIG. 2 is a figure depicting the structure of the
Escherichia coli expression vector pMALPEC60 of the present
invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0017] The present invention is embodied in more detail by the
following examples, but this embodiment is not intended to restrict
the present invention. The basic operations and the enzyme
reactions with regard to the DNA recombination are carried out
according to the literature [Molecular Cloning. A Laboratory
Manual", Cold Spring Harbor Laboratory, 1989]. Unless otherwise
stated, restrictive enzymes and a variety of modification enzymes
to be used were those available from TAKAPA SHUZO. The
manufacturer's instructions were used for the buffer composition as
well as for the reaction conditions, in each of the enzyme
reactions. The cDNA synthesis was carried out according to the
literature [Kato, S. et al., Gene 150: 243-250 (1994)].
EXAMPLES
[0018] Preparation of Poly(A).sup.+ RNA
[0019] After 1 g of a human stomach cancer tissue was homogenized
in 20 ml of a 5.5 M guanidinium thiocyanate solution, 750 .mu.g of
mRNA was prepared according to the literature [Okayama, H. et al.,
"Methods in Enzymology" Vol. 164. Academic Press, 1987]. This was
subjected to oligo(dT)-cellulose column chromatography washed with
a 20 mM Tris-hydrochloric acid buffer solution (pH 7.6), 0.5 M
NaCl, and 1 mM EDTA to obtain 10 .mu.g of a poly(A).sup.+ RNA
according to the literature mentioned above.
[0020] Construction of cDNA Library
[0021] Ten micrograms of the above described poly(A).sup.+ RNA were
dissolved in a 100 mM Tris-hydrochloric acid buffer solution (pH
8), one unit of an RNase-free, bacterial alkaline phosphatase was
added, and the reaction was run at 37.degree. C. for one hour.
After the reaction mixture was subjected to phenol extraction
followed by ethanol precipitation, the pellet was dissolved in a
solution containing 50 mM sodium acetate (pH 6), 1 m E,T.A, 0.1%
2-mercaptoethanol, and 0.0% Triton X-100. Thereto was added one
unit of a tobacco acid pyrophosphatase (Epicentre Technologies) and
a total 100 .mu.l volume of the resulting mixture was reacted at
37.degree. C. for one hour. After the reaction mixture was
subjected to phenol extraction followed by ethanol precipitation,
he pellet was dissolved in water to obtain a solution of a decapped
poly(A).sup.+ RNA.
[0022] The decapped poly(A).sup.+ RNA and 3 mmol of a chimeric
DNA-RNA oligonucleotide
(5'-dG-dG-dG-dG-dA-dA-dT-dT-dC-dG-dA-G-C-A-3') were dissolved in a
solution containing 50 mM Tris-hydrochloric acid buffer (pH 7.5),
0.5 mM ATP, 5 mM MgCl.sub.2, 10 mM 2-mercaptoethanol, and 25%
polyethylene glycol, whereto was added 50 units of T4RNA ligase and
a total 30 .mu.l volume of the resulting mixture was reacted at
20.degree. C. for 12 hours. After the reaction mixture was
subjected to phenol extraction followed by ethanol precipitation,
the pellet was dissolved in water to obtain a chimeric-oligo-capped
poly(A) RNA.
[0023] After digestion of a vector pKA1 (Japanese Patent Kokai
Publication No. 1992-117292) developed by the present inventors
with KpnI, about 60 dT tails were added using a terminal
transferase. A vector primer to be used below was prepared by
digestion of this addition product with EcoRV to remove a dT tail
at one side.
[0024] After 6 .mu.g of the previously-prepared
chimeric-oligo-capped poly(A).sup.+ RNA was annealed with 1.2 .mu.g
of the vector primer, the resulting mixture was dissolved in a
solution containing 50 EM Tris-hydrochloric acid buffer (pH 8.3),
75 mM KCl, 3 nM MgCl.sub.2, 10 mM dithiothreitol, and 1.25 mM dNTP
(DATP+dCTP+dGTP+dTTP), 200 units of a transcriptase (GIBCC-BRL)
were added, and the reaction in a total 20 .mu.l volume was run at
42.degree. C. for one hour. After the reaction mixture was
subjected to phenol extraction followed by ethanol precipitation,
the pellet was dissolved In a solution containing 50 mM
Tris-hydrochloric acid buffer (pH 7.5), 100 mM NaCl, 10 mM
MgCl.sub.2, and 1 mM dithiotreitol. Thereto were added 100 units of
EcoRI and a total 20 .mu.l volume of the resulting mixture was
reacted at 37.degree. C. for one hour. After the reaction mixture
was subjected to phenol extraction followed by ethanol
precipitation, the pellet was dissolved in a solution containing 20
mM Tris-hydrochloric acid buffer solution (pH 7.5), 100 mM KCl, 4
mM MgCl.sub.2, 10 mM (NH.sub.4).sub.2SO.sub.4, and 50 .mu.g/ml of
the bovine serum albumin. Thereto were added 60 units of an
Escherichia coli DNA ligase and the resulting mixture was reacted
at 16.degree. C. for 16 hours. To the reaction mixture were added 2
.mu.l of 2 mM dNTP, 4 units of an Escherichia coil DNA polymerase
I, and 0.1 unit of an Escherichia coli DNase H and the resulting
mixture was reacted at 12.degree. C. for one hour and then at
22.degree. C. for one hour.
[0025] Next, the cDNA-synthesis reaction solution was used for
transformation of an Escherichia coli DH12S (GIBCO-BRL). The
transformation was carried out by an electroporation method. A
portion of the transformant was sprayed on the 2xYT agar culture
medium containing 100 .mu.g/ml ampicillin and the mixture was
incubated at 37.degree. C. overnight. A colony formed on the agar
medium was picked up at random and inoculated on 2 ml of the 2xYT
culture medium containing 100 .mu.g/ml ampicillin. After incubation
at 3700 for 2 hours, the mixture was infected with a helper phage
MK13KO7 (Pharmacia) and incubated further at 37.degree. C.
overnight. The culture solution was centrifuged to separate the
mycelia and the supernatant, wherein a double-stranded DNA was
isolated from the mycelia by the alkaline hydrolysis method and a
single-stranded plasmid DNA from the supernatant according to the
conventional method. After double digestion with EcoRI and NotI,
the double-stranded plasmid DNA was subjected to 0.8% agarose gel
electrophoresis to determine the size of the cDNA insert. On the
other hand, after the sequence reaction using an M13 universal
primer labeled with a fluorescent dye and a Taq polymerase (a kit
of Applied Biosystems), the single-stranded phage DNA was examined
with a fluorescent DNA sequencer (Applied Biosystems) to determine
the about 400 bp base sequence at the 5'-terminus of the cDNA. The
sequence data were filed as the Homo-Protein cDNA Bank
database.
[0026] cDNA Cloning
[0027] The base sequencing of the clones selected at random from
the above-mentioned cDNA library was carried out and the obtained
base sequence was converted to three frames of the amino acid
sequence, which were subjected to a search of the protein data
base. he analysis software used was GENETYX-MAC (Software
Development). As the result, a protein encoded by a plasmid
pHP00839 contained in the clone HP00839 was revealed to be highly
homologous to the porcine PEC-60 amino acid sequence. The structure
of this plasmid is depicted in FIG. 1. The structure consisting of
a 42-bp 5'-nontranslation region, a 261-bp open reading frame, and
a 95-bp 3'-nontranslation region (Sequence No. 2) was found from
the determination of the whole base sequence of the cDNA insert.
The open reading frame codes for a protein consisting of 86 amino
acid residues and the search of the protein data base using this
sequence revealed such a high 73.3% homology to the porcine PEC-60
amino acid sequence over the whole regions. Table 1 shows the
comparison between the amino acid sequence of the human PEC-60-like
protein of the present invention (HS) and that of the porcine
PEC-60 (SS). Therein, the marks of * (asterisk) and . (dot)
represent an amino acid residue identical with the protein of the
present invention and an amino acid residue similar to the protein
of the present invention, respectively.
1TABLE 1 HS MAVRQWVIALALAALLVVDREVPVAAGKLDFSRMPICEH-
MVESPTCSQMSNLVCGTDGLT **** **.*******..*******.*.*
**********.***.**.. . ******.* SS MAVRLWVVALALAALFIVDREVP-
VSAEKQVFSRMPICEHMTESPDCSRIYDPVCGTDGVT HS YTNECQLCLARIKTKQDIQIMKDGKC
*..**.******..******.***.* SS YESECKLCLARIENKQDIQIVKDGEC
[0028] Protein Synthesis by in vitro Translation
[0029] The vector pHP00839 having the cDNA of the present invention
was used for in vitro translation with a TNT rabbit reticulocyte
lysate kit (Promega). In this case, [.sup.35S] methionine was added
to label the expression product with a radioisotope. Each of the
reactions was carried out according to the protocols attached to
the kit. Two micrograms of the plasmid pHP00839 was reacted at
30.degree. C. for 90 minutes in a total 100 .mu.l volume of the
reaction mixture containing 50 .mu.l of the TNT rabbit reticulocyte
lysate, 4 .mu.l of a buffer solution (attached to the kit), 2 .mu.l
of an amino acid mixture (Met-free), 8 .mu.l of
[.sup.35S]methionine (Amersham) (0.37 Mbq/.mu.l), 2 .mu.l of T7RNA
polymerase, and 80 U of RNasin. To 3 .mu.l of the resulting
reaction mixture was added 2 .mu.l of the SDS sampling buffer (125
mM Tris-hydrochloric acid buffer, pH 6.8, 120 mM 2-mercaptoethanol,
2% SDS solution, 0.025% bromophenol blue, and 20% glycerol) and the
resulting mixture was heated at 95.degree. C. for 3 minutes and
then subjected to SDS-polyacrylamide gel electrophoresis.
Determination of the molecular weight of the translation product by
carrying out the autoradiography indicated that the cDNA of the
present invention yielded the translation product with the
molecular mass of about 10 kDa. This value is consistent with the
molecular weight of 9,454 predicted for the putative protein from
the base sequence represented by Sequence No. 2, thereby indicating
that the cDNA certainly codes for the protein represented by
Sequence No. 2.
[0030] Expression of Fusion Protein by Escherichia coli
[0031] Two strands of an oligonucleotide primer PR1 (5'-
2 GCGGTCTCGGGAAAGCTCCCTTTCTCAAG-3')
GCGTCGACTCAGCATTTGCCATCTTTCA-3')
[0032] and PR2 (5'-
[0033] were synthesized using a DNA synthesizer (Applied
Biosystems) according to the attached protocol.
[0034] PCR was carried out using 1 ng of the plasmid pHP00839 as
well as 100 pmole each of primer PR1 and primer PR2 to amplify a
region encoding a maturation protein consisting of C-terminal 60
amino acid residues. After phenol extraction and ethanol
precipitation, the pellet was digested with 20 units of Bsa1 (New
England Biolabs), treated with Klenow enzyme, and then digested
with SalI. The reaction product was subjected to 1.5% agarose gel
electrophoresis, cutting-off of an about 200-bp DNA fragment from
the gel, and purification.
[0035] Then, after 1 .mu.g of pMALTM-c2 (New England Biolabs) was
digested with 20 units of XmnI (New England Biolabs) and SalI, the
product was subjected to 0.6% agarose gel electrophoresis followed
by cutting-off of a 6.7-kbp DNA fragment from the gel. The vector
fragment and the cDNA fragment were ligated by using a ligation kit
and then Escherichia coli JM109 was transformed. Plasmid pMLPEC was
prepared from the transformant and the objective recombinant was
identified by the restriction enzyme cleavage map. FIG. 2 depicts
the structure of the obtained plasmid.
[0036] A suspension of 10 ml of an overnight-incubated liquid of
pMALPEC60/JM109 in 500 ml of the Rich culture medium (contains 10 g
of triptone, 5 g of yeast extract, 5 g of NaCl, and 2 g of glucose
per one liter) was incubated in a shaker at 37.degree. C. and
isopropylthiogalactoside was added so as to make 1 mM when
A.sub.600 reached about 0.5. After further incubation at 37.degree.
C. for 3 hours, the mycelia collected by centrifugation were
suspended in 25 ml of a column buffer for amylose column (10 mM
Tris-hydrochloric acid, pH 7.4, 200 mM NaCl, and 1 mM EDTA). After
sonification, the suspension was centrifuged and the supernatant
was charged into an amylose column (New England Biolabs) with a
3.5-ml head volume. After the column was washed with an 8-fold
column volume of the column buffer, a maltose-binding
protein/?EC60-like protein fusion protein was eluted with 20 ml of
the column buffer containing 10 mM maltose to afford 15.3 mg of the
fusion protein. The SDS-polyacrylamide electrophoresis of this
fusion protein indicated a single band at the position of about 50
kDa. This molecular mass value is consistent with the molecular
weight predicted for the ma ose-binding protein/PEC60-like protein
fusion protein.
[0037] To 200 .mu.l of the column buffer containing 100 .mu.g of
the maltose-binding protein/PEC60-like protein fusion protein was
added 1 .mu.g of factor Xa and the reaction was run at 4.degree. C.
for 12 hours. The reaction solution was subjected to the
amylose-column chromatography and the fraction eluted straight was
collected to afford 16 .mu.g of a PEC-60-like protein. The
SDS-polyacrylamide electrophoresis of this protein indicated a band
at the position of about 7 kDa corresponding to the PEC-60-like
protein of the present invention.
[0038] Probable Industrial Applicability
[0039] The present invention provides a human PEC-60-like protein,
a DNA encoding said protein, and a human cDNA encoding said
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. Said DNA can be used for the expression of a large amount
of said protein.
Sequence CWU 1
1
3 1 180 DNA Homo sapiens 1 ggaaagctcc ctttctcaag aatgcccatc
tgtgaacaca tggtagagtc tccaacctgt 60 tcccagatgt ccaacctggt
ctgcggcact gatgggctca catatacgaa tgaatgccag 120 ctctgcttgg
cccggataaa aaccaaacag gacatccaga tcatgaaaga tggcaaatgc 180 2 398
DNA Homo sapiens 2 gcaggcccca gccagctcag gctacactat cccaggatca
gcatggccgt ccgccagtgg 60 gtaatcgccc tggccttggc tgccctcctt
gttgtggaca gggaagtgcc agtggcagca 120 ggaaagctcc ctttctcaag
aatgcccatc tgtgaacaca tggtagagtc tccaacctgt 180 tcccagatgt
ccaacctggt ctgcggcact gatgggctca catatacgaa tgaatgccag 240
ctctgcttgg cccggataaa aaccaaacag gacatccaga tcatgaaaga tggcaaatgc
300 tgatcccaca ggagcacctc aagccatgaa gtgtcagctg gagaacagtg
gtgggcatgg 360 agaggatatg acatgaaata aaagatccag cccaactg 398 3 86
PRT Homo sapiens 3 Met Ala Val Arg Gln Trp Val Ile Ala Leu Ala Leu
Ala Ala Leu Leu 1 5 10 15 Val Val Asp Arg Glu Val Pro Val Ala Ala
Gly Lys Leu Pro Phe Ser 20 25 30 Arg Met Pro Ile Cys Glu His Met
Val Glu Ser Pro Thr Cys Ser Gln 35 40 45 Met Ser Asn Leu Val Cys
Gly Thr Asp Gly Leu Thr Tyr Thr Asn Glu 50 55 60 Cys Gln Leu Cys
Leu Ala Arg Ile Lys Thr Lys Gln Asp Ile Gln Ile 65 70 75 80 Met Lys
Asp Gly Lys Cys 85
* * * * *