Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-04-30
2004-02-17
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S069100, C435S069500, C435S252300, C435S320100, C436S501000, C530S300000, C530S350000, C536S023500
Reexamination Certificate
active
06692926
ABSTRACT:
TECHNICAL FIELD
The present invention relates to recombinant human SM-11044 ((L)-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serine pyrrolidine amide hydrobromide)-binding receptor proteins exhibiting ligand-binding activities (abbreviated as SMBP hereinafter), and their uses. More particularly, it relates to transformed cells that are designed to express a recombinant human SMBP at an elevated level to the extent that its ligana-binding activity can be measured by deleting the polythymidine sequence from the base sequence of the 3′-untranslated region, and cellular membrane fractions thereof, to recombinant human SMBPs isolated from the transformed cells or the cellular membrane fractions thereof, to a screening system for discovering human SMBP agonists/antagonists characterized by utilizing the transformed cells, the cellular membrane fractions thereof or the isolated recombinant human SMBPs, and to human SMBP agonists or antagonists obtainable by the screening system.
BACKGROUND ART
SM-11044 ((L)-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluoro-phenyl)propyl] serine pyrrolidine amide hydrobromide)-binding receptor protein (SMBP) was discovered as a new protein that is bound by SM-11044, which is an agonist for &bgr;-adrenergic receptors, and by iodocyanopindolol, which is an antagonist for &bgr;-adrenergic receptors (Sugasawa, T. et al., J. Biol. Chem. 272, 21244-21252 (1997)). SMBP is a membrane protein resided at lung, ileum, and eosinophil membrane, and is believed to act as a receptor for SM-11044. SM-1T1044 was known to have activities to down-regulate the depolarization-mediated contraction of intestine and to inhibit migration of eosinophils, and has been believed to exert such SM-111044's functions via SMBP (Sugasawa, T. et al., J. Biol. Chem., 272, 21244-21252 (1997)).
Although the cDNA of a human SMBP was recently cloned (International Publication No. WO 98/26065), it was not reported that SM-11044 binds to any recombinant protein translated from the cDNA. In other words, there has been no report showing that a human SMBP is expressed at an elevated level to the extent that its ligand-binding activity can be measured, and, therefore, any SMBP has not yet been established in its particular availability.
DISCLOSURE OF THE INVENTION
The present invention aims to provide a recombinant human SMBP exhibiting ligand-binding activities, and its use. More particularly, it aims to provide transformed cells that are designed to express a recombinant human SMBP at an elevated level to the extent that its ligand-binding activity can be measured by deleting the polythymidine sequence from the base sequence of the 3′-untranslated region, cellular membrane fractions thereof, and recombinant human SMBPs isolated from the transformed cells or the cellular membrane fractions thereof, as well as a screening system for discovering human SMBP agonists/antagonists characterized by utilizing the transformed cells, the cellular membrane fractions thereofor the isolated recombinant human SMBPs, and human SMBP agonists or antagonists obtainable by the screening system.
As mentioned above, SMBP has been believed to be a receptor that mediates actions to down-regulate the depolarization-mediated contraction of intestine and to inhibit migration of eosinophils. Accordingly, it is expected that substances exhibiting an agonistic activity for SMBP would bind to SMBP to exert the functions as mentioned above, thereby leading to pharmaceutical compositions for treating inflammatory diseases involving eosinophil infiltration, asthma, or bowel diseases.
The present inventors attempted to construct a screening system for discovering efficiently ligands binding to SMBP, which comprises using SMBP in view of the development of such pharmaceutical compositions. International Publication No. WO 98/26065 describes that western blotting with use of anti-human SMBP antibody revealed that a SMBP protein was expressed by COS cells transformed with the recombinant human SMBP cDNA. However, the SMBP protein was expressed in a quite small amount, and, consequently, it has no t been reported that any human SMBP is expressed in a sufficiently high level to construct screening systems. In fact, the inventors obtained the relevant human SMBP cDNA fragment (SEQ ID NO: 1) from the applicant of the International Application (WO 98/26065), Vetigen, and transformed the cDNA into COS-1 cells or CHO-K1 cells. Then, the inventors determined a ligand-binding activity of the transfectants, but found no activity. Due to these facts, the inventors believed either of 1) that any protein had not translated from the human SMBP cDNA, or 2) that, even if a protein was translated, it had not been expressed at an elevated level to the extent that its ligand-binding activity can be measured.
The present inventors presumed that drawbacks involving the structure of the cDNA would cause no or little expression of the protein. Restudy of the base sequence of SEQ ID NO: 1 revealed that the 3′-untranslated region contains a polythymidine sequence consisting of a consecutive sequence of as many as 37 thymidines, and that the polyuridine sequence of the mRNA corresponding to the polythymidine sequence binds to the polyadenine tail (poly-A) residing at the 3′-terminus of the SMBP mRNA to form certain secondary structure, which would thereby restrain translation into proteins.
On the basis of the above presumption, by deleting the polythymidine sequence from the 3′-untranslated region in the base sequence of the human SMBP cDNA depicted in SEQ ID NO: 1, the inventors have successfully expressed a recombinant human SMBP at an elevated level to the extent that its ligand-binding activity can be measured for the first time. Further, the inventors have successfully established, for the first time, a screening system for discovering SMBP agonists/antagonists that are effective in a human, owing to the availability of such measurement of ligand-binding activity.
The present invention has been completed on the basis of the findings as described above.
Thus, the present invention relates to:
(1) A process for expressing a recombinant protein at an elevated level, which comprises deleting a sequence comprising the polythymidine sequence from the base sequence of the 3′-untranslated region;
(2) A process for expressing a recombinant human SMBP at an elevated level, which comprises;
(a) preparing a DNA wherein a sequence comprising the polythymidine sequence is deleted from the 3′-untranslated region in the base sequence of SEQ ID NO: 1;
(b) introducing the DNA of the above (a) into an expression vector;
(c) transforming a host cell with the expression vector of the above (b); and
(d) culturing the transformed cells of the above (c) under an appropriate condition;
(3) A DNA encoding a recombinant human SMBP, which is characterized in that;
(e) a sequence comprising the polythymidine sequence is deleted from a 3′-region from position 1875 in the base sequence of SEQ ID NO: 1; and
(f) the recombinant human SMBP that is a translation product of the DNA can be expressed at an elevated level to the extent that its ligand-binding activity can be measured;
(4) The DNA of the above (3) wherein a sequence comprising all or part of the base sequence from positions 1899 to 1935 of SEQ ID NO: 1 is deleted;
(5) The DNA of the above (4) wherein the portion of the base sequence from positions 1.875 to 2072 of SEQ ID NO: 1 is deleted.
(6) The DNA of the above (5), which consists of the base sequence of SEQ ID NO: 3.
(7) An expression vector which carries the DNA of any one of the above (3) to (6).
(8) A transformed cell expressing a recombinant protein at an elevated level to the extent that its ligand-binding activity can be measured, which is obtainable by the process of the above (1) or (2), or a cellular membrane fraction thereof.
(9) A transformed cell expressing a recombinant human SMBP at an elevated level to the extent that its ligand-binding activity can be measured, which is obt
Hidaka Jun
Kawakami Hajime
Sugasawa Toshinari
Brannock Michael
Eyler Yvonne
Sumitomo Pharmaceuticals Company Limited
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