IDENTIFICATION OF AN IgE-BINDING PROTEIN BY MOLECULAR CLONING

Abstract: A polypeptide which selectively binds IgE having a molecular weight of about 31,000 daltons, characterized in that it reacts with anti- epsilon BP serum, binds with IgE-sepharose 4B, has 5 methionine residues, and is devoid of potential N-glycosylation sites, its cDNA and a method of preparing the same.

Description: IDENTIFICATION OF AN IgE-BINDING PROTEIN BY MOLECULAR CLONING Field of the Invention This invention is in the field of recombinant f 5 DNA technology and more particularly to the production of IgE specific protein having an apparent molecular weight of about 31,000 daltons. Background of the Invention Both the production and function of 10 immunoglobulin E (IgE) molecules are intimately dependent on IgE-binding proteins. One type of IgE-binding proteins includes the cell surface receptors of mast cells, basophils, lymphocytes and other cell types (1,2). The receptors on mast cells and basophils are responsible for 15 IgE-mediated immediate hypersensitivity reactions (3,4), while those on lymphocytes play important roles in the regulation of IgE antibody responses (5,6). Another type of IgE-binding proteins includes the lymphokines which function in either potentiating or suppressing IgE 20 antibody production (5) . In order to further understanding of the IgE system, it is important to establish the structural relatedness of these proteins, the structure-function relationship of each of these proteins, and the regulation of their gene expression. To 25 this end, this invention's primary objective is to initiate molecular cloning of DNA for these relevant proteins. Rat basophilic leukemia (RBL) are cells which have been used extensively to study a high affinity IgE .30 receptor present on basophils and mast cells and from "* which IgE-binding proteins have already been isolated and Λ characterized (reviewed in 1,4,7). One well-studied IgE- binding protein is a glyσoprotein of M = 55,000 which has been studiedin a number of laboratories (8,9,10). It 35 has been definitively established that this protein is expressed on the surface of RBL cells, mast cells and basophils, and is responsible for the high-affinity IgE-binding property of these cells. The biochemistry of the high-affinity igE receptor became somewhat complex when a multi-subunit nature of the receptor was revealed by the identification of other protein components in the efforts to purify the receptor from RBL cells. One group of scientists has isolated two additional proteins, M = 33,000 (11,12) and M = 10,000 (13), in the affinity purification of IgE- receptor complexes. They also demonstrated that these two proteins could be chemically cross-linked to the M = approximately 55,000 glycoprotein by treating RBL cells or cell lysates' with bifunctional cross-linking reagents, and therefore designated them as the β and γ subunits, respectively, of the high-affinity IgE receptor, with the M = approximately 55,000 glycoprotein being designated as the α subunit. Another group of scientists has isolated a M = 30,000 - 33,000 protein in addition to the M = 55,000 glycoprotein, in the purification of RBL IgE receptor by repetitive affinity chromatography with IgE immunoadsorbent (14,15,16). Although 'this protein shares certain similarities with the protein designated as the β subunit, their exact relationship remains to be firmly established. Furthermore, it remained to be determined whether the isolation of either protein can be attributed to its ability to bind IgE independently. More recently, proteins of M = 30,000 - 33,000 which are distinct from the β subunit but which possess an IgE-binding property have been identified in RBL cells (17). The primary object of the present invention is to create and culture recombinant DNA molecules which produce an IgE specific protein having an approximate molecule weight of about 31,000 daltons. The long-range objective is to use this in vitro derived protein to establish the structural relatedness of the IgE system proteins, the structure-function relationship of each of these proteins, and the regulation of their gene expression. References The content of the following references is incorporated into the foregoing specification as fully as though set forth therein as a background for those skilled in the art. 1. Froese, A. (1980) CRC Crit. Rev. Immunol. 1, 79-130. 2. Spiegelberg, H.L. (1984) Adv. Immunol. 35, 61-88. 3. Ishizaka, K. and Ishizaka, T. (1978) ' Immunol. Review 41, 109-148. 4. Metzger, H. (1983) Contemp. Top. Mol. Immunol. , 115-145. 5. Ishizaka, K. (1984) Ann. Rev. Immunol. 2_, 159-182. 6. Katz, D.H. and Marcelletti, J.F. (1983) Prog. Immunol. V, 465-482. 7. Metzger, H., Kinet, J.-P., Perez-Montfort, R., Rivnay, B. and Wank, S.A. (1983) Prog. Immunol. V, 493-501. 8. Conrad, D.H. and Froese, A. (1976) J. Immunol. 116, 319-326. 9. Kulczycki, A., Jr., McNearney, T.A. and Parker, C.W.. (1976) J. Immunol. 117, 661- 665. 10. Kanellopoulos, Jr., Rossi, G. and Metzger, H. (1979) J. Biol. Chem. 254, 7691-7697. 11. Holowka, D., Hartman, H., Kanellopoulos, J. and Metzger, H. (1980) J. Recept. Res. 1, 41-68. 12. Holowka, D. and Metzger, H. (1982) Molecular Immunol. 19, 219-227. 13. Perez-Montfort, R., Kinet, J.-P. and Metzger, H. (1983) Biochemistry 22, 5722- 5728. 14. Kulczycki, A., Jr. and Parker, C.W. (1979) J. Biol. Chem. 254, 3187-3193.. 15. Hempstead, B.L., Parker, C.W. and Kulczycki, A., Jr. (1981) J. Biol. Chem. 256, 10717-10723. 16. Hempstead, B.L., Parker, C.W. and Kulczycki, A., Jr. (1983) Proc. Natl. 5 Acad. Sci. USA 80, 3050-3053. 17. Holowka, D. and Baird, B. (1984) J. Biol. Chem. 259, 3720-3728. 18. Kulczycki, A., Jr., Isersky, C. and Metzger, H. (1974) J. Exp. Med. 159, 600- 10 616. 19. ' Dunn, T.B. and Potter, M. (1957) J. Natl. Cancer Inst. 18, 587-595. 20. Bazin, H. (1982) In Protides of the Biological Fluids-29th Colloquium 1981 15 (Peeters H. ed.), Pergamon Press, Oxford and New York, 615-618. 21. Liu, F.-T., Bonn, J.W., Ferry, E.L., Yairramoto, H. , Molinaro, C.A., Sherman, L. , Klinman, N.R. and Katz, D.H. (1980) J^ 20 Immunol. 124, 2728-2737. 22. Liu, F.-T. and Orida, N. (1984), J. Biol. Chem. 259, 10649-10652. 23. Liu, F.-T., Albrandt, K. , Sutcliffe, J.G. and Katz, D.H. (1982) Proc. Natl. Acad. 25 Sci. USA 79, 7852-7856. 24. Parnes, J.R., Velan, B. , Felsenfeld, A., Ramanathan, L. , Ferini, U., Appella, E. and Seidman, J.G. (1981) Proc. Natl. Acad. Sci. USA 78, 2253-2257. 30 25. McConahey, P.J. and Dixon, F.J. (1966) Int. Arch. Allergy Appl. Immunol. 29, 185- 189. 26. Laemmli, U.K. (1970) Nature (London) 227, 680-685. 35 27. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205. 28. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 29. Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. 30. Helm, R.M., Conrad, D.H. and Froese, A. (1979) Int. Archs. Allergy Appl. Immunol. 5_8, 90-98. 31. Kanellopoulos, J.M., Liu, T.Y., Poy, G. and Metzger, H. (1980) J. Biol. Chem. 255, 9060-9066. 32. Pecoud, A.R., Ruddy, S. and Conrad, D.H. (1981) J. Immunol. 126, 1624-1629. 33. Goetze, A., Kanellopoulos, J., Rice, D. and Metzger, H. (1981) Biochemistry 20, 6341-6349. 34. Rivnay, B., Wank, S.A., Poy, G. and Metzger, H. (1982) Biochemistry 21, 6922- 6927. 35. Segal, D.M., Sharrow, S.O., Jones, J.F. and Siraganian, R.P. (1981) J. Immunol. 126, 138-145. 36. Mayrhofer, G., Bazin, H. and Gowans J.L. (1976) Eur. J. Immunol. 6_, 537-545. 37. Suzuki, T., Taki, T., Hachimine, K. and Sadasivan, R. (1981) Mol. Immunol. 18, 55- 65. 38. Huff, T.F., Uede, T. and Ishizaka, K. (1982) J. Immunol. 129, 509-514. 39. Hempstead, B.L., Kulczycki, A., Jr. and Parker, C.W. (1981) Bioche . Biophys. Res. Commun. 98, 815-822. Summary of the Invention Recombinant DNA molecules are produced which have the translatable end-product of an apparently 31,000 dalton IgE specific protein. For molecular cloning, double-stranded cDNA was synthesized from sucrose gradient-fractionated RBL mRNA, inserted into vector plasmids and used to transform recipient host organisms. By screening transformants using a hybridization-selection /in vitro translation procedure, several clones containing cDNA which hybridized to mRNA coding for a 31,000 IgE-binding protein were identified. The DNA sequence of one of these cloned cDNAs was determined and the amino acid sequence corresponding to the part of the protein was deduced. This cloned cDNA most likely codes for the 31,000 IgE-binding protein identified in RBL, cells, which appears to be uniquely related to the IgE-binding phenotype of the cells and which may have a significant role in the IgE mediated activation of basophils and mast cells. The invention includes a method of producing a cDNA molecule which has, as a translational product, a polypeptide which selectively binds IgE, the method comprising of isolating the genetic material^-'coding for IgE-binding polypeptide which has a molecular weight of about 31,000 daltons, is immunoprecipitated from translational products of RBL mRNA, and . is not precipitated by normal rabbit serum; incorporation of the genetic material into a vector; transfer of the vector into a recipient organism; selection and cloning of the host cell carrying the IgE-binding protein expression; producing clones; and collecting the protein produced. An IgE-binding protein produced by the aforesaid method reacts specifically with the anti-ε BP antiserum and binds with IgE-Sepharose 4B. The IgE-binding protein produced by the aforesaid method is characterized as a molecule with at least 5 methionine residues; a molecule which is devoid of potential N-glycosylation sites; and a molecule which can be isolated independently from a 55,000 Dalton IgE- binding. protein. The invention also includes cDNA characterized by the production of an approximately 31,000 Dalton IgE-specific binding protein. The cDNA is characterized by the production of a translational product, wherein said translational product is: an approximately 31,000 Dalton IgE-binding protein; a molecule with at least five methionine residues; a molecule having no N-glycosylation sites; and a molecule which can be isolated independently from a 55,000 Dalton IgE protein. The cDNA may also be characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, characterized in that it is: a molecule having 570 base pairs; a molecule that reacts specifically with antiepsilon BP antiseru ; and a molecule . which hybridizes specifically with mRNA from RBL cells. The cDNA characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons characterized in that said cDNA's carboxy-terminal half amino acid-coding sequence is substantially: ACA CTG ACA GTG CCC TAC GAT ATG CCC TTG CCT GGA GGA GTC ATG CCT CGC ATG CTG ATC ACA ATC ATA GGC ACA ' GTG AAG CCC AAC GCA AAC AGT ATC ACT CTG AAT TTC AAG AAA GGG AAC GAC ATC GCC TTC CAC TTT AAC CCC CGC TTC AAT GAG AAC AAC AGA AGA GTC ATC GTG TGC AAC ACG AAG CAG GAC AAT AAC TGG GGA AGG GAA GAA AGA CAG TCA GCT TTC CCC TTT GAG AGC GGC AAA CCA TTC AAA ATA CAG GTC CTG GTT GAA GCC GAC CAC TTC AAG GTT GCG GTC AAT GAT GTT CAT CTG TTG CAG TAT AAC CAT CGG ATG AAG AAC CTC AGG GAA ATC AGC CAA CTG GGG .ATC ATT GGT GAC ATA ACC CTC ACC AGC GCT TCC CAC GCC ATG AJTC TAA GCC AGA AGG GGT GGG CCG GCA CCA GAA CTG CCC TGT GTG TTA TGA GCG GGA AAC TTT GCA TTT CTC TCT CCT TAT ACT TCT TGT AAG ACA TCC ATT TAA TAA AGT CTC GTG CTG AGA GAA AAA AAA ACC CCC CCC CCC CC The above-characterized cDNA encodes the following protein: THR LEU THR VAL PRO TYR ASP MET PRO LEU PRO GLY GLY VAL MET PRO ARG MET LEU ILE THR ILE ILE GLY THR VAL LYS PRO ASN ALA ASN SER ILE THR LEU ASN PHE LYS LYS GLY ASN ASP ILE ALA PHE HIS PHE ASN PRO ARG PHE ASN GLU ASN ASN ARG ARG VAL ILE VAL CYS ASN THR LYS GLN ASP ASN ASN TRP GLY ARG GLU GLU ARG GLN SER ALA PHE PRO PHE GLU SER GLY LYS PRO PHE LYS ILE GLN VAL LEU VAL GLU ALA ASP HIS PHE LYS VAL ALA VAL ASN ASP VAL HIS LEU LEU GLN TYR ASN HIS ARG MET LYS ASN LEU ARG GLU ILE SER GLN LEU GLY ILE ILE GLY ASP ILE THR LEU THR SER ALA SER HIS ALA MET ILE ### The cDNA molecule is characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, said cDNA also being characterized in that it is a molecule with 121 nucleotides in a 3' untranslated region; a molecule with a typical AATAAA sequence 15-21 bases before the poly (A) addition site; a molecule with a sequence devoid of any transmembrane sequences; a molecule with a sequence devoid of any N-linked carbohydrate attachment sites; and a molecule which displays hydrophilic characteristics. The cDNA molecule is also characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons wherein said molecule reacts specifically with an RNA species from • RBL cells. Detailed Description of the Drawings Figure 1 is a representation of NaDodSo.-PAGE analysis of IgE-binding proteins isolated from the in vitro translation products of RBL mRNA. A) RBL poly(A) RNA was fractionated on a 10-30% sucrose gradient (see ref. 22 for the RNA sedimentation coefficient in each fraction) and individual fractions were translated in vitro. IgE-binding proteins in the translation products were then isolated using IgE-Sepharose 4B. B) In vitro translation products from sucrose gradient-fractionated RNA (pool of fractions 13-16) were subjected to immunoprecipitation with rabbit anti-εBP serum (lane a) or normal rabbit serum (lane b). The proteins were analyzed on 10% polyacrylamide gels. Figure 2 is a representation of NaDodSO.-PAGE analysis of the in vitro translation product from mRNA hybridized to a cloned cDNA. mRNA hybridizing with 136C9.13 cDNA immobilized on nitrocellulose filters was translated in vitro and the translation products were reacted with Sepharose 4B conjugated with BSA (lane a), mouse monoclonal IgG, (lane b) or mouse monoclonal IgE. (lane c), or were subjected to immunoprecipitation with rabbit anti-εBP serum (lane d) , or normal rabbit serum (lane e) . Isolated proteins were analyzed on 10% polyacrylamide gels. Figure 3 is a representation of the nucleotide sequence of cloned cDNA 136C9.13. The amino acids predicted from the nucleotide sequence are presented below the coding sequence. Figure 4 is a reproduction of Northern blot hybridization analysis of RNA from various cell lines with cloned 136C9.13 cDNA probe. Total poly(A) + RNA (5 μg each) from 5 cell lines - a mouse IgE-secreting hybridoma (26.82, lane a), a mouse IgG,-secreting hybridoma (109.3, lane b) , RBL (lane c), mouse mastocytoma P815 (lane d) and rat lymphoma IR983F (lane e) were glyoxylated, electrophoresed on 2% agarose gels, transferred to nitrocellulose filters and hybridized with 32P-labeled 136C9.13 probe. Hybridization conditions: 2x Denhardts/25 mM PIPES, pH 6.8/25 raM EDTA/0.75 M NaCl/50% formamide/O.2% NaDodS0 4 /50 μg/ml heat-denatured tRNA/50 μg/ml heat-denatured salmon sperm DNA; 42° C, 16 hr. Washing conditions: 15 mM NaCl/1.5 mM Na citrate/0.1% NaDodS0 4 at 51°C. Figure 5 is a representation of NaDodSO.-PAGE analysis of 125I-labeled IgE-binding protein from RBL cells. (A) Cell lysates from RBL (lane a), P815 (lane b) or rat lymphoma (lane c) were subjected to repetitive affinity purification with IgE-Sepharose 4B as described in Example 1. The proteins were labeled with 125I at the end of the first cycle of affinity purification. (B) RBL cell lysates were subjected to affinity purification with IgE-Sepharose 4B and the bound proteins were iodinated, eluted and treated with lentil lectin-Sepharose 4B. The unbound material was then i) reacted with IgE-Sepharose 4B (lane a); or ii) subjected to immunoprecipitation with rabbit anti-εBP '(lane b) or normal rabbit serum (lane c). Isolated proteins were analyzed on 10% polyacrylamide gels Detailed Description of the Invention This invention involves the production of a IgE binding protein, and the production of a cDNA molecule capable of use in recombinant DNA technology. More particularly, this invention involves the formation of a cDNA that produces a IgE binding protein having an approximate molecular weight of about 31,000 daltons, and the production of host/recipient organisms which are capable of producing that protein. Identification of 31K IgE-Binding Protein in the In Vitro Translation Products of RBL mRNA. Previously, we have demonstrated the expression of IgE-binding activity on the surface of Xenopus oocytes injected with RBL mRNA and have isolated from those oocytes a 31K translation product using an IgE immunoadsorbent (22). The extraordinary intensity of the 3IK protein band on NaDodSO.-PAGE gels in comparison to the presumed α subunit of the IgE receptor suggested that this protein might have an intrinsic IgE-binding activity. Sucrose gradient- fractionated RBL mRNA was subjected to in vitro translation in the rabbit reticulocyte lysate system. The translation products (composed of numerous translated proteins — data not shown) were examined by affinity purification with IgE immunoadsorbent. As shown in Figure 1A, a 31K protein was isolated virtually free of other proteins from the translation products of 13-14S RNA. A M = 30,000 - 33,000 protein, in addition to another higher molecular weight protein, was isolated from RBL cell lysates using IgE immunoadsorbent (14-16). It has been reported that polyclonal rabbit antisera raised against these proteins appeared to react with both protein components (15). It was postulated that the 31K protein detected above most likely corresponded to the small M = 30,000 - 33,000 protein and that the antisera would be useful to facilitate the study of the 31K protein. Therefore, the reactivity of this translation product with the serum was examined. (For simplicity, this antisera is designated as anti-IgE binding proteins; anti-eBP). As shown in Figure IB, a 31K protein was immunoprecipitated from the translation products of RBL mRNA by anti- εBP serum (lane a) and not by normal rabbit serum (lane b) . It should be noted that the 31K protein band is clearly the major protein in immunoprecipitates and is the only protein which was immunoprecipitated specifically. Cloning of cDNA Coding for the 3IK IgE-Binding Protein. RBL mRNA from sucrose density gradient fractions 14-16 was used to generate a cDNA library which was screened by a hybridization-selection/in vitro translation procedure for clones coding for the IgE-binding protein. From about 1000 clones, one positive clone (136C9.13) was identified. As shown in Figure 2, cDNA from this clone hybridized to mRNA from RBL cells which could be translated to a 31K protein which binds IgE (lane c) but not BSA (lane a) or IgG, (lane b) . Furthermore, the translated protein was shown to react specifically with the anti-εBP antiserum (lane d vs. lane e). Insert analysis indicated that the cloned cDNA was about 570 bp in length. The nucleotide sequence of the 136C9.13 cDNA was determined and a single open reading frame was identified. As shown in Figure 3, this cDNA contains a coding sequence for 138 amino acids representing approximately the carboxy-ter inal half of the protein. The cDNA sequence also contains 121 nucleotides of 3' untranslated region and a typical AATAAA sequence 15-21 bases before the poly(A) addition site. Homology searches of the nucleic acid database demonstrated that this sequence has not been previously described and the there is no significant homology to other known protein or DNA sequences. The protein appears rather hydrophilic as indicated by a hydropathicity plot (29); inspection of the sequence does not identify any potential transmembrane sequences nor any N-linked carbohydrate attachment sites. The 31K IgE-Binding is Expressed Uniquely by RBL Cells and Not by Cells Lacking Receptors. In order to relate the 136C9.13 cDNA to the IgE-binding property of RBL cells with confidence, one should first demonstrate that this cDNA detects a mRNA species present in RBL cells but not in other cells which lack the IgE receptor. Northern blot hybridization experiments were performed the results are shown in Figure 4. The 136C9.13 cDNA hybridized specifically with mRNA of sizes 1.1 kb and 1.6 kb from RBL cells (lane c). Under more stringent conditions, only the 1.1 kb mRNA band was detected. The 1.1 kb band but not the 1.6 kb band was also detected with much lower intensity in mRNA from mouse mastocytoma line P815 (lane d) . Neither of these two mRNAs were found in a rat lymphoma (lane e) nor in either of two mouse hybridomas secreting IgE (lane a) and IgG, (lane b) , respectively. A 1.1 kb mRNA has also been detected in mouse mastocytoma cell line CXBG ABMCT-1 but not in mouse macrophage line P388D1 (data not shown). The discovery of an RBL mRNA coding for a 31K IgE-binding protein prompted us to document that this protein was actually translated in vivo in certain cell types which bind IgE. When an RBL cell lysate was adsorbed with IgE-Sepharose and the bound proteins were iodinated, eluted and readsorbed with IgE-Sepharose, a 31 K protein was indeed isolated in addition to a protein of M = ~55,000 which has been designated as the α subunit of the IgE receptor (Figure 5, lane a). The two-cycle affinity purification procedure was found to be necessary to eliminate proteins binding to IgE nonspecifically (14,15). It was convenient, in conjunction with this procedure, to radiolabel the protein at the end of the first cycle of affinity purification, yielding proteins of high specific radioactivity. Neither protein was isolated from mouse mastocytoma P815 cells (lane b) nor from rat B lymphoma cells (lane c), indicating that both proteins are unique to RBL cells. Next,' it was desired to establish that the RBL cell-derived 31K protein detected above is an IgE-binding protein. Since it may be argued that in the above experiments, the 31K protein was isolated from RBL cells by virtue of its association with the M = "55,000 M protein, it is necessary to demonstrate that this protein can be isolated independently JLrom. the higher molecular weight component. This was approached by employing a lectin adsorbent since the latter protein (α subunit), which is highly glycosylated, is known to bind to various lectins (30), while the 31K protein, which is not (or lightly) glycosylated should bind to lectins only weakly or not at all. Thus, the protein bound to and subsequently eluted from the first IgE-Sepharose 4B described above was allowed to react with lentil lectin-Sepharose 4B and the unbound material was then treated with IgE immunoadsorbent'. As shown in Figure 5B (lane a), the 31K protein was indeed the major protein isolated by IgE-Sepharose 4B. This result indicates that the 31K protein found in RBL cells is an IgE-binding protein. The success of the above experiment also established that the isolated 3IK protein, alone, reacts with the anti-εBP antiserum, as is the case for the in vitro translated 31K protein. Thus, supernatants obtained following the absorption with lentil lectin-Sepharose 4B described above were subjected to immunoprecipitation with anti- e BP serum and the 31K protein was • found to be specifically immunoprecipitated (Figure 5B, lane b vs. lane c) . This result strongly supports the relatedness of this protein to the one identified in the in vitro translation products. EXAMPLE I The example of the invention is divided into the following stages: I. Preparation of RNA and In Vitro Translation of RNA II. cDNA Cloning and Screening III. Immunoprecipitation (A) Isolation of an IgE-binding protein with IgE-Sepharose 4B; one cycle procedure used for in vitro translation products. (B) Isolation of an IgE-binding protein by repetitive affinity purification with IgE-Sepharose 4B; used to cell lysates. (C) Immunoprecipitation with rabbit anti- eBP serum. (D) Electrophoresis, Northern Blot hybridization and DNA sequence analysis. Cell Lines and Reagents. Cell lines used include RBL (18, kindly provided by Dr. H. Metzger of the National Institutes of Health), mouse mastocytoma P815 (19), rat lymphoma IR983F (20, originally from Dr. H. Bazin and provided by Dr. M. Zanetti of the Medical Biology Institute) , and mouse hybridomas secreting either antidinitrophenyl (DNP) IgE or IgG, (21). Mouse monoclonal IgE (HI-DNP- ε-26.82) and IgG- j^ (HI-DNP-αl-109.3) specific for DNP (21), goat anti-rabbit IgG (GARG) antibodies (21) and rabbit antiserum prepared against proteins affinity- purified from RBL cells by repetitive chromatography on IgE immunoadsorbent (anti-εBP; 15) have been described previously. Immunoadsorbents were prepared by conjugating 10 mg of protein to 1 g (dry weight) of CNBr-activated Sepharose 4B (Pharmacia) . Preparation of RNA and In Vitro Translation of RNA. Extraction of total cytoplasmic RNA from various cell sources by the standard phenol/chloroform method, isolation of poly(A) RNA by oligo(dT)-cellulose affinity chromatography and sucrose density gradient fractionation of RBL poly(A) RNA have all been described (22). In vitro translation of mRNA was performed using the rabbit reticulocyte lysate system (New England Nuclear) in the presence of [ 35S]methionine. cDNA Cloning and Screening. Double-stranded cDNA was prepared from sucrose gradient-fractionated RBL mRNA, inserted into the Pst I site of pBR322 and used to transform E. coli C600 (23). A hybridization-selection/in vitro translation procedure was used to screen for positive clones (24). Briefly, pools of 12 chimeric plasmids were bound to nitrocellulose filters, the filters were incubated with total RBL poly (A) RNA and the hybridized RNA was eluted and translated in vitro. The translation products were subjected to immunoprecipitation (see below) to detect an IgE-binding protein. A single positive clone was identified by subjecting the individual clones of a positive pool to the same procedure. This cell line, identified as 136C9.13, is on deposit in the American Type Culture Collection, accession No. and will be available under the terms of the Budapest Treaty. Immunoprecipitation. (A) Isolation of an IgE-binding protein with IgE-Sepharose 4B; one cycle procedure used for in vitro translation products. The translation mixture (20 μl) was diluted with an equal volume of 2 x TENT (1 x TENT = 20 mM Tris-HCl, pH 7.5/10 mM EDTA/150 mM NaCl/1% Trasylol) containing 2% Triton X-100 and mixed with 50 μl (packed volume) of rabbit gamma-globulin (RγG)-Sepharose 4B for 1 hour at 4°C. The supernatant was then mixed with 50 μl of IgE-Sepharose 4B for 3 hours at 4°C. The beads were washed four times with 4 ml of TENT-1% Triton X-100, and one time with 4 ml of TENT-0.05% NaDodS0 4 . The bound protein was eluted with 75 μl of 62.5 mM Tris-HCl, pH 6.8/2% NaDodSO 4 /10% glycerol/5% 2-mercaptoethanol by boiling for 3 minutes. (B) Isolation of an IgE-binding protein by repetitive affinity purification with IgE-Sepharose 4B; used for cell lysates. This procedure is essentially the same as previously reported (14,15) with slight modifications as described (22), hereby incorporated by 7 reference. Briefly, the cell lysate (typically 5 x 10 cells) was subjected to the first cycle of affinity purification as described in (A) , and the bound proteins were eluted three times each with 200 μl of 0.5N acetic acid/1% Triton X-100, neutralized with 75 μl of 2 M Tris-HCl, pH 8.8/1 Triton X-100 and subjected to a second cycle of affinity purification. In this study, at the end of the first cycle of affinity purification, proteins were radiolabeled while still bound to IgE-Sepharose 4B by the chloramine-T method (25) using 1 mCi of Na 125I and 10 μg of chloramine-T in 30 μl of 0.1 M sodium phosphate, pH 7.5, for 60 seconds at 4°C. The beads wre further washed three times with 4 ml of TENT-1% Triton X-100 and the bound proteins were eluted as described above. (C) Immunoprecipitation with rabbit anti- εBP serum. To in vitro translation products (20 μl) or proteins isolated from RBL cells using IgE-Sepharose 4B (200 μl) were added 3 μl of normal rabbit serum (NRS). T mixture was incubated for 1 hour and then reacted with GARG-Sepharose 4B (50 μl) for 1 hour. The supernatants were reacted with3μi ' of rabbit anti-εBP serum for 1 hour and next mixed with GARG-Sepharose 4B (50 μl) for 1 hour. The beads were washed and the bound proteins were eluted as described above. All the above procedures were performed at 4°C. (D) Adsorption with lentil lectin-Sepharose 4B. Proteins bound to IgE-Sepharose 4B, iodinated and eluted as described in procedure (B) above were mixed with lentil lectin-Sepharose 4B (Pharmacia, 100 μl of a 1:1 suspension in,10 mM Tris-HCl, pH 7.4/0.15 M NaCl/1 Triton X-100) for 1 hour at 4°C. (E) Electrophoresis, Northern Blot Hybridization and DNA Sequence Analysis. NaDodS0 4 /poly- acrylamide gel electrophoresis analysis of proteins was conducted using the Laemmli system (26) hereby incorporated by reference. Gels containing S-labeled proteins were treated with autoradiography enhancer 3 (EN HANCE, New England Nuclear), dried and fluorographed; gels contai .ni.ng 125I-labeled proteins were dried and autoradiographed. For Northern blot analysis, RNAs were glyoxylated, electrophoresed on 2 % agarose gels, transferred to nitrocellulose filters and hybridized with cDNA probe labeled with 32p by nick-translation (27) hereby incorporated by reference. The DNA sequence was determined by the dideoxy primer-extension method._(28) hereby incorporated by reference. Industrial Applications Applicability has been discussed in the text of the preceding sections of this specification. Specifically, it has been discovered that: (1) a 31K IgE-binding protein can be translated in vitro from RBL mRNA and has been identified; (2) cDNA coding for this protein has been found to be novel; and (3) this protein has been shown to be expressed specifically in certain cell types known to possess IgE-specific receptors. Studies using the cDNA probe as well as antibodies against synthetic peptides corresponding to specific parts of the predicted sequence should further establish the expression of the described gene specifically in IgE-receptor positive cells, the localization of the protein in these cells and the role this protein may play in IgE-mediated activation of basophils and mast cells. Thus, the protein may be used as an IgE regulant or to absorb IgE, in effect an "IgE sponge", in the handling of allergenic reactions. Modifications obvious to one with ordinary skill in the art may be made without departing from the scope of the present invention. For example, the genetic material used to form the cDNA maybe induced by inserting other human cell lines containing the IgE-specific binding receptors or with other cell material other than RBL mRNA. While the invention has been described in terms of rat IgE-binding protein, the methods are equally applicable to producing cDNA's useful in detecting related human equivalents or siderophones.

Claims: CLAIMS WHAT IS CLAIMED IS: 1. A method of producing a cDNA molecule which has, as a translational product, a polypeptide which selectively binds IgE, the method comprising of: isolating the genetic material coding for IgE-binding polypeptide which has a molecular weight of about 31,000 daltons, is immunoprecipitated from translational products of RBL mRNA, and is not precipitated by normal rabbit serum ; incorporation of the genetic material into a vector; transfer of the vector into a recipient organism; selection and cloning of the host cell carrying the IgE-binding protein expression; producing clones; and collecting the protein produced. 2. An IgE-binding protein, produced by the method of Claim 1, which reacts specifically with the anti-εBP antiserum and binds with IgE-Sepharose 4B. 3. An IgE-binding protein produced by the method of Claim 1, characterized as: a molecule with at least 5 methonine residues; a molecule which is substantially devoid of potential N-glycosylation sites; and a molecule which can be isolated independently from a 55,000 Dalton IgE protein. 4. A cDNA characterized by the production of an approximately 31,000 Dalton IgE-specific binding protein. 5. A cDNA characterized by the production of a translational product, wherein said translational product is: an approximately 31,000 Dalton IgE-binding protein; a molecule with at least five methionine residues; a molecule having no n-glycosylation sites; and a molecule which can be isolated independently from a 55,000 Dalton IgE protein. 6. A cDNA characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, characterized in that it is: a molecule having at least 570 base pairs; a molecule that reacts specifically with antiepsilon BP antiserum; and a molecule which hybridizes specifically with mRNA from RBL cells. 7. A cDNA characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, characterized in that said cDNA's carboxy-terminal half amino acid sequencing is substantially: ACA CTG ACA GTG CCC TAC GAT ATG CCC TTG CCT GGA GGA GTC ATG CCT CGC ATG CTG ATC ACA ATC ATA GGC ACA GTG AAG CCC AAC GCA AAC AGT ATC ACT CTG AAT TTC AAG AAA GGG AAC GAC ATC GCC TTC CAC TTT AAC CCC CGC TTC AAT GAG AAC AAC AGA AGA GTC ATC GTG TGC AAC ACG AAG CAG GAC AAT AAC TGG GGA AGG GAA GAA AGA CAG TCA GCT TTC CCC TTT GAG AGC GGC AAA CCA TTC AAA ATA CAG GTC CTG GTT GAA GCC GAC CAC TTC AAG GTT GCG GTC AAT GAT GTT CAT CTG TTG CAG TAT AAC CAT CGG ATG AAG AAC CTC AGG GAA ATC AGC CAA CTG GGG ATC ATT GGT GAC ATA ACC CTC ACC AGC GCT TCC CAC GCC ATG ATC. 8. A protein translated from the cDNA characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, characterized in that said protein's carboxy-terminal half amino acid sequencing is substantially: THR LEU THR VAL PRO TYR ASP MET PRO LEU PRO GLY GLY VAL MET PRO ARG MET LEU ILE THR ILE ILE GLY THR VAL LYS PRO ASN ALA ASN SER ILE THR LEU ASN PHE LYS LYS GLY ASN ASP ILE ALA PHE HIS PHE ASN PRO ARG PHE ASN GLU ASN ASN ARG ARG VAL ILE VAL CYS ASN THR LYS GLN ASP ASN ASN TRP GLY ARG GLU GLU ARG GLN SER ALA PHE PRO PHE GLU SER GLY LYS PRO PHE LYS ILE GLN VAL LEU VAL GLU ALA ASP HIS PHE LYS VAL ALA VAL ASN ASP VAL HIS LEU LEU GLN TYR ASN HIS ARG MET LYS ASN LEU ARG GLU ILE SER GLN LEU GLY ILE ILE GLY ASP ILE THR LEU THR SER ALA SER HIS ALA MET ILE. 9. . A cDNA molecule characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons, said cDNA characterized in that it is: a molecule with 121 nucleotides in a 3' untranslated region; a molecule with a typical AATAAA sequence 15-21 bases before the poly (A) addition site; a molecule with a sequence devoid of any transmembrane sequences; a molecule with a sequence devoid of any N-linked carbohydrate attachment sites; and a molecule which displays hydrophilic characteristics. 10. The cDNA molecule characterized by the production of an IgE binding protein having an approximate molecular weight of about 31,000 daltons wherein said molecule reacts specifically with an RNA species from RBL cells.

Inventors: LU FU-TONG, LU, FU-TONG
Applicants: MEDICAL BIOLOGY INST
Application year: 1986
Main classification: C12N15
IPC codes: C07K14, C12N15, C12P21, C12R1