DD263538A5 - Method for producing a human Fc deep receptor - Google Patents

Abstract

The invention relates to a process for the preparation of a low affinity human Fce receptor and its water-soluble portion beginning with the amino acids of about 50 to about 150 of the low affinity human Fce receptor, preferably the N-terminal Met-Glu -Leu-Gln-Val-Ser-Ser-Gly-Phe-Val, their isolation and purification. The recovered low affinity human Fce receptor, preferably its water-soluble portion, is useful in the treatment of local and allergic reactions induced by the expression of IgE and may be incorporated into the appropriate pharmaceutical compositions.

Description

an Fc _E receptor prepared according to claim 17 is subjected to a trypsin or lysyl endopeptidase treatment and subsequently separated by means of HPLC.

20. The method according to claim 1, characterized in that for the preparation of the DNA sequence, the oligonucleotide of the formula

3'-TtI ACC DAY TT ^ AA ^ GT-5 'C _A GG

is synthesized by adding the corresponding nucleotides by means of an oligosynthesizer. For this 26 pages drawings.

Field of application of the invention

The invention relates to a method for producing a human Fce receptor with low affinity, civ: isolation, recombinant production and purification.

Characteristic of the known state of the art

Receptors for the Fc part of immunoglobulin (FcR) are expressed by different blood cell cell lineages and form an important link between antibody molecules and their effector functions, such as internalization of ligand-receptor complexes, antibody-induced cytolysis of target cells and the Release of inflammatory carriers. Expression of Fc receptors has also been demonstrated on T and B lymphocytes, suggesting a possible role of FcR in both the regulation of immune response and the involvement of immunoglobulin (Ig) binding factors, such as IgE, IgA and IgG-binding factors in the isotype-specific regulation of the antibody reaction.

At present, neither the Fc receptors nor the genes coding for the Fc receptors are isolated, although two types of Fc receptors for IgE (Fc _c R) are known which differ in their structure and function, namely

a) Fc _c high affinity receptors for basophils and mast cells and

b) Fcc receptors with low affinity for lymphocytes and monocytes.

Object of the invention

The aim of the invention is to obtain expressed polypeptides for the treatment of local and systemic IgE allergy reactions as an active ingredient for use in pharmaceutical compositions in an economical manner.

Explanation of the essence of the invention

It is an object of the present invention to provide a process for preparing a low affinity human Fc _e receptor whose water-soluble portion starting with amino acids from about 50 to about 150 of the entire Fc _c receptor, preferably with the N- Termina! Met-Glu-Leu-Gln-Val-Ser-Ser-Gly-Phe-Val-, as well as its glycosylated derivatives, their isolation and purification.

According to the invention, the object is achieved by the steps

a) isolation and purification of the water-soluble part of the lymphoblastic cells secreted or secreted by IgE-binding factor (Fc _c R);

b) partial sequencing of the water-soluble portion of low affinity human Fc _E receptor isolated by hydrolysis to isolate the fragments thus obtained and to determine the sequences of fragments so obtained;

c) Preparation of two hybridization probes, namely

Probe 1 for the production of Fc / L cell strain-specific cDNA by multiple cyclic subtraction with Uk "|

Cell mRNA; ; |

and probe 2 for the preparation of an oligonucleotide encoding one of the isolated partial sequences, preferably I

a mixture of oligonucleotides of the following formula j

3'-TT ^ ACC TAG TT ^ AA ^ GT-5 'j

^C A ^GG J

that for the amino acid sequence of the formula

Lys-Trp-Ile-Asn-Phe-Gin encoded; !

d) Isolation and Identification of Cxpresslone Vehicles Encoding Gene Coding for Low Affinity Humfn Fc _C Receptor {

contained by the following steps';

Synthesis of cüNA from sinar RNA matrix derived from Fcc receptor mRNA-producing lymphoblastoid cells i. )

Incorporation of this synthesized cDNA into expression vehicles to form an expression vehicle bank,

Hybridization of this incorporated cDNA to identify those expression vehicles which are Fc _r receptor; carrying coding gene, with two labeled probes containing cDNA specific for Fc _t R ⁺ L cells and one containing the gene of the low affinity, low affinity receptor oligonucleotide, and

- replication of the thus obtained Fc _E -receptor;

e) expression of FccR cDNA;

f) Determining the human Fcc low affinity gene encoding gene using the isolated cDNA sequence obtained from the vehicles of step e) according to Sanger et al. and the chemical fission method of Maxam and Gilbert; ;

g) expression of Fc _t R mRNA; and

h) Preparation of an expression vector containing the coding for a water-soluble fragment of the Fc _c receptor DNA sequences, their O-glycosylated derivatives and the expression of a soluble fragment in microorganisms or in mammalian cells.

The individual stages will be explained in more detail below.

a) Isolation and purification of the water-soluble part of Fc _c R

Human B-lymphoblastoid cells, such as RPMI-8866 cells, secrete Fc _c R of about 46 kd on their surface i

and release a species of about 25 kd into the culture supernatant. It was found that the Fc _c R activity, the clutch;

lymphoblastoid cells, e.g. RPMI-8866 cells secreted or secreted by the ELISA method (Figure 1) using two different monoclonal antibodies (see EP-A-No. 86110420.6 of the same Applicant, filed July 29, 1986). was higher in the concentrated culture supernatant compared to NP-40 detergent-solubilized membrane receptors, although an equal number, eg 10 ⁵ cells / ml, was used to produce Fc _c R. Furthermore, when affinity purified supernatants were chromatographed on SDS-PAGE under nonreducing conditions and Fc _c R activity was eluted from portions of the gel corresponding to the defined molecular mass, activity was observed in the 45-46kd and 24-25kd regions (Figs ). In fact, the concentrated culture supernatants contained a higher level of activity in the 25kd range. Therefore, serum-free culture supernatants were used as the receptor source.

For the sequential immunoaffinity purification of Fc _t R with a molecular mass of about 25 kd, immunoaffinity columns were used, which according to the manufacturer's description were prepared from 10 mg / ml purified monoclonal antibody coupled with Sepharose 4B beads (Pharmacia, Piscataway, New York). were manufactured. The sequential adsorption of 200-250X concentrated culture supernatant on BSA-Sepharose, transferrin-Sepharose and normal mouse IgG-Sepharose gave about 70% of the original activity but without improving the specific activity. Ddrgestellt as shown in Table 1., Was left to the Rezeptormal ^ rhl nich the Präbdsorption bind 4-16 hours to 3-5 sepharose column, reduces the overall activity of the Essigsäureeluats \> λ de, the specific activity was 83 units / pg and the purity increased 190 times. Further purification of the receptor by HPLC reverse phase chromatography on C-4 column using a linear gradient of 0-65% acetonitrile and 0.1% trifluoroacetic acid increased the specific activity to 1630 units / pg and the receptor was purified 371 fold. However, the final extraction was only 33% of the original. As can be seen from Table 1, a similar purification scheme was used for detergent-solubilized membrane Fc _t R, but the specific activity recovered was significantly lower than that observed in the culture supernatants. It is important to note that the choice of elution buffer played a role in the degree of recovery, 2.5% acetic acid elution with rapid neutralization was important for the final yield of the receptor.

As can be seen from Table 1, immunoaffinity purification of Fc _E R by means of specific monoclonal antibodies in the solid phase was not sufficient to obtain a pure receptor, as measured by a single band on SDS-PAGE.

Therefore, freshly eluted Fc ₁ R was further purified by reverse phase HPLC purification. The fresh, eluted Fc ₁ R was preparatively applied to a C-4 column and chromatographed by a linear gradient of 0-65% acetonitrile; the chromatography profile is shown in FIG. the fractions obtained at different retention times were checked for Fc _c R activity by ELISA. It was found that most of the Fc _E R was eluted by acetonitrile at a concentration between 44 and 45%. When 0.5 ml samples were collected corresponded to the Fc _t R activity to the hatched peak shown in FIG. J. Rechromatographic analysis of the active fraction showed a single peak, indicating the presence of a homogeneous receptor mixture. The fractions recovered at different retention times were also monitored by SDS-PAGE analysis.

Therefore, the concentrated culture supernatant (crude supernatant) containing the putative Fc _c R and the material eluted from the immunoaffinity and C-4 HPLC column was subjected to dialysis and lyophilization on a 10% SDS-PAGE as follows tested. The results show the presence of multiple bands in the strip of crude, concentrated supernatant. There is a 22-24 kd corresponding broadband to see.

The receptor component collected after sequential purification on non-specific and specific immunofixin gels still shows multiple bands, although the activity of these eluates is significantly higher than that of the raw material. The Fc _c R activity (Figure 4) obtained after C-4 HPLC purification shows a single band corresponding to 25kd, and the material thus purified exhibited very high activity in the ELISA method using monoclonal antibodies. It is important to note that the 25kd brad corresponds to the smaller species of Fc _c R detected after surface iodination and immunoprecipitation of Fc _c R using the same antibodies, suggesting that the 46 and 20 kD components are antigen are identical and the latter is the water-soluble part of Fc _E R.

Since purification of IgE-binding activity from RPMI-3866 cell culture supernatants requires many steps, one had to check the immunological activity of the putative Pc ₁ R at the different purification steps. Equal amounts of HPLC-purified Fc _c R were reapplied to specific 3-5 immunoaffinity and nonspecific NMIg Sepharose gels and the activity was monitored in the effluent and eluent of these gels. The results shown in Table 2 indicate that ilas purified material apparently has binding to the specific column compared to the nonspecific gel in which the available activity in the effluent and a small proportion in the eluate were found, and almost all of them Activity is recovered in the eluate.

It also becomes clear that a good portion of the activity is loosely bound to the nonspecific gel and lost during the wash.

b) Partial sequencing of the water-soluble portion of Fc _c R

Fc _c R, which was prepared after sequential purification of concentrated cell culture supernatant using immunoaffinity gels and C-4 HPLC, corresponded to a single band of SDS-PAGE and was active in the ELISA procedure, was subjected to amino acid sequencing. Trypsin and lysyiendopeptidase were used to prepare two different proteolytic preparations. Overall, 11 or 12 fragments were obtained by trypsin and lysyl endopeptidase treatment. The HPLC profile of lysyl endopeptidase defragmentation shown in Figure 5 has 12 major peaks corresponding to the defined fragments of Fc _c R. The following selected fragments were obtained: Met-Glu-Leu-Gln-Val-Ser-Ser-Gly-Phe-Val- (N-Tarminal),

Gly-Glu-Phe-Ile-Trp-Val-Asp-Gly-Se.-His-Val-Asp-Tyr-Ser-Asn-Trp-Ala-Pro-Giy-Glu-Pro-Thr-, Lys-His- Ala-Ser-His-Thr-Gly-Ser-Trp-Ile-Gly-Leu-Arg-Asnl.su-Asp-Leu-Lys and Lys-Trp-Ile-Asn-Phe-Gln-.

c) Preparation of two hybridization probes

Probe I (CLiNA specific for Fc _t R-positive L cells): ·

L-cells lacking thymidine kinase (Tk), Ltk 'cells, were co-transfected with high molecular weight DNA from RPMI-8866 cells and with the herpes simplex virus-derived Tk gene. After HAT selection, the Tk-positive transformants were stained with biotinylated anti-Fc _c R antibody (8-30) and FITC-avidin and sorted by a cell sorter. FceR positive L cells were enriched by several sorting cycles. Two L-cell transformed lines, LV-8-30 and L-VI-8-30 expressing Fc _e R, which is detected by anti-Fc _t R (8-30), were constructed from two independent transfection experiments. Pig.3 estimates the FACS analysis of these two transformed lines stained with both anti-Fc _c R and human IgE. All RNA was prepared from LV-8-30 cells by the guanidine isothiocyanate-cesium chloride method (See Biochemistry 18, 5294 (1979)) DNA complementary to mRNA from LV-8-30 cells was removed Synthesis of the enzyme reverse transcriptase on an oligo (dt) primer, the cDNA was labeled by incorporation of α- ³² P -deoxy-CTP into this reaction The ³² P-labeled cDNA was mixed and hybridized with a 10-fold excess of poly ( A) ⁺ RNA derived from Ltk "cells. This mixture was loaded onto a hydroxyapatite column and the unbound single-stranded cDNA was re-hybridized with poly (A) * RNA from Ltk 'cells. positive L-cell transformant was used as a probe for the detection of the gene for Fc _e R in Xgt-10 library (Figure 7)

 Probe II: A mixture of oligonucleotides of the following formula:

3'-TT ^ ACC TAG TT ^ AA * GT-5 ' ^C A ^GG ·

was synthesized by means of an ADI synthesizer at the 0.2 pmol level by known methods. The recovered oligonucleotide partially encoding the amino acid sequence of the following formula

Lys-Trp-Ile-Asn-Phe-gin,,

was used as a labeled probe for the detection of the gene for the Fc _t receptor in an expression vehicle.

d) Isolation and identification of expression vehicles containing gene coding for low affinity human Fc _c R

The exponentially growing RPMI-8866 cells were disrupted in guanidine isothiocyanate solution. The mRNA is isolated by centrifugation on a cesium chloride gradient, phenol extraction and ethanol precipitation. Subsequently, poly (A) * RNA is isolated by oligo- (dt) -cellulose chromatography.

The construction of the double-stranded cDNA is carried out according to Gubler and Hoffman (Gene 25,263 [1983]). The poly (A) ⁺ RNA is used as a matrix for the preparation of a single-stranded cDNA by means of reverse transcriptase and an oligo- (dt) starter. After treatment with RNase H, the second strand of the DNA is synthesized by means of DNA-polymerase I. The synthesis of the first and second strand of DNA was carried out in a solution containing a mixture of deoxynucleotide triphosphate of adenosine, thymidine, guanosine and cytidine. The recovered double-stranded cDNA-Gnmisch was then modified by the enzyme T4-DNA polymerase to remove any small remaining 3 'protruding ends from the first strand of the cDNA. The EcoRI linkers were added and ligated to the double-stranded cDNA by enzyme T4 ligase. cDNA which was not longer than 10P ¹¹¹ V was fractionated and the excess linker was removed by column chromatography with Bio-Gel A-50m The dota-fractionated cDNA was ligated to EcoRI-digested A.gt-10. The cDNA-containing AgMO vectors were packaged in vitro and infected with a strain of Escherichia coli 0600hfl, Among the plaques thus obtained, those containing sequences specific for Fc _t R were identified by colony hybridization two different probes were used:

1. FcER ^ transformant-specific cDNA.

2. Radioactively labeled synthetic Oiigi nucleotide of the following formula

3'-TTp ACC DAY TT ^ AA ^ GT-5 'C _A GG

Twenty-three out of about 300,000 clones that hybridized with both probes were identified and all cDNA inserts hybridized together. From the cDNAs of these clones, the larger cDNA insert (about 1600 kb) was selected. The EcoRI insert from the AgMO recombinant DNA clone was labeled by "nick" translation using a- ^3J P-dCTP and by Northet n hybridization with mRNA from various cells, including RPMI-866, Daudi, CEM, Fc _E analyzed R ⁺ L cells and Ltk- ~ cells. The insert hybridized only with mRNA from Fc _t R positive cells such as RPMI-8866 cells and Fc _e R ⁺ L cells. This insert was subcloned into an EcoRI site of cloned pGEM4 vector (Promega biotec), designated as pFc _c R-1 and propagated.

e) Expression of the Fc _c R cDNA:

The Eco RI insert containing the Fc _E R cDNA isolated from the AgMO clone described above was ligated to Eco RI-purified pGEM ™ 4 plasmid vector (see Figure 9), designated LE392, and expressed in E. coli am 1 August 1986 under the number FERM BP-1116 (Fermentation Research Institute, Agency of Industrial Science and Technology, Japan). Since this vector contains both SP6 and T7 promoters in opposite orientation, Fc _E R cDNA can be transcribed into mRNA by either SP6 or T7 RNA polymassase.

Therefore, the pGEM4DNA containing the pFc _E R cDNA was digested with BamHI, and the recovered plasmid DNA was used as a template to synthesize the mRNA by SP6 RNA polymerase, and the resulting RNA (5pg) was injected into Xenopus oocytes. After a two-day incubation period, the oocytes were lysed and the presence of pFc _E R was confirmed by an enzyme-linked immunosorbent assay (ELISA) using two anti-Fc _t R antibodies, 3-5 and 8-30, respectively Epitopes on Fc _c R recognize detected. As shown in Figure 9, the lysate of 10 oocytes injected with the RNA transcript of Fc _c R-1 exhibited Fc _E R values equal to those of 1 χ 10 ⁵ RPMI-8866. Cell-derived words were comparable. On the other hand, the lysate of Oozytun, di? sham-injected, no activity on. This result indicates that the product of Fc _c R-1 cDNA tailed the two different antigenic determinants with Fc _e R recognized by the monoclonal antibodies.

Another expression vector, for example, pDE 2 (see JA Patent Publication 1986/88879 of May 7, 1986) was used which contains two early promoters SV40 in opposite directions to each other to ensure cDNA expression in each orientation (Figure 8) to confirm that pFc _e R-1 includes the entire coding sequence of Fc _c R. The DNA tag between the two early promoters SV40 was removed by EcoRI digestion and replaced with the insert cDNA of pFc _e R-1 (pDE2-Fc _E R-1). Cos7 cells were transferred by the DEAE-dextran method with 2pg / ml Fc _E R cDNA-containing pDE2. After two days of culture, cells were stained twofold with anti-Fc _c R and human IgE and analyzed on a double laser FACS. As shown in Figure 8, approximately 30% of the cells transfected with pDF.2-Fc _c R-1 were labeled by both anti-Fc _t R and human IgE. In addition, staining was well correlated with anti-Fc _E R and human IgE, demonstrating that both anti-Fc _E R and IgE bind to the same molecule (s) the surface of the transfected cells was re-expressed (s). In fact, cells transfected with IFN-βcDNA-containing control vector pDE2 stained neither anti-Fc _E R nor human IgE. These results confirm that the isolated cDNA actually encodes the Fc _E R molecule.

f) Determination of the complete nucleotide sequence of the Fc _t R cDNA and the deduced protein sequence

The complete nucleotide sequence of the Eco RI insert from Fc ₁ RI was determined by the dideoxy termination method (see Sanger et al., InProc Natl Acad, See, USA 74, 5463-5467, 1977) and the chemical cleavage method (Sishe Maxam and Gilbert, Proc. Natl. Acad., U.S.A., 74, 560-564 [1977]). The complete nucleotide sequence and the deduced amino acid sequence are shown in Table 3, the coding sequence having the formula:

Glu Glu Gly 5 Tyr Ser Glu He 10 Glu Leu Per bad 15 mead GAG GAA GGT Gin DID TCA GAG ATC Glu GAG CTT CCC AGG bad ATG CTC CTT CCA CAA ATA AGT CTC DAY GAG CTC GAA GGG TCC AGG TAC GTT CTC TCC Cys Cys bad 20 Gly Thr Gin He 25 Leu Leu Gly Leu 30 bad TGT TGC AGG bad GGG ACT CAG ATC Val CTG CTG GGG CTG Val CGG ACA ACG TCC CGT CCC TGA GTC DAY GTG GAC GAC CCC GAC GTG GCC GCA CAC CAC

Ai a Ala Leu 35 Ala Gly Leu Leu 40 Leu Leu Leu -26- 263 338 GCC GCT CTG Trp GCT GGG 'VTG CTG Thr CTG CTT CTC 45 Thr CGG CGA GAC TGG CGA CCC GAC GAC ACT GAC GM GAG Leu Trp ACC ACC TGA CTG TGG TGG Trp Asp Thr 50 Gin Ser Leu Lys 55 Leu Glu Uiu GAC ACC TGC GAC ACC Thr CAG AGT CTA AAA gin CTG GM GAG 60 His ACC CTG TGG ACA GTC TCA GAT TTT CAG GAC CTT CTC bad Ala CAC TGT GTC AGG GCT GTG bad Asn Val 65 Gin Val Ser Lys 70 Leu Glu Ser TCC CGA CGG AAC GTC Ser CAA GTT TCC AAG Asn TTG GM AGC '75 Ala GCC TTG CAG TCT GTT CAA AGG TTC MC MC CTT TCG HIs His GCC AGA TTG CAC CAC CGG Asp gin mead 80 Gin Lys Ser Gin 85 Thr Gin He GTG " GTG GAC CAG ATG Ala CAG AT THE TCC CAG Ser ACG CAG ATT 90 Gly CTG GTC TAC GCG GTC TTT AGG GTC TCC TGC GTC TAA Ser gin GGT CGC AGG TCA CAG CCA Leu Glu Glu 95 bad Ala Glu Gin 100 bad Leu Lys AGT GTC CTG GAG GM Leu CGA GCT GM CAG Gin AGA TTG AT THE 105 Glu GAC CTC -CTT CTT GCT CGA CTT GTC CAG TCT MC TTT Ser gin GM GM GTC TCT CAG CTT Leu Glu Leu 110 Trp Asn LBU Asn 115 Leu Gin Ala AGA GTC TTG GAG CTG Ser. TGG MC CTG MC Gly CTT CM GCA 120 Asp AAC CTC GAC TCC ACC TTG GAC TTG GGG GAA GTT CGT Asp Leu GAC AGG CCC GAT CTG CTG Ser Phe Lys 125 Gin Giu Leu Asn 130 bad Asn Glu CTA GAC AGC TTC MG Ser CAG GAA TTG MC Glu AGG MC GM 135 Ser TCG AAG TTC TCC GTC CTT MC TTG GAG TCC TTG CTT Ala Ser AGC AAG CTC GCT TCA TCG Leu Leu Glu 140 Leu bad Glu Glu 145 Thr Lys Leu CGA AGT TTG CTG GM bad CTC CGG GAG GAG Val ACA MG CTA 150 Asp MC GAC CTT AGA GAG GCC CTC CTC GTG TGT TTC GAT bad mead GAi TCT CAC AGG ATG CTA Leu Gin Val 155 Ser Gly Phe Val 160 Asn - Cys TCC TAC TIG CAG GTG Ser AGC GGC TTT GTG Cys MC AAG TGC 165 Glu MC GTC CAC TCC TCG CCG AT THE CAC TGC TTG TGC ACG Per Glu GAG AGG ACG CCT GM CTC Trp He Asn 170 Gin bad Lys Cys 175 Tyr Ph " Gly GGA CTT TGG ATC AAT Phe CAA CÜü AAG TGC Tyr TAC TTC GGC 180 Lys ACC DAY TTA TTC GTT GCC TTC ACG TAC ATG MG CCG Lys Gly MG MG ATG AAG GGC TTC Lys Gin Trp 185 His Ala bad Tyr 190 Cys Asp Asp TTC CCG AAG CAG TGG Val CAC GCC CGG DID Ala TGT GAC GAC 195 Thr TTC GTC ACC GTC GTG CGG GCC ATA GCC ACA CTG CTG mead Glu ACC CAG CGG ATG GAA TGG Gin Leu Val 200 He His Ser Per 205 Glu Gin aap TAC CTT CAG CTG GTC Ser ATC CAC AGC CCG Glu GAG CAG GAC 210 Gly GTC GAC CAG AGC DAY GTG TCG GGC GAG CTC GTC CTG Phe Leu GGG TCG CTC TTC CTG CCC Lys His Ala 215 His Thr Gly Ser 220 Ho Gly Leu AAG GAC MG CAT GCC Ser CAC ACC GGC TCC Trp ATT GGC CTT 225 Thr TTC GTA CGG AGC GTG TGG CCG AGG TGG TM CCG GAA bad Asn ACC TCG ACC CGG AAC TGG Asp Leu Lys 230 Glu Phe He Trp 235 Asp Gly Ser GCC TTG GAC CTG AAG Gly GAG TTT ATC TGG Val GAT GGG AGC 240 Leu CTG GAC TTC GGA CTC AAA DAY ACC GTG CTA CCC TCG His Val TTG CCT CAC CAT GTG MC GTA CAC

Tyr Ser Asn 245 Ala Per Gly Olu 250 Thr Ser -9 - 263 538 TAC AGC AAC Trp GCT CCA GGG GAG Per ACC AGC 255 Asp ATG TCG TTG TGG CGA GGT CCC CTC CCC TUG TCG Arg. Ser Gin GAC ACC • GGG CGG AGC CAG CTG Glu Asp Cys 260 mead mead bad Gly 265 Gly bad GCC TCG GTC GAG GAC TGC Val ATG ATG CGG GGC Ser GGT CGC ? 70 Gly CTC CTG ACC GTG TAC TAC GCC CCG TCC CCA GCG Trp A * 5p Asp GGC CAC AGG TGG AAC GAC CCG Phe Cys Asp 275 Lys Leu Gly Ala 280 Val Cys ACC TTG CTG TTC TGC GAC bad AAG CTG GGC GCC Trp GTG TGC 285 Ala AAG ACG CTG CGT TTC GAC CCG CGG TGG CAC ACG Asp bad Leu GCC GCA ACC GAC CGG CTG CGG Thr Cys Thr 290 Per Aid 3 Glu 295 Ser Ala CTG GCC GAC ACA TGC ACG Per CCA GCC AGC GAA Gly TCC GCG 300 Ala TGC ACG TGC CCG GGT CGG TCG CTT GGT AGG CGC Glu Ser mead GCC GGC CCA GAG TCC ATG CGG Per Asp Ser 305 Per Asp Per Asp 310 bad Leu CTC AGG TAC CCT GAT TCA bad CCA GAC CCT GAC Gly CGC CTG 315 Gly GGA CTA AGT AGA GGT CTG GGA CTG GGC GCG GAC Per Thr Per PGI Ala Per Leu TCT Ser CCG CCC ACC CCC CCT GCC CCT CTC His TCT TGA GGG TGG GGG Ser CGG GGA GAG CAC AGA ACT TCT GTG AGA

Dor's single large open reading frame begins at position 183 and spans 963 nucleotides, encoding 321 amino acids. The isolated partial amino acid sequences of three peptide fragments and the isolated N-terminal Met-Glu-Leu-Gln-Val-Ser-Ser-Gly-Phe-Val of purified Fc _£ R confirm that the longest raster is the coding sequence of Fc _t R Protein is. The 21 amino acid hydrophilic N-terminal sequence is followed by a hydrophobic region consisting of 26 uncharged amino acids (22-47). The signal sequence in which regol located in the N-terminus of most membrane-bound or secretory proteins was not found. Therefore, the hydrophobic stretch of 26 amino acids is likely to be a membrane-embedded Hegion, as the subsequent residues are mostly hydrophilic and no other part of the sequence appears to cross the membrane. The N-terminal hydrophilic stretch is terminated by a cluster of very basic amino acid residues (Arg-Arg-Arg-Cys-Cys Arg-Arg). This cluster of basic amino acids, which is typically found on the cytoplasmic side of the membrane proteins, is known as a stop-transfer sequence that plays an important role in integrating into the lipid bilayer (see Blobel in Proc. Natl. Acad. USA 77, 1496-1500 [1980]) and Schneider et al., Nature 311, 675-678 [1984]). There is a putative N-linked carbohydrate addition site at position 63 which would be located in the extracellular membrane protein region. All these results show that Fc _t R is aligned with the N-terminus on the cytoplasmic side and the C-terminus on the outside of the cell. Relatively large amounts of soluble 25 kd Fc _c R were found in the culture supernatant of RPMI-8866 cells. The N-terminal amino acid residue (Met) of the soluble Fc _e R was found at position 150, and the previous residue, arginine, is a common target for trypsin-like proteases. The C-terminal region (150-321) contains two cysteine clusters (160,163,174 and 191 and 259,273,282 and 288) that are likely to form disulfide bonds and give a tightly folded structure that is resistant to proteolytic enzymes. The C-terminal region (150-321 '/ corresponds to the soluble Fc _£ R, which is a product of the proteolytic cleavage of the membrane-bound Fc ₁ R

 g) Expression of FcjR mRNA:

The poly (A) * RNA was prepared from various cell types and analyzed by Northern blotting for the expression of Fc _E R mRNA, with a major band of 1700b in B-cell lymphoblastoid lines (RPMI-8866, RPMI-1788). , was detected in the Epstoin-Barr virus-transformed pre-B-cell line of the fetal liver (FL 8-2) and in two Fc _E R * L-cell transformants, but not in Fc _c R ~ cells, including two Burkitt lymphoma lines (Daudi and Jijoye), a T cell line (CEM), and an L cell transformant expressing another B cell antigen (CD20) Further, Fc _t R mRNA was not detected in normal T cells are detected, whereas normal B cells express comparable levels of FC _£ R mRNA as B-lymphoblastoid lines.

h) Preparation of an expression vector containing the DNA sequences coding for a water-soluble fragment of the Fc _c receptor

The codopes encoding the water-soluble portion of the Fc _E receptor, desirably the codons for the amino acids of about 50 to 150, preferably about 150, were removed using a suitable endonuclease extract for the FCc receptor (see Table 3). Upon introduction of the resulting truncated Fc _c receptor into the organisms under conditions leading to a high yield, the desired water-soluble polypeptides were recovered without the aforementioned amino acids. The water-soluble portion of the Fc _E receptor therefore contains at least the following sequence:

Leu TTG AAC Gin CAG GTC VaI GTG CAC Ser TCC AGG Ser AGC TCG GIy GGC CCG Phe • TTT AAA VaI GTG CAC Cys TGC ACG Asn AAC TTG ' Thr ACG TGC - 10- 263 538 Met ATG TAC Trp TGG ACC lie ATC DAY Asn AAT TTA Phe TTC AAG In CAA GTT Arg CGG GCC Lys AAG TTC Cys TGC ACG Tyr TAC ATG Tyr TAC ATG Pho TTC AAG GIu CAA CTT GIu GAG CTC Lys AAG TTC Gin CAG GTC Trp TGG ACC VaI GTC CAG His CAC GTG AIa GCC CGG Arg CGG GCC Tyr TAT ATA AIa GCC CGG Cys TGT ACA Asp GAC CTG Cys TGC ACG Pro CCT GGA GIy GGC CCG VS AAG TTC Gin CAG GTC Leu CTG GAC VaI GTC CAG Ser AGC TCG Hey ATC DAY His CAC GTG Ser AGC TCG Pro CCG GGC GIu GAG CTC GIu GAG CTC GIn CAG GTC GIy GGC CCG Lys AAG TTC GIu GAA CTT Thr ACC TGG Lys AAG TTC His CAT GTA Ala GCC CGG Ser AGC TCG His CAC GTG Thr ACC TGG GIy GGC CCG Ser TCC AGG Trp TGG ACC He ATT TAA GIy GGC CCG Asp GAC CTG Met ATG TAC Leu CTG GAC GIy GGG CCC Asp GAC CTG Leu CTG GAC Lys AAG TTC GIy GGA CCT GIu GAG CTC Phe TTT AAA Hey ATC DAY Trp TGG ACC VaI GTG CAC Asp GAT CTA GIy GGG CCC Asp GAC CTG Phe TTC AAG Asn AAC TTG Thr ACC TGG Tyr TAC ATG Ser AGC TCG Asn AAC TTG Trp TGG ACC AIa GCT CGA Pro CCA GGT GIy GGG CCC GIu GAG CTC Pro CCC GGG Thr ACC TGG Ser AGC TCG Leu CTT GAA Arg CGG GCC VaI GTG CAC Leu TTG AAC GIu GAG CTC Asp GAC CTG Cys TGC ACG VaI GTG CAC Met ATG TAC Met ATG TAC Arg CGG GCC GIy GGC CCG Ser TCC AGG GIY GGT CCA Arg CGC GCG Ser AGC TCG His CAT GTA U in CAG GTC Asp GAC CTG Phe TTC AAG Cys TGC ACG Asp GAC CTG Arg CGT GCA Lys AAG TTC Leu CTG GAC GIy GGC CCG AIa GCC CGG Trp TGG ACC VaI GTG CAC Cys TGC ACG Arg CGG GCC Ser AGC TCG Asp GAC CTG GIy GGC CCG Thr ACA TGT Cys TGC ACG Thr ACC TGC Pro CCG GGC Pro CCA GGT AIa GCC CGG Ser AGC TCG GIu GAA CTT GIY GGT CCA Ser TCC AGG Ah GCG CGC Trp TGG ACC Asn AAC TTG Leu CTC GAC Ala GCC CGG Pro CCT GGA Asp GAT CTA Ser TCA AGT Arg AGA TCT Pro CCA ÜGT Asp GAC CTG Pro CCT GGA Asp GAC CTG GIy GGC CCG Arg CGC 3CG Leu CTG GAC Asp GAC CTG Arg CGG GCC Mot ATG TAC Ala GCC CGG Ala GCC CGG Pro CCT GGA Leu CTC GAG His CAC GTG Ser TCT AGA TGA ACT GIu GAG CTC Ser TCC AGG Pro CCC GGG GIy GGA CCT Pro CCC GGG Thr ACC TGG Ser TCT AGA

In addition, it has been found that the membrane-spanning region of Fc _c R does not function as a signal sequence in the conventional recombinant processes, and therefore, the secretion of a water-soluble receptor protein from a suitable host requires the use of a suitable eukaryotic signal sequence.

Such a signal sequence may additionally be provided in a position in front of the cDNA encoding di-n water-soluble portion.

A preferred embodiment of the invention is therefore a novel water-soluble recombinant fragment having at least one O-glycosylation site, preferably a fragment accompanied by native O-glycosylation and its production, the novel PUmids containing these DNA sequences and their production (see, for example Fig. 18: Schemes of pFc _E R-1 (see also Fig. 17) and psFc _e R-1 [see also Fig. 19]).

A preferred O-glycosylated water-soluble Fc.R fragment contains amino acids 150 to 321 as shown in Table 3.

In a novel cDNA sequence according to the invention, the cDNA for at least part of the amino acids 1 to 148 of the Fc _c R, in particular the coding sequence for the N-th rminale cytoplasmic region is missing, so that, for example, only the coding sequence for the membrane-Umspanner ^ s part of the protein and r.s fraction coding for the water soluble part of the cDNA of the whole receptor.

In this novel cDNA sequence, at least a portion of the coding for the amino acids 1 to 148 cDNA Sequcnz by a suitable cDNA fragment encoding a eukaryotic signal sequence, exchanged by means of suitable restriction endonucloases and ligases.

For example, a plasmid containing a cDNA insert encoding Fc _e R is prepared by exchanging at least a portion of the

j coding sequence for amino acids 1 to 148, for example amino acids 1 to 134, by a eukaryotic cDNA

Signal sequence, eg an interleukin cDNA signal sequence, e.g. B. by the BSF-2 Sipnalsequenz modified. So wurdfi in the following example described a corresponding plasmid, z. B. plasmid LE 392 or pGEM 4 (pFc _e R-1) (see Fig. I 7),

described in CeI ¹ 47,659 (1986) digested with HindIII to obtain a HindIII fragment of I.Okbp containing the coding sequence for amino acids 134 to 321 of the complete Fc _c R cDNA. The recessed 3'-ends of this' fragment were then filled in with the Klenow fragment of the DNA and the DNA was Poiymerase anschlioße'id with PstI

digested. The recovered fragment was then in a: ι. suitable vector, preferably with a BamHI Pstldigerierten pBSF2-L8geclont. Dar vector is conveniently prepared as follows:

The EcoRI-BamHI 1.2kbp BSF-2 cDNA insert was prepared by digesting pBSF-2.38 (see Nature 324.73-7611986!) With HindIII and BamHI. The recovered fragment, which contained a full length of BSF-2 cDNA, was then digested and the recessed 3 'end was filled in with Klenow fragment of DNA polymerase. After the Kpnl

ι Digestion was the recovered fragment Kpnl Hinfl 1OObp containing the DSF-2 leader sequence in the multiple;

Cionierstelle pGEM 4, which had been previously digested with Kpnl and SmaI cloned. One of the selected clones was propagated and designated pBSF2-L8 (see Fig. 2u).

pBSF2-L8 was digested with BamHI and the recessed 3 'ends were filled in with Klenow fragment of DNA polymerase. After filling in the BamHI site, the above-mentioned HindIII-PstI Fc _e cDNA was cloned into pBSF'2-L8 digested BamHI-Pst-1, as previously mentioned. One of the selected clones was propagated and designated as psFc _c R-1 (see Figure 19).

To compare the biological activity of the proteins produced by clones pFc _c R-1, psFc _c R-1, pAnFc _c R-1 (Example C) and pANFc _c R-2 (Example D) (see Figure 18) Plasmids were linearized by digestion with suitable enzymes, for example pFc _c R-1 and psFc _c R-1 with BamHI and pANFc _e R-1 and pANFc _c R-2 with EcoRI, and the recovered fragments were used as template for the synthesis of mRNA with SP6 RNA polymerase used. In recovered mRNAs were injected into oocytes of Xenopus leavis. After a two-day incubation period, the Fc _t R activities in the culture supernatants and lysates of the oocytes were submitted by an enzyme-linked immunosorbent assay (ELISA) using AnUt-Fc ₄ R antibodies 3-5 and 8-30 (see EU-PA 86111488.2 on August 19, 1986), which recognize two different epitopes on Fc _t R. As shown in Figure 21, the Fc _e R activity was determined for the oocyte NP-40 lysate, oocyte PBS lysate, and oocyte culture supernatant. It can be seen that while in PBS lysate and culture supernatant no activity could be detected with transcripts of pFc _t R-1, pANFc _t R-1, and pAFc _t R-1 and pANFc _£ R-2 injected oocytes, Fc _e R activity was detected in the supernatants and PBS lysates after injection with fragments of psFc _t R-1. Further, by a modified ELISA method with Anit-FccR antibody 3-5, IgE and AP anti-human IgE, it was found that the Fc _c R water-soluble fragment secretion product from the oocytes has the IgE-binding property PsFCcR-1 mRNA-injected oocytes were incubated on S-δ antibody-coated plates, which were then incubated with human IgE and finally with AP-Anit-IgE. The results clearly indicated that the Fc _c R secreted by the oocytes formed a complex rr.lt IgE (see Figure 22). As a control, binding was performed with untransformed oocyte supernatant buffer and RPMI-8866 supernatant.

To assess the IgE-binding property of the soluble FCcR fragment recovered from oocytes injected with psFc _c R-1 mRNA, the oocyte supernatant was further tested for its ability to inhibit rosetting between ORBCs conjugated to human IgE and SKW6. CL4 cells bearing Fc _c R on their surface were coated. The presence of soluble Fc _c R reduced the number of rosetting cells to which more than 20 ORBCs were bound, indicating that the soluble receptor competes with the cell membrane Fc _c R for IgE bound to the surface of ORBC (see Figure 23).

The corresponding genes obtained according to the invention can be introduced into organisms under conditions which lead to high yields, as already mentioned above. Those skilled in the art will appreciate useful means and vectors, for example, refer to European Patent Publication 0093619 of November 9, 1983.

In general, prokaryotes are preferred for expression. For example, E. coli K12 strain 294 (ATCC No. 31446)

especially useful. Other vulnerable microbial strains are, for example, E. coli X1776 (ATCC No. 31537). The mentioned

ι strains and E. coli W3110 (F ", lambda", protoxic, ATCC No. 27325), bacilli such as Bacillus subtilus, and others

Enterobacteriaceae such as Salmonella typhimurium or Serratia marcenses and various Pseudomonas species can be used.

In general, plasmid vectors containing replicon and control sequences derived from species compatible with these host cells are used in conjunction with these hosts. The vector usually carries a replication site as well as marker sequences capable of selecting a phenotype in transformed cells! perform. For example. E. coli usually with pBR322, one derived from an Escherichia coli species

Plasmid (Bolivar, et al., Gene 2:95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance, providing a simple means of identifying transformed cells. The pBR322 plasmid or other microbial plasmid must also contain promoters that can be used by microwell organisms for expression, or these plasmids must be modified to contain these promoters.

The most commonly used promoters for the construction of recombinant DNA are the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 [19781; Itakura et al., Science 198, 10556 [1977]; Goeddel et al., Nature 281) , Π44 [1979]) as well as the tryptophan (trp) Promotoi system (Goeddel et al., Nucleic Acids Res.8,4057 [1980], EP-A-O036776). These are the most commonly used promoters, but other microbial promoters have been discovered and used.

For example, the genetic sequence for Fc _c R can be placed under the control of the leftward promoter bacteriophage lambda (Pi.). This promoter is one of the strongest known promoters that can be controlled. The control is exercised by the lambda repressor and adjacent restriction sites are known. A temperature sensitive allele of this repressor gene can be placed on the vector containing the complete Fc _c R sequences. If the temperature is increased to 42 ⁰ C, the repressor is inactivated, and the promoter is expressed on its Maximolwert. The mRNA generated under these conditions would be sufficient to give a cell containing about 10% of its fresh

contains synthesized RNA from the P _L promoter. According to this scheme, it is possible to construct a clone bank in which a functional Fc _t R sequence is adjacent to a ribosome binding sequence and at different distances from the lambda P _L promoter. These clones can be screened to select the one with the highest yield. i

The expression and translation of the Fc _t R-Seque, tz can also be placed under the control of other regulons which may be "homologous" to the organism in its untransformed state. For example, lactose-dependent E.colichromosomal DNA contains a lactose or lac -Operone, which effects the lodeotigestion by expression of the enzyme beta-galactosidase The Lac control factors can be obtained from bacteriophage lambda plaC5 which is infectious to E. coli The phage lac operon can be transduced from the same bacterial species Suitable regulons for the process according to the invention can be obtained from the plasmid's own plasmid DNA The Lac promoter-operator system can be induced by IPTG.

Other promoter-operator systems or parts thereof may also be used. For example, arabinose operator, colicin eroperator, galactose operator, alkali phosphatase operator, trp operator, xylose A operator, tac promoter, and the like can be used.

The genes are most advantageously expressed in Escherichia coli when the promoter-operator system of the plasmid pER 103 (see Rastl-Dworkin et al., Gene 21, 237-248 and EP-A-0115.613), deposited at the German Collection of Microorganisms, Grisebachs'rasse 8, D-3400 Göttingen on 27 October 1983 under DSM 2773 under the Budapest Treaty. A corresponding expression plasmid expressing, for example, the water-soluble portion of the low affinity human Fc _E receptor having amino acids 150 to 321 (see Table 3) can be prepared as follows:

A plasmid containing the mentioned prmotor-operator system, for example the plasmid pKh 100 (see Example B) was digested with SstI. Subsequently, the 3 'protruding ends were removed and the linearized plasmid was dephosphorylated. After purification, for example by phenol / chloroform extraction, electrophoresis, electroelution and precipitation, the linearized vector was ligated with an insert obtained as follows: A plasrnid containing portion of the low affinity human Fct receptor gene, for example, pGEM 4-Fc _c R, which is obtained by digestion of the EcorRI insert of plasmid LE392 with HindIII and re-isertion of the larger EcorRI / HindIII fragment in pGEM 4 (FIG. Promega Biotec, Madison, WI 53711, USA) was digested with EcoRI and HindIII. Then the!

5 'protruding ends by addition of Klenow fragment of DNA polymerase I smoothed, and all four dXTP's and the I

5'-phosphate groups were removed. After purification of the recovered fragment, this was reconstituted with Sau 3A

cut out (recut) and the larger fragment was isolated. Since this method not only the "upstream" 5 'region, j

but also removes the nucleotides for the first 18N-terminal amino acids of the desired water-soluble part, j

were two oligodeoxynucleotides of the formulas i

EBI-496:

5 'GAACTGCAGGTGAGCTCTGGTTTCGTTTGCAACACTTGCCCGGAAAAATG 3';

EBI-497:

3 'CTTGACGTCCACTCGAGACCAAAGCAAACGTTGTGAACGGGCCTTTTTAC CTAG 5'

Sau3A

synthesized to recover the corresponding codons of the following formula:;

GluLeuGlnValSerSerGlyPheValCysAsnThrCysProGluLysTrp

5 'GAACTGCAGGTGAGCTCTGGTTTCGTTTGCAACACTTGCCCGGAAAAATG 3'

3 'CTTGACGTCCACTCGAGACCAAAGCAAA ₁ CGTTGTGA-ACGGGCCTnTTAC CTAG 5'

Sow 3 A

(without the ATG codon, this codon is contained in the promoter / operator / linker system of the plasmid pER 103).

Each of the oligodeoxy nucleotides produced was annealed and ligated to the above-mentioned water-soluble Fc _c R fragment. After heat denaturation of the T4 DNA ligase used, T4 polynucleotide kinase and ATP were added to phosphorylate the 5 'ends of the DNA.

After cleaning the insert durcl. Agarose gel electrophoresis was ligated this inset with the linearized Vsktor DNA.

The recovered ligation solution was transformed into E. coli HB101 and some of the ampicillin-resistant colonies were isolated. These plasmids were examined for correctness of their construction by restriction enzyme analysis. A plasmid was selected and designated pRH 246 (see FIG. 16).

In addition to prokaryotes, eukaryotic microbes such as yeast cultures can also be used. Among the eukaryotic microorganisms, Saccharomyces cerevisiae is most commonly used, although a number of other species are commonly available. For expression in Saccharomyces, the following plasmids are commonly used: plasmid YRp7 (Stinchcomb, et al., Nature 282, 39 [1979]; Kingsman et al., Gene 7, 417 [1979]; Tschumper, et al., Gene 10,157 [1980]), plasmid YEp 13 (Bwach et al., Gene 8, 121-133 [1979]) and plasmid pJDB207 (see VDBDggs et al. In Gene cloning in yeast), ed. R. Williamson, Genetic Engineering Vol 2,175-203 (1981 !, Academic Press, London; deposited December 28, 1984 under DSM3181 at German Collection of Microorganisms, Grisebachstr. 8, D-3400 Gottingen, under the Budapest Treaty) YRp7 contains the gene TRP1, which is a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076. The presence of TRP 1 lesion as a characteristic of the yeast host cell genome then provides an efficient In the same way, the plasmid YEp 13 contains the yeast LEU 2, which can be used for the complementation of a LEU 2 minus mutant strain.

Suitable supportive sequences in yeast vectors include the 5 'flanking region of the genes for ADHI;

(Ammerer, G., Methods of Enzymology 101, 1992-201, 1933J), 3-phosphoglycerate kinase (Hitzemann, et al., I. Biol. Chem. 255, 2073 [1980]) or other glycolytic enzymes (Kawasaki and Fraenkel, BBRC 108, 1107- 1112 (1982)) such as enolase,

Glyceraldehyde-S-phosphate dehydrogenase, hexokinase, pyruvic acid, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase and glucokinase In the construction of suitable expression plasmids, the termination sequences associated with these genes also become this expression vector-3 Other promoters having the added advantage of growth-condition-controlled transcription are the promoter regions of the genes for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, with which Nitrogen metabolism related degradative enzymes, the aforementioned glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for maltose and galactose utilization, promoters regulated by the yeast "mating" site, such as the promoters of the BARI genes, MR-Alpha-1, STE 2, STE 3 and STE 5 can be used for a temperature-regulated system by means of temperature-dependent Siv mutations (Rhine, PhD, University of Oregon, Eugen, Oregon [1979], Herskowitz and Oshima in The Molecular Biology of the Yeast Saccharomyces ?, Part 1,181-209 [1981] , Cold Spring Harbor Laboratory). These mutations directly affect the expression of the silent "mating" cassettes of the yeast and therefore indirectly the "mating" dependent promoters.

In general, however, any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

The genes, preferably the water-soluble portion of the low affinity human Fc _c receptor, having amino acids 1SO to 321 (see Table 3) can best be expressed in yeast when the ADHI promoter is co-administered with the ADHI promoter. Terminator is used. For example, the preparation of a suitable yeast expression vector can be accomplished by preparing:

a) a plasmid containing the yeast ADHI terminator,

b) a plasmid containing the yeast ADHI promoter and the yeast ADHI terminator (see J. Biol. Chem. 257, 3018-3025 (19821),

c) a plasmid containing the yeast ADHI promoter, a gene coding for the yeast "mating" factor α Lesder peptide (MFa leader sequence) (see Cell 30, 933-943 [1982] ), a multicloning site and the yeast ADHI terminator,

d) a plasmid containing: the ADHI promoter, the coding sequence for the water-soluble portion of the low affinity human Fc _c receptor and the ADHI terminator,

e) a plasmid containing the yeast ADHI promoter, a yeast mating factor a leader gene, the water soluble portion of the low affinity human Fc _t receptor, a mulitcloning site and the yeast ADHI terminator, as well

f) transforming the recovered plasmid DNA into a suitable yeast vector, whereby a plasmid containing the expression cassette without the MFα leader sequence and a plasmid containing the MFa leader sequence expression cassette are recovered.

Description of the methods a to f:

a) The ADHI terminator may be isolated from a plasmid containing this promoter, for example from plasmid pJD14 (see I.L. Bennetzen et al., J. Biol. Chem. 257, 3018-3025 [1982]). The ADHI terminator was subcloned into plasmid pHC18 (Pharmacia P-1, No. 27-4949-01) as a HindIII Sphi fragment. During the subclone, the HindIII site was destroyed by a fill-in reaction, and after the addition of salt linker, the plasmid pWS214S4 was recovered. After digestion of pWS214S4 with Sphl and Sa 11, the smaller fragment was isolated by agarose gel electrophoresis, electroelution and precipitation by known methods.

The isolated fragment containing the ADHI terminator was ligated with vector DNA, preferably by digestion of Bluescribe M13 + (See Stratagene, San Diego, California 92121, USA) with Sa11 and Sphl and after purification of the recovered vector DNA by agarose gel. Electrophoresis, Elektroeluticn and precipitation was obtained by known methods. The recovered ligase solution was transformed into E. coli JM101 and the plasmid of one of the ampicillin-resistant colonies was designated pRG241 (see Fig. 10) which contained an approximately 340 bp fragment containing the ADHI terminator.

b) The ADHI terminator can be isolated from a plasmid containing this promoter, for example from a plasmid pY-JDB-HulFNOmega 1 (see Example A and DE-PA P 3635867.3, filed October 22, 1986) then introduced into a suitable vector, such as the plasmid pRH 241.

The required insert was prepared by digesting the plasmid pY-JDB-HulFN-omega 1 with Sphl with the 3 'end removed using E. coli DNA polymerase I in the presence of dGTP and recut with Xhol. After isolation of the fragments obtained by agarose gel electrophoresis, electroelution and precipitation by known methods, the blunt end of the 400 bp fragment was ligated by ligation with the adapter pair of the following formula

ΕΒΙ <410: 5 'AATTGGAAGGATC 3'

EBI-429: 3 'CCTTCCTAG-p 5 ¹

and the sticky end by ligation with the adapter pair of the following formula

EBI-418: 5 'P-TCGAGCCACGTGGTAC3'

EBI-424: 3 'CGTGCAC 5'

transformed.

The ligations were carried out simultaneously and after purification of the ligation product by agarose gel electrophoresis, it was ligated with a suitable linearized vector DNA, preferably with linearized vector DNA recovered by digesting the plasmid pRH241 with EcoRI and KpnI. The obtained ligase solution was transformed into E. coli, and the resulting colonies were examined by known methods for the presence of a plasmid with the correct construction. A plasmid designated plasmid pRH 242 (see Figure 11) was selected.

c) Following chemical synthesis of the yeast "mating" factor a-leader peptide (see J. Kurian et al., Cell 30, 933-943 [1982]), the insert was prepared following synthesis of the following oligodeoxynucleotides as follows:

Name sequence

MF1 TCGAGCCTCATATCAATGAGATTCCCATCTATTTTCACTGCTGTTTTGTT (50 mer)

MF2 AGCAGCGAACAAACAGCAGTGAAAATAGATGGGAATCTCATTGATATGAGGC (53 mer)

MF3 CGCTGCTTCCTCCGCTTTGGCTGCTCCAGTCAACACTACTACTGAAGACGAAACTGCTOAAATTCCAGCT (70mer)

MF4 CAGCTTCAGCTGGAATTTGAGCAGTTTCGTCTTCAGTAGTAGTGTTGACTGGAGCAGCCAAAGCGGAGGA (70mer)

MF 5 GAAGCTGTCATCGGTTACTCTGACTTGGAAGGTGACTTCGACGTTGCT (48 mer)

MF6 GCAAAACAGCAACGTCGAAGTCACCTTCCAAGTCAGAGTAACCGATGA {48 mer)

MF7 GTTTTGCCATTCTCCAACTCCACTAACAACGGTTTGT TCATTAACACTACTATTGCATCGATTGCT (69 mer)

MF8 CCTTAGCAGCAATCGATGCAATAGTAGTGTTAATGMCAACAAACCGTTGTTAGTGGAGTTGGAGAATG (69mer)

MF9 GCTAAGGAAGAAGGTGTTTCTTTGGACAAGAGGCCTCTGCAGGAATTCT (49mer)

MF10 CTAGAGAATTCCTGCAGAGGCCTCTTGTCCAAAGAAACACCTTCTT (46 mer)

Each of the oligonucleotides MF2 to MF9 was phosphorylated. Upon termination of the reactions by heat, the following mixtures were prepared: MF1 and MF2; MF3undMF4; MF5undMF6; MF7 and MF8 as well as MF9 and MF10. Then, after heating and cooling, the resulting five solutions were taken together and ligated. To this solution was added linearized vector DNA, which was preferably obtained by digestion of pRH 242 with Xhol and XbaI after purification by electrophoresis, electroelution and precipitation by known methods. This ligation solution was used to transform E.coli JM101, the plasmids of the resulting colonies were assayed by digestion with Xhol and XbaI, those plasmids containing an insert of about 290 bp (see Fig. 12) further subcloned by the Inserts in M13 mp8 and by sequencing according to the dideoxy chain termination method of Sanger (see Proc Natl Acad Sci., 74, 5463-5467, [1977]). A plasmid containing the correct insert was selected and designated pRH243 (see Figure 13).

d) The coding sequence for the water-soluble portion of the low affinity human Fc _c receptor was isolated from the plasmid pGEM4 by digestion with Hind III and EcoR I, in addition, the sticky ends were cloned with the Klenow fragment of E. coli DNA. Polymerase I and the four deoxynucleoside triphosphates filled. The larger fragment was dephosphorylated and purified by known methods.

Since HindIII cuts the Fc _c R cDNA in the 5'-direction (upstream) of the first amino acid, the insert is recut with Sau3A, since in this method not only the 5'-upstream region but also the 5'-region is cut Nucleotides for the first 18N-terminal amino acids of the water-soluble fragment, starting with amino acid 150 of the complete Fc _c receptor, were eliminated, two oligodeoxynucleotides of the following formulas

5 'TCGAGCTCATATACAATGG ATG GAA TTG CAA GTT TCC TCT GGT TTC GTT TGT AAC ACT TGT CCA GAA AAG TG

3 ¹ CGAGTATATGTTACC TAC CTT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT TTC AC C DAY

Sau3A

synthesized the complete gene by means of the yeast codon usage of the following formula

mead

5 'TCGAGCTCATATACA ATG 3' CGAGTATATGT TAC

XhoI VaI GTT CAA 155 Ser TCC AGG Ser TCT AGA GIY GGT CCA Phe TTC AAG VaI GTT CAA 160 Cys TGT ACA As η AAC TTG Thr ACT TGA Cys TGT ACA Pro CCA GGT 165 GIu GAA CTT GIu GAA CTT Leu GIn TTG CAA AAC GTT 3 '5' Lys AAG TTC Trp TG ACC DAY

Sau3A

restore.

The prepared oligodeoxynucleoiides were annealed, the S &sub3; A-EcoRI fragment was added and ligated by T4 DNA ligase and the resulting fragment was purified by electrophoresis, elec- trolysis and precipitation by known methods.

-15- 263 53β

This insert was ligated with a suitable linearized vector DNA, preferably with the larger fragment obtained by digestion of pRH242 with XbaI (fill) and XhoI and additional purification by known methods.

The obtained ligation solution was transformed into E. coli JM101, the plasmids of some of the resulting colonies were examined with various restriction enzymes by known methods. A plasmid containing the correct insert was selected and designated pRH 244 (see Figure 14).

e) The coding sequence for the water-soluble portion of the low affinity human Fc _c receptor was isolated from the plasmid pGEM 4-Fc _t R by digestion with Hind III and EcoR I, in addition the sticky ends were cleaved with the Klenow fragment of E . coli DNA polymerase I and the four deoxynucleotide triphosphates. The smaller fragment thus obtained was dephosphorylated and purified by known methods.

Since Hind III cuts the Fc _e R DNA about 60 bp upstream of the first amino acid, the insert is rescored with Sau 3 A. In this method, not only the 5 'upstream region, but also the nucleotides for the first 18N-terminal amino acids of the water-soluble fragment were eliminated, two were oligodeoxynucleotides of the following formulas

5 'ATGGAATTGCAAGTTTCCTCTGGTTTC ATG GAA TTG CAA GTT TCC TCT GGT TTC GTT TGT AAC ACT TGT CCA GAA AAG TG

3 'TACCTTAACGTTCAAAGGAGACCAAAG TAC CTT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT TTC AC C DAY 5' Sau3A

synthesized the complete gene by means of the yeast codpn usage of the formula

mead

5 'ATG 3' TAC

GIu Leu Gin VaI Ser Ser GIy Phe VaI Cys Asn Thr Cys Pro GIu GAA TTG CAA GTT TCC TCT GGT TTC GTT TGT AAC ACT TGT CCA GAA CTT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT

Lys Trp

MG TG 3 '

TTC ACC DAY 5 '

Sau3A

restore.

Both oligodeoxynucleotides were annealed, the Sau3A-EcoRI fragment was added and ligated by T4 DNA ligase and the resulting fragment was purified by electrophoresis, electroelution and precipitation by known methods.

This insert was ligated with a suitable linearized vector DNA, preferably with the larger fragment obtained by digesting plasmid pRH243 with EcoR I and Stu I and by additional purification by electrophoresis, electroelution and precipitation by known methods.

The obtained ligation solution was transformed into E. coli JM101. The plasmids of some of the resulting colonies were examined with various restriction enzymes by known methods. A plasmid containing the correct insert was selected and designated pRH 245 (see FIG. 15).

f) The expression cassettes of plasmids pRH244 and pRH245, consisting of ADHI promoter, MFa leader gene (pRH245 only), FccR-water-soluble gene and ADHI terminator, were isolated by digestion with Hind III and BamH I and with a yeast plasmid ligated, for example with a suitable vector DNA such as the plasmid pJDB207 or YEp 13. In addition to microorganisms, cell cultures derived from multicellular organisms can also be used as the host. In principle, any cell culture, whether from vertebral or invertebral cultures, can be used. However, the interest in vertebral cells is greatest and the reproduction of vertebral cells in culture (tissue culture) has become a routine task in recent years (Ticsue Culture, Academic Press, Kruse and Patterson, ed. [1973]). Examples of such useful host cell lines are VERO cells and HeLa cells, Chinese hamster ovary cell lines (CHO) and W 1-3b, BHK, COS-7 and MDCK cell lines. Expression vectors for these cell lines will typically contain (if necessary) an origin of replication, in front of the promoter gene to be excised, along with necessary ribosome binding sites, RNA splice sites, polyadenylation site and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by a viral genome. For example, the promoters commonly used are derived from Polyoma, Adenovirus 2, and most commonly Simian Virus 40 (SV40). The first and last promoters of SV40 are particularly useful because both are readily allowed to recover from the virus as a fragment that also contains the origin of replication of virus SV40 (Fiers et al., Nature 273, 1113 [1978]).

Smaller or larger SV-40 fragments may also be used, provided they contain the approximately 250 bp sequence extending from the Hind HI site to the Bgl I site in the vital origin of replication. Furthermore, it is also possible and often desirable to use promoter or control sequences normally associated with the desired gene sequence, provided that these control sequences are compatible with the host cell systems.

< An origin of replication may be due either to the construction of the vector that contains an exogenous ion like it

for example, derived from SV40 or other viral sources (eg, polyoma, adeno, VSV, BPV, etc.) or may be provided by the host cell's chromosome replication mechanism. When the vector is incorporated into the chromosome of the host cell, the latter is often sufficient. *)

However, it is best to use a Cloner vehicle (shuttle plasmid) that allows replication in both eukaryotes and prokaryotes. The ability of the plasmid to replicate in prokaryotes provides a convenient means of manipulating the DNA sequence and achieving high levels of plasmid DNA needed for transfection into mammalian cells. Such a shuttle plasmid contains both prokaryotic DNA structural principles and DNA sequences derived from Eurokaryotes. The prokaryotic part of the plasmid consists of an origin of replication, which is usually derived from the plasmid pBR 322 (Mulligan RC et al., Proc. Natl. Acad. Sei. USA 78, 2072-2076 [1981]), and a marker gene, which allows selection on an antibiotic-containing medium. The most commonly used genes for selection are those which confer resistance to either ampicillin, tetracycline or chloramphenicol (Mulligan R.C., et al., Proc. Natl. Anad., U.S.A., 1978, pp. 2072-2076 (19811).

The eukaryotic portion of the shuttle plasmid must contain an origin of replication that is typically derived from viral genomes, such as Simian 40 virus (Mulligan RC, et al., Proc. Natl. Acad., U.S.A., 78, 2072-2076 (U.S. 1981]) or bovine papilloma virus (DiMaio D. et al., Mol. Cell., Biol., 4,340-350 (1984)). Second, a selectable marker gene that allows for the shuttle plasmid-accepting cells is required under selective conditions This marker gene can be of either prokaryotic or eukaryotic origin (e.g., prokaryotic genes: gene gpt encoding xanthine-guanine phosphoribosyltransferase (Mulligan RC et al., Proc. Natl. Acad. See, USA 78, 2072-2076 (1981), Mulligan RC et al., Science 209, 1422 (1980)), gene neo, which encodes a bacterial phosphatase that induces resistance to neomycin derivative G418 (Southern P. et al., J. Mol Appl. Genet., 1,327 (1982 ), Scholer U. et al. in Cell 36, 1422 (19841), CAT gene encoding chloramphenicol acetyl transferase (Gorman C. in Mol. Cell Biol., 2, 1044 (1982)); eukaryotic genes: genes encoding thymidine kinase (Wigler M. et al., Cell 11, 233 (1977).) The third eukaryotic DNA structural principle, which allows the expression of a cloned gene of interest, is a promoter sequence which may be either constitutive or inducible (eg, constitutive promoter: Simian 40 Virus or Rous sarcoma virus (Mulligan RC et al., Science 209, 1422 [1980], Laimons L et al., Proc Natl Acad, U.S.A., 79, 6453, 1182]); Inducible Promoter: Viral Drug Tumor Virus Promoter in the Mouse (Chapman A, B. et al. In Mol. Cell. Biol. 3,1421-

f 1429, heat shock protein promoter (Pelham H. et al. in EMBO J. 1,1473 [1932]), metallothionein promoter (Mayo K. et al. in Cell

29.99 [1982], Karin M. et al. in Nature299, 1997 [1982]).

One way to obtain relatively large amounts of the soluble portion of the Fc _c receptor protein in higher eukaryotes) is to "anneal" the soluble portion of the Fc _c receptor gene to the SV-40 promoter (constitutively) and this hybrid gene into a plasmid I

to clone which contains the gene coding for the dihydrofolate reductase (dhfr). Under selective pressure, the number of '

dhfr gene and the adjacent DNA sequences up to a thousand times orhöht, whereby not only dio yield of the dhfr gene but ι also that of the soluble portion of the Fc receptor _c increases (EP-AO 093 619).

i) The prepared low affinity Humah Fc _c receptor prepared according to the invention, preferably;

water-soluble portion beginning at about amino acid 50 of the full-length Fc _c receptor and extending to about 150 is suitable for the treatment or prophylaxis of local and allergic reactions induced by IgE and may be incorporated into suitable pharmaceutical compositions such as solutions or sprays be incorporated. As :

Plasmids pANFc _c RI and pANFc _E R-2 are prepared as follows: The plasmid LE392 or pGEM4 (pFc _c R-1) (see FIG. 17) was digested with Hind III and EcoR I. The isolated cDNA fragment,!

starting with nucleotide 584 and shown in Figure 17 was cloned into a Hind III and EcoR I digested pGEM4. One i

the selected clone was propagated and designated pANFcc R-1. , ;

The abovementioned plasmid LE392 or pGEM4 (pFc _c R-1) was digested with EcoRI and a fragment covering the entire length of the

Fc _t R-cDNA of -1.7 Kbp was recovered. The recovered EcoPI fragment was then partially with Sau3A:

to remove the cDNA sequence coding for the putative cytoplasmic domain, e.g. For amino acids 1 to 23, and a cDNA fragment beginning with nucleotide 254 as shown in Fig. 17 was recovered, which was labeled with a '

palindromic 26mer linker of the formula

5'-GATCTGAGTCATGGTACCATGACTCa-S '

was ligated to restore an ATG start codon at the 5 'end and a Kpn I restriction site. The resulting ligated fragment was digested with Kpn I and cloned into Kpn I and EcoRI digested pGEM4. Only those clones were cut by collagen hybridization in which the region lacked the putative cytoplasmic domain. One clone was selected and propagated and named pANFc _t R-2.

The invention will be illustrated with reference to the following non-exhaustive examples with reference to the drawings. :

In the accompanying drawings show:

Fig. 1: derived from NP-40 detergent-solubilized RPMI-8066 cells and serum-free culture supernatants (0 0)

Fcc activity, cell lysate of culture supernatant (Δ Δ).

Figure 2: Immunoaffinity purified soluble Fc _c R. Immunoaffinity purified Fc ₁ R derived from RPMI-8866 culture supernatants was analyzed under non-reducing conditions by NaDodSO ₄ / PAGE. After electrophoresis, 4mm strips were cut, minced, and eluted overnight at room temperature in lysis buffer. The Fc _e R activity was assessed by an ELISA method using two specific monoclonal antibodies. Figure 3: Purification of water-soluble Fc _c R. Serum-free culture supernatants from RPMI-8866 cells were concentrated 200X on Amicon YM 10, sequentially pre-adsorbed onto BSA-Sepharose, transferrin-Sepharose, NMIg-Sepharose, followed by specific immunoaffinity chromatography on 3 -5-Sepharose. The eluate was applied to HPLC C-4 column and treated with:

a linear gradient of acetonitrile of 0-65%, containing 0.1% trifluoroacetic acid, eluent. The Fc _t R activity containing fractions are indicated by hatching.

Fig. 4: the purified water-soluble Fc _e receptor having a molecular weight of about 25 kd. The purified soluble Fc _c R was examined by SDS-PAGE analysis. A photograph of the silver-colored gel is shown. i

Fig. 5: the peptide map of the water-soluble Fc _c R after lysyl endopeptidase digestion obtained after extensive pre-adsorption immunoaffinity chromatography and HPLC. ;

FIg.6: the FACS analysis of l-cell transformants. The two independent L-strain transformants, LV-8-30 (A) and L-VI-8-30 (B), were challenged with biotinized control antibody-human IgG (a, d), human IgE (b, e) and anti-Fc _e R 8-30 (c, f) stained and developed with FITC-avidin. Uncolored cells had the same pattern as those stained with control antibodies (a and d), the x-axis and the y-axis represent log fluorescence strength and relative cell number, respectively. Figure 7: cDNA cloning strategy

Probe I Cellular DNA from RPMI 8866

Probe II culture supernatant from RPMI 8866

Tranefektion J Cleaning

Ltk "cells purified soluble

I FACS sorting I

-Traneformant j L-ZeIlen

Aminosäureneequenzen

cDNA mRNA ^

Synthetic Oligonucleotides (17mer)

Tranformantenspezifische cDNA

\ l

RPMI 8866-i> mRNA-> cells

cDNA library in il, gt10

Koloniehybridieeierung

cDNA clone for

Rg.8: the EcoRI insert in plasmid pDE2, referred to as pDE2-Fc _e R-1 vector.

8 ': the expression of Fc _t R cDNA in transfected Cos-7 cells. The transfected Cos-7 cells were stained with phycocyanin-conjugated anti-FctR antibody (3-5) and biotinized IgE, developed with FITC-avidin, and analyzed by double laser FACS; a) cells transfected with human IFN-β cDNA-containing pDE 2; b) cells transfected with pDE-2 Fc _E R-1. Contour pictures represent the correlated expresspricr. of "White surface determinants, by showing pealines containing the same percentage of cells, with the two parameter vertices, put x-axis and y-axis

the green and red log fluorescence strengths, respectively.

Figure 9: the EcoRI insert in plasmid pGEM-4.

Figure 9 ': the expression of an Fc _c R cDNA in Xenopus oocytes. The oocytes injected with mRNA transcript from pFc _t R-1, control mRNA or mRNA from 8866 cells were incubated for 2 days and lysed. The Fc _c R values in the lysates were determined by

ELISA measured.

Fig. 14-16: 1 - soluble fragment "2, Klenow filling Fig. 17: 1 scheme of pcFcJM Fig. 19: 1-amino acids Fig. 19: Scheme of psFc _c R-1 Fig. 20: Scheme of pBSF2-L8

Fig. 21: Xenopus oocytes were injected with mRNA transcripts of pFc _£ R-1, ρΔΝ-FCtR-i, pAN-Fc _E R-2 and psFccR-1. After a two-day incubation period, FCjR activity in PBS lysate, NP-40 lysate and culture supernatant was determined by an ELISA method using anti-FceR antibodies 3-5 and 8-30.

FIG. 22: the IgE binding of the soluble Fc _c R derived from oocytes injected with psFc _t R-1 mRNA. The culture supernatant of these oocytes was incubated on the antibody 3-5 coated plate, it was closed human IgE and finally AP anti-IgE

Fig.23: the inhibition of IgE rosetting. Supernatants or control supernatants from oocytes injected with psFc _t R-1 mRNA were probed with human IgE-coated ORBC and SKW-C14 cells (Exp. II) and RPMI-8866 cells (Exp. I and III). incubated. X axis and y axis represent the number of OFtBC bound to cells or the relative number of cells

rosette-forming lines.

Fig. 24: SDSA-PAGE analysis of the NP-40 lysates and culture supernatants of the oocyte exposition of pFc _t R-1, pAN-Fc _c R-1, pAN-Fc _c R-2 and psFc _t R-1. The molecular mass marker is shown on the left.

General materials and methods:

Monoclonal anti-FCcR antibodies 3-5 (γ, γ) and 8-30 (μ) were isolated by hybridization of P3U1 myeloma with spleen cells from Ba! B / c mouse monone to RPMI-8866 cells (see EU). No. 86110420.6 of the same applicant, filed on July 29

1986). Antibody 8-30 recognizes the epitope at the IgE-binding site of Fc _c R and may block the binding of IgE to 8866-type lymphoblastic cells. Antibody 3-5 recognizes another epitope on Fc _c R and can not effectively block IgE binding to its receptors. These antibodies precipitate 46 kd and 25 kd polypeptides under reducing and non-reducing conditions. The monoclonal antibodies were purified from the ascitic fluid by 50% saturated ammonium sulfate precipitation followed by gel filtration using Sepharose 6B (Pharmacia Fine Chemical, Uppsala, Sweden) for IgM class or ion exchange chromatography using QAE-Sephadex (Pharmacia Fine Chemical). for IgG 1. The polyclonal Mauc IgG was isolated in the same way. The anti-mouse IgM alkaline phosphatase conjugate was obtained from Tago (Burlingame, California).

Reverse phase HPLC was performed using a Waters HPLC system with a Hi-Pore, RP-304 (250 χ 4.6 mm) column (Bio-Rad). The immunoaffinity-purified Fc _c R was added to the reverse phase column equilibrated with water containing 0.1% trifluoroacetic acid and eluted with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid (TFA). A flow rate of 0.5 ml / min and a linear gradient (from 0% to 65% in 60 minutes) of acetonitrile was used. The eluted material was frozen and lyophilized before its activity was tested by ELISA.

NaDodSCVPAGE

Crude, immunoaffinity-purified and HPLC-purified fractions of Fc _c R were analyzed by electrophoresis on a 1%

NaDodSO ₄ -10% polyacrylamide gel (Figure ₄ ) was analyzed and the proteins were determined by silver staining using j

Daiichikagaku silver stain (Daiichi-Kogaku, Tokyo). To measure the Fc _c R activity, the gel was electrophoresed in

Cut 4 mm thick slices, size-reduced with lyse puff * eluted overnight at room temperature with shaking, this! eluiei te material was collected after centrifugation and tested for activity by ELISA. <The enzyme reactions were carried out by standard protocols (see Molecular cloning - a laboratory manual, (Molecular cloning, - a manual for the laboratory), T. Maniatis et al. (1982), ed. Cold Spring Harbor or according to the instructions of the supplier J. The restriction maps The described plasmids and reaction schemes are not drawn to scale The oligodeoxynucleotides were synthesized using an Applied Biosystem üNA Synthesizer Model 381A I and polyacrylamide gel electrophoresis (12% acrylamide, 0.6% bisacrylamide, 8M urea, 1 x TBE Buffer) elution and desalting using a Sephadex G-25 column j

Example A!

Preparation of plasmid pY-JDB-HulFN-Omega 1 j

a) Preparation of the H6fe ADHI Promoter Fragment j 80pg of the plasmid pES 103 ϊη'δΟΟμΙ were digested with Bamhi and Xhol. The about 1450 base pair (bp) promoter fragment is separated on a 1% agarose gel from the vector, isolated by electroelution from the gel and precipitated. The fragment was suspended in ϊη40μΙ TE buffer (10 mM Tris, 1 mM EDTA, pH 8). The pES 103 used was prepared by insertion of the approximately 1450 bp BamHI-Xhol fragment of plasmid AXa11 (see G. Ammerer in Methods Enzymology 101, 1992-201 [19831 ) in pUC18 (see Pharmacia PL, No. 27-4949-01).

b) Preparation of the vector pJDB 207

10 μg of pJDB 207 in 100 μl solution are cut with BamH I and linearized. To inhibit re-ligation, the 5'-terminal phosphate groups are removed by calf intestinal phosphatase (CIP) treatment. The linear form is from:

remaining undigested plasmid on a 1% agarose gel, isolated by electroelution and precipitated. The precipitated vector DNA is dissolved in 20 μM TE buffer.

c) expression vector for IFN-omega 1

50μg of the plasmid pRHW12 (see EP-A-0.170.204) in 600μΙ solution are linearized by cleavage with HindIII. The resulting staggered ends are transformed into blunt ends by addition of Klenow fragment of DNA polymerase 1 (10 units) and 25 μΜ each of the four deoxynucleoside triphosphates and by incubation at room temperature. The linear form was purified by electrophoresis on agarose gel and subsequent isolation. The fragment was suspended in 50μΙ TE buffer.

To obtain ends compatible with the promoter fragment, an Xhol linker was added to the ends of the linear pRHW12. 3μΙ Xhol linkers (0.06 OD _2Mnm , Pharmacia PL, No.27-7758-01, Formula d [OCTCGAGG]) in 20μΙ solution are assayed using

5 units of T4 polynucleotide kinase and ιΑΤΡ phosphorylated. After inactivation of the enzyme by heating to 70 ° C for 10 minutes 5μΙ of this solution are combined with 10μΙ of linear pRHWi2 and ligated in a total of 20μΙ reaction solvent T4 DNA ligase (16 hours at 14 ⁰ C). The ligase is then inactivated by heating to 70 ⁰ C for 10 minutes and the reaction volume is made up to 150μΙ with 1 χ medium buffer (1 OmM Tris, pH 7,5,5OmMNaCI, 1OmM MgCl ₂ ). The Xhol-specific, tacky 5 'ends are generated by treatment with 100 units of xhol. The linear pRHW12 with Xhol ends is purified by electrophoresis on agarose gel and electroeluted from the gel. Before precipitation, 5μΙ promoter fragment [from part al) is added. After precipitation, the DNA is suspended in 14.5μΙ TE buffer, ligase buffer and 5 units of T4 DNA ligase are added and ligation is performed at 14 ° C for 16 hours. After enzyme inactivation, the volume is made up to 200 μΙ and the DNA is cleaved with the aid of BamHI. The DNA is recovered by purification on agarose gel and dissolved in 20 μM TE buffer.

d) Ligation of the fragments

The final expression vector is prepared by treating 5 μΙ of the BamH I fragments (promoter and IFN-omega 1 gene) with 1 μΙ of the linearized pJDB 207 vector (yeast 2 μ terminator and yeast 2 origin of replication, Leu 2 marker, E .coli origin of replication, ampicillin resistance gene) was recovered in 10μΙ solution in the presence of 1 unit of T4 DNA ligase.

e) transformation

10μΙ of competent E. coli HB101 cells were transformed by addition of 5μΙ ligase solution and added to LB agar containing 100 μg / ml ampicillin. 1 ? the resulting clones were selected and the plasmids were isolated. After cutting the plasmids with various restriction enzymes and electrophoresed on agarose gel, a plasmid was selected which had the correct structure; it was named pY-JDB-HUIFN-Omega 1.

Example B

Construction of the Expression Plasmid pRH 100

7pg of the plasmid pER 103 (see Eva Dworkin-Rastl et al., Gene 21,237-248 [1983] and EP-A-0115,613) were linearized in 50μΙ the reaction medium with the restriction endonuclease Hind III. After incubation at 37 ° C. for ¹ hour, 50 μl of 2 × CIP buffer were added (2 × CIP buffer = 20 mM Tris, pH = 0.2 mM EDTA). After the addition of 2 units of calf intestinal alkaline phosphatase (CIP), the 5'-terminal phosphate residues were removed; incubation was for 30 minutes at 4 ~. The reaction was terminated by the addition of 4μΙ 0.5 EDTA solution and the addition of 10μ11M Tris, pH = 8.0. The proteins were removed by extraction twice with phenol and once with phenol / chloroform. The DNA was precipitated from the aqueous phase after the addition of 0.1% by volume of 3 M sodium acetate solution pH 5.5 and 250 μl of ethanol, and the DNA precipitate was washed once with 70% strength ethanol solution after centrifuging. The DNA was dried and the pellet was then dissolved in 20 MlTE buffer (10 mM Tris, pH = 8.01 mM EDTA).

Ι-μΙ portions of the synthetic oligodeoxynucleotides d (AGCTTAAAGATGAGCT) and d (CATCTTTA) were phosphorylated in 10 μΙ of the reaction solution with addition of 10 units of T4-PNK (polynucleotide kinase) and 1 mM rATP. The reaction took place at 37 ° C and lasted 45 minutes. The reaction was stopped by heating to 70 ⁰ C for zmin minute.

5μΙ of the plasmid solution and the phosphorylated oligonucleotide were mixed together and heated to 70 ° C for 5 minutes. The solution was then cooled to ⁰ ° C. and 2 μl of 1 × 10 1 ligase buffer (500 mM Tris, pH = 7.5), 10 mM MgCl ₂ ,.

20 mM DDT (dithiothreitol), 1 mM rATP, 500 μg / ml BSA (bovine serum albumin), 2 μl water and 10 units T4 DNA ligase were added. The reaction lasted 40 hours and was carried out at 4 ° C. It was stopped by heating at 70 ° C for ten minutes.

2μΙ this ligase reaction were (New England Biolabs) digested at 37 ⁰ C for 3 hours in total 30μΙ solution with 10 units of restriction endonuclease SacI. The reaction was stopped by ten-minute heating at 70 C ^9. 5μΙ of this reaction mixture was ligated in a total of 30μΙ by addition of 10 units of T4-PNK for 16 hours at 14 ° C.

200 μl of competent E. coli HB101 were mixed with 10 μl of this ligase reaction. The bacteria were kept on ice for 45 minutes and then heated to 42 ^° C. for 2 minutes for DNA uptake. The bacterial suspension was further incubated at ⁰ ° C. for 10 minutes. Finally, the transformed bacteria were incubated on LB agar containing 50 μl. Ampicillin.

Twelve of the bacterial colonies were randomly selected and the flaskides thereof were subsequently isolated on a romatic scale (see Birnboim et al., Nucl. Acids Res. 7, 1513-1523 [1979]). The resulting DNA was cut with the restriction endonuclease SacI and the DNA was separated on an agarose gel (1%, 1 χ TBE buffer). The migration of DNA as a linear 4,400 pb molecule actuated that a SacI recognition site had been incorporated into the plasmid One of these plasmids was randomly selected E. coli HB101 was re-transformed with the DNA from the corresponding mini-preparation From the resulting transformed bacteria, a colony was selected which was grown to a larger scale, and the plasmid isolated therefrom was cut with the restriction endonucleases EcoR I and BamH 1, the DNA was separated on a 1% agarose gel, and the smaller Fragment was isolated by electroelution from dom gel This EcoRI-BamHI DNA fragment of about 460 bp length was prepared according to Sanger (see Sanger et al., Proc. Natl. Acad.

Sequence 74, 4663-5467 (19771). The plasmid thus analyzed was designated pRH100.

Examples

Construction of the plasmid pANFc _E R-1

10 μg of pFcJM DNA were incubated with 20 units of HindIII in 50 μl of medium salt buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM MgCl ₂ , 1 mM DDT) for 1 hour and then with 20 units of EcoR I in 100 μl of a high saline buffer (10 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1 mM MgCl ₂ , 1 mM DDT) for 1 hour to recover the HindIII-EcoRI fragment containing a 1 kbp region of the soluble Fc _c R cDNA (see Fig. 17: start nucleotide 584). The digested DNA was subjected to 1% preparative agarose electrophoresis and the approximately 1 kbp Hind HI-EcoRI fragments were electroeluted from minced gels and in 70% ethanol at -80 ⁰ C for 1 hour precipitated long. 1 \ ig pGEM4 (Promega Biotec) as described above with 2 units of Hind III and 2 units of EcoR I digested, phenol extracted and precipitated for 1 hour at -8O ⁰ C with ethanol. The pGEM4 digested by HindIII-EcoRI fragment and HindIII-EcoRI was digested with 200 units / T4 ligase in 10 μΙ of a ligation buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM MgCl ₂ , 10 mM DDT, 1 mM spermidine, 1 mM ATP, 0.1 mg / ml BSA) was incubated for 16 hours at 4 ° C. and transfected into E. coli (MC 1065). One clone was solubilized, propagated, and named pANFc _E R-1 after its construction.

Example D

Construction of the plasmid pANFc _E R-2

200 ^ g pFc _e R-1 were digested with 400 units of EcoR I in 200μΙ of a high salt buffer for 2 hours at 37 ⁰ C and then were electrophoresed on a 1% preparative agarose gel. An approximately 1.7 kb EcoRI fragment was electroeluted and precipitated long C for 1 hour at -80 ⁰ with ethanol. 4μς of the fragment were digested at 37 ⁰ C for 3 hours with 10 units Accl in 40μΙ a little salt-containing buffer and partially digested at 37 ⁰ C for 20 minutes with 0.4 units Sau3A, extracted with phenol and ethanol precipitated to sow 3 A-EcoR I fragment (see FIG. 17: start nucleotide

254). The DNA fragment was ligated to 1.7 μg of synthetic linker (5'-GATCTGAGTCATGGTACCATGACTCAGATCGTGCTG-S ') with 200 units of T4 / ligase in 10 μl of a ligation buffer for 16 hours at 4 ° C, then extracted with phenol and precipitated with ethanol. The fragments were dissolved, digested 3 hours at 37 ⁰ C with 24 units Kpn I in 40μΙ a little salt-containing buffer, extracted with phenol and precipitated with ethanol. The excess linkers were removed by gel chromatography with a Biogel A50m column. The fragments were precipitated with ethanol.

Separately, 1 ug pGEM 4 were digested with 8 units of Kpn I in 20 .mu.l of a little salt-containing buffer for 2 hours at 37 ⁰ C and then incubated with 8 units of EcoRI in 40 .mu.l of a high saline buffer for 2 hours at 37 ⁰ C incubated.

The previously ligated to synthetic linker fragments were then ligated for 16 hours by incubating with 200 units of T4 ligase in 10μΙ a ligation buffer at 4 ⁰ C and transfected into E. coli (MC 1065) in digested by Kpn 1-EcoR I pGEM. 4 By colony hybridization, the clone pANFc _c R-2, which lacks only one cytoplasmic domain, was isolated and propagated.

Example 1 j

a) Isolation of crude Fc _t R from the culture supernatant. RPMI-8866 cells were incubated in RPMI 1640 medium supplemented with 10% fetal CSF serum and antibiotics (100 units / ml of penicillin i and 100pg / ml streptomycin) Density of 1x10 * cells / ml and a cell viability of 95-99% in] Spinner bottles cultivated. The cells were harvested by centrifugation at 15minutiger 5000UmIn ^'1, washed three times with Hanks BSS j and cultured for 48 hours at the same density in serum-free medium in spinner flasks. Culture supernatant was collected and supplemented with phenylmethylsulfonyl fluoride (PMSF) (1mM), 0.02% sodium azide (NaN ₃ ), and 200 J units / ml Aprotenin. The culture supernatants were kept at 4 ° C during concentration. Concentration of the RMPI-0866 culture supernatants was done with an Amioon filter system using a DIAFLO-YM10-j filter. The 200 to 300 times concentrated material was then centrifuged for 40 minutes at 4 ° C and 85,000 χ G to remove the soluble material to recover the crude FceR preparation. <

b) Isolation of Fc _c R from Cell Lysates RPMI 8866 cells (2 × 10 ⁹ cells) were washed four times with PBS and dissolved in 10 ml lysis buffer (PBS containing 0.5% nonindot P-40 [NP-40] i) and lysed 1 mM PMSF for 45 minutes on ice with regular vortexing. An additional 10 ml lysis buffer j was added and lysis was continued for an additional 30 minutes. The lysate was "centrifuged for 45 minutes at 37000U min ¹ and 4 ° C." The lipid layer was sorgfältip removed and the supernatant was collected and stored at -7O ⁰ C.

Example 2 Immunoaffinit & tsreinigung

Culture supernatant concentrate (see Example 1 a), corresponding to 5 to 10 liters of culture, was sequentially gated on 2 ml of BSA-Sepharose gels, human transferrin Sepharose gels and normal mouse Ig (NMIg) Sepharose gels for one hour absorbed at 4 ° C under rotation. The effluent collected from the last immunoaffinity gel was then added to 2 ml of anti-Fc _e R (3-5) coupled to Sepharose. The immunoadsorption was allowed to proceed at 4 ° C with rotation between 4 and 16 hours. The gel was poured into a column, the effluent was collected, and the gel was sequentially washed with 150 ml of Buffer A (Tris-HCl, 10 mM, pH 7.5, NaCl, 0.5M, NP-40, 0, 5%), 160 ml Buffer B (Tris-HCl, 10 mM, pHV, 5, NP-40.0.1%), 150 ml Buffer C (Tris-HCl, 10 mM, pH 7.5) and washed with 25 ml 2.5% acetic acid (v / v). eluted and immediately neutralized in Tris-HCl, 2M, pH 8.0.

The material was, if it was not immediately further purified by HPLC, stored at -8O ⁰ C.

The eluate was then fractionated by reverse phase HPLC on a C4 column at a linear gradient of 0-65% acetonitrile containing 0.1% trifluoroacetic acid.

Example 3 Enzyme bound immunoadsorbent assay (ELISA)

The activity of Fc _c R was measured by its ability to bind to monoclonal antibodies 3-5 and 8-30 and monitored by an enzyme-linked immunosorbent assay with two antibodies (ELISA). 96-well microtiter plates were first coated with monoclonal antibody 3-5 (ΙΟΟμΙ / well) (10pg / ml) in coating buffer (NaHCO ₃ 0.1 M, pH 9.6) and incubated overnight at 4 ° C. The flatten are then washed four times with rinsing buffer,

ie, Dulbecco's, phosphate buffer pH with 0.05% Tween 20, and then 100 μΙ of test sample diluted with dilution buffer (Tris-HCl 0.05M, pH 8.1, MgCl ₂ 1 mM, NaCl 0.15M, Tween 20 0.05 % IV / VI, NaN ₃ 0.02%, BSA 1%) was added. The microtiter plates were incubated for 2 hours at room temperature, washed four times with rinse buffer, and then 100 μΙ of pretreated and diluted goat anti-mouse IgM alkaline phosphatase conjugate was added. The plates were incubated for two hours at room temperature and washed four times with rinse buffer. In the final step, 100 μM of substrate, p-phenylphosphate disodium (1 mg / ml) in substrate buffer (NaHCO ₃ 0.05M, pH 9.8, MgCl ₂ .6H ₂ .0.1mM) was added and the colorimetric reaction was repeated for ₂ hours Measured at 405 and 620nm for 30 minutes.

Example 4 Hydrolysis of the Fc _c receptor by lysyl endopeptidase and separation of peptides

The purified Fc _c R was in 5OmM Tris-HCl buffer, pH, 9,5,6 hours at 35 ⁰ C at an enzyme-substrate ratio of 1: 500 was digested (M / M) with Achromobacter lyticus. The lyophilized peptide fragments were purified by HPLC.

Separation of peptides by reverse phase HPLC

The peptides were separated by HPLC using a C4 column on a Hitachi 655 liquid chromatograph equipped with a 655-60 gradient processing section and a 100 μΙ sample loop Rheodyne autosampler. Elution of peptides was performed with a linear gradient of 2-propanol: acetonitrile = 7: 3 (v / v) of 0-35% in one hour and in 0.1% trifluoroacetic acid at a flow rate of 1.0 ml / min. The fractionated peptides were collected manually by observing the optical density at 215nm.

Amino acid analysis and sequence determination

The amino acid analyzes were performed with an amino acid analyzer Hitachi 835-5 after hydrolysis of the peptide fragments in 5.7HCl at 110 ° C in evacuated, sealed tubes for 22-24 hours. Automated Edman degradation was performed with a 4 ?0A protein sequencer (Applied Biosystems, Inc. California) using a standard sequencing program. The phenylthiohydantoin (PTH) amino acids are determined by reverse phase HPLC with isooratic elution (see Tsunasawa et al., J. Biochem., 97, 701-704 (19851).

The following amino acid sequences were detected.

Met-Glu-Leu-Gln-Val-Ser-Ser-Gly-Phe-Val,

Gly-Glu-Phe-Ile-Trp-Val-Asp-Gly-Ser-His-Val-Asp-Tyr-Ser-Asn-Trp-Ala-Pro-Gly-Glu-Pro-Thr,

Lys-His-Ala-Ser-His-Thr-Gly-Ser-Trp-lle-Gly-Leu-Arg-Asn-Leu-Asp-Leu-Lys-and

Lys-Trp-Ile-Asn-Phe-Gln-.

Example 5

Preparation of Oligoniicteotids the formula

3'-TtJ ACC TAG TT ^ AAq GT-5

The oligonucleotide was synthesized with an ADI synthesizer at the 0.2 pmole level.

The resulting oligonucleotide was demethylated with thiophenol / triethylamine / dioxane (1: 2: 2) at room temperature within 90 minutes and methanol (10 χ 1 ml of methanol) was washed.

After removal of the demethylated oligonucleotide from the controlled porosity glass (CPG) and cleavage of the.

Concentrated ammonia protecting group at room temperature for 1 hour and at 5B ⁹ C for 16 hours, the oligonucleotide was dried using Speedvac®.

Further purification was carried out over 20% acrylamide / 8 moles of urea gel with TBE buffer (10.9 g tris [hydroxymethyl] aminomethane, 5.5 g boric acid and 9.3 g ethylenedinitrilo-tetraacetic acid disodium salt in 11).

The subsequent gel electrophoresis (40 cm χ 25 χ 0.1 cm) was performed at 50 watts. The 17-mer band was excised, eluted with water and desalted in water on a 0.9 x 13 cm Sephadex G-25 medium column.

The fractions containing 17-mer were taken together and dried.

Yield: 7.7 OD ₂₆₀ (~ 285ug).

Examples

Set up Fc ₁ R ⁺ L cell transformants

One million Uk cells were transfected with 150 ng herpes simplex virus derived from tk gene and 20 pg high molecular weight genomic DNA from RPMI-8866 cells (see Wigler et al., Cell 16,777-785 (1978) co After the cells had been stored in HAT medium for 10 days, they were sslidated to HAT-resistant L cells were collected with biotinized anti-Fc _e R (8-30) and fluorescein isothiocyanate (FITC). conjugated d avidin stained and sorted by FACS For each transfection several sorting cycles were performed, two transformants, LV-8-90 and L-VI-8-30, had become independent transfections.

Example 7

Cloning of Fc _e R cDNA

a. Probe I: Total RNA was isolated from the Fc _t R ⁺ L cell transformants LV-8-30 by the guanidium / cesium chloride method (see Chirgwin et al., Biochemistry 18, 5294-5299 [1979]). Poly (a) ⁺ RNA was prepared by oligo-dT cellulose chromatography. Radioactively labeled cDNA was synthesized from poly (A) ^v RNA using ³² P-dCTT (see Davis et al., InProc Natl Acad., U.S.A. 81,2194-2198 [1984]). The labeled cDNA was annealed with an excess of poly (A) ⁴ RNA from untransformed Uk cells and applied to a hydroxyapatite column. The single-stranded cDNA was collected and used as probe I.

b. Probe II: The 17-mer oligonucleotides complementary to the mRNA sequence encoding the amino acid fragment Lys-Trp-Ile-Asn-Phe-Gln containing a mixture of 24 possible sequences were probed with γ- ³² Ρ-ΑΤΡ radiolabeled by T4 polynucleotide kinase.

Double-stranded cDNA was synthesized from poly (A) ⁺ RNA derived from RPMI-8866 cells using the Amersham cDNA Synthesis System (Amersham, UK) (see Gubler and Hoffman in Gene 25, 263-269 [1983]). After treatment with EcoRI methylase and T4 DNA polymerase, the double-stranded cDNA, which was longer than 1000 bp, was cloned by EcoRI linker into the EcoRI site of Xgt 10 and packaged by Gigapsck in vitro (vector cloning systems). Two sets of replica filters of phage plaques were made. One set was hybridized with ^M P-labeled, 17-base oligonucleotides (probe II) (see Wood et al., Proc. Natl. Acad., USA 83, 8585-1588 [1985]). Another set of filters was hybridized with ^w P-labeled subtracted cDNA specific for Fc _£ R positive L cells (probe I) overnight in 6 χ SSC at 68 ° C and with 0.1 χ SSC and Wash 0.1% SDS for 2 hours. The plaques that hybridized with both probes were isolated.

Example 8

Expression of Fc _c RcDNA

a) For expression of Fc _t R cDNA in pGEM4 by SP6 promoter mRNA with SP6 RNA polymerase using BamHI digested pFc _e R-1 as template (see Melton et al., Nucl. Acids. Res. 12, 7035-7056 [19841]. 10ng of mRNA were injected into an oocyte and as a negative control, murine BSF-1 mRNA was similarly prepared and injected into another oocyte.

After two days of incubation in Barth nutrient medium at 20 ° C, the oocytes were lysed in 50μΙ lysis buffer (10 mM Tris-HCl, pH 7.5, 0.5M NaCl, 0.5% NP40). The oocyte lysates were assayed for Fc _e R activity by ELISA method consisting of a "sandwich" method and using two anti-Fc _c R antibodies, 3-5 and 8-30 as a positive control concentrated culture supernatant from RPMI-8866 cells.

b) For expression of Fc _c R cDNA in Cos-7 cells, the insert of pFc _c R-1 was cloned into the EcoRI site of the pDE2 vector (see JA Patent Publication 1986/88879 of May 7, 1986). Cos 7 cells (5 x 10 ⁵ ) were seeded on 60 mm plates one day before transfection. The transfection was performed with 2 μl of plasmid DNA in 1 ml of 25 mM Tris-HCl (pH 7.5), 137 mM NaCl, 5 mM KCl, 0.6 mM Na ₂ HPO ₄ , C, 5 mM MgCl ₂ , 0.7 mM CaCl ₂ and 500 \ ig DEAE-dextran (Pharmacia Fine Chemical) performed. After one hour incubation at 37 "C the solution with DMEM containing 10% ^p CS and 150μΜ chloroquine was exchanged, incubated for 3 hours at 37 ⁰ C and exchanged then containing 10% FCS fresh DMEM. 48 hours later, the cells were incubated with Phycocyanin-conjugated anti-Fc _t R antibody (3-5) and biotinized IgE stained, developed with FITC-avidin and by double-l.aser-FACS (see Kikutani et al., J. Exp. Med. In press [1936]). ) developed.

Example 9

Determination of the nucleotide sequence of the Fc _c -RcDNA

The insert of pFc _c R-1 was digested with HindIII and Pvuii restriction enzymes. The digested DNA was subcloned into M13

and sequenced (see Sanger et al in Proc Natl Acad, U.S.A., 74, 5463-5467) and confirmed by the method of Maxam and Gilbert (see Proc Natl Acad., U.S.A., 74, 560-564 [1977]. ).

Example 10

, Northern blof-ant. Lysis of Fc _t R mRNA

3 micrograms of poly (A) ⁺ RNA from different cells were separated on a 1% agarose gel, transferred to nitrocellulose paper and incubated with a pFc, R-1 insert from "nick" translation (see Thomas et al., Proc. Natl. Acad. U.S.A. 77, 5201-5205 [19801] For BSF-1 induction, one hundred million B or T cells were Λ or 10pg / ml IgE and 50μ / ιηΙ recombinant human BSF-1 for 24 hours cultivated (see Yokota et al in Proc. Natl. Acad., USA in Press [1986]) and 10pg of total RNA were extracted and analyzed by Northern blotting as described above.

Example 11 Expression of the water-soluble portion of human low-affinity Fc _t receptor in yeast

Part One:

Preparation of plasmids pJDB-244 and pJDB-245

a) Preparation of the plasmid pRH 241 containing the yeast ADHI terminator: vector preparation

10pg BluescribeM 13 ⁺ (Strategists, San Diego, California 92121, USA) were double-digested with 20 units each of Sail and Sphl. The reaction was stopped by adding 1 / 25VoI of 0.5 M EDTA (ethylenedinitrile tetraacetic acid disodium salt) and heating at 70 ° C for 10 minutes. The excised vector was purified by agarose gel electrophoresis (1% agarose, 1 χ TBE buffer: 6.05 g / l tris (hydroxylmethylaminomethane), 3.1 g / l boric acid, 0.37 g / l EDTA) 0.5pg / ml Containing ethidium bromide; Electrophoresis at 4V / cm). After localization of the DNA band by UV light (254 nm), the DNA was electro-eluted with DE 81 paper and finally purified by ethanol precipitation. The DNA was dissolved in 10 μM TE (10 mM Tris, pH 8.0, 1 mM EDTA), inserin

Ten pWS 214S4 were digested using 20 units each of Sphl and Sail. The smaller fragment containing the ADHI terminator was isolated by agarose gel electrophoresis, electroelution and precipitation. Subsequently, the DNA was dissolved in 10μΙ TE.

1 μΙ vector DNA and 1 μΙ insert DNA were ligated into 10μΙ solution by T4 DNA ligase, 3μΙ of the ligation solution was used to transform E. coli JM101 (supE, thi, del (lacproAB), F '[traD36, proAB , laclq, lacZ-delM 15) lambda minus). Some of the resulting colonies were cultured to the size of 1 ml after ampicillin selection. The plasmids were isolated (see Birnboim H. et al., In Nucl. Acids Res., 7, 1513 [1979]) and digested with SphI and Sail. The fragments were separated on an agarose gel. The. Presence of the ADHI terminator fragment was confirmed by an approximately 340bp (base pair) long DNA fragment. One of the plasmids was selected for further use and designated pRH 241 (restriction map: see FIG. 10).

b) Preparation of the plasmid pRH containing the yeast ADHI promoter and the yeast ADHI terminator (see J.L. Bennetzen et al., J. Biol. Chem. 257, 3018-3025 [1982]):

vector production

10pg pRH241 were double digested with EcoRI and KpnI. The vector portion was released from the small fragment by anarose gel electrophoresis, electroelution and precipitation. Finally it was solved in 10μΙ TE. insert manufacturing

10 _M g of plasmid pY-JDB-Hu-IFN-omega 1 (see Example A and DE-PA P 3635867.3, filed October 22, i986) were cut with SphI. The 3 'protruding end was removed by addition of 5 units of E. coli DNA polymerase I in the presence of dGTP. The DNA was further cut with Xhol and the resulting fragments were isolated by agarose gel electrophoresis, electroelution and precipitation. The smooth end of the 400 bp fragment containing the ADHI promoter was converted by ligation of the adapter pair:

EBI-410: 5 'AATTGGAAGGATC 3' EBI-429: 3 ¹ CCTTCCTAG-p 5 '

The sticky end of the restriction fragment was converted using the adapter pair:

EBI-418: 5'p-TCGAGCACGTGGTAC 3 'EBI-424: 3' CGTGCAC 5 '

After simultaneous ligation of the two adapters, the ADHI fragment was again purified by agarose gel electrophoresis. The DNA was dissolved in 5μΙ TE.

1 μΙ vector and 5μΙ insert were ligated in a total of 10μΙ solution using T4 DNA ligase. By ligating the insert into the vector, the EcoRI and KpnI sites are destroyed. 3μl of the ligase solution was used to transform E. coli JM101. Several colonies were examined microscopically for the presence of a plasmid of the desired construction by restriction of the plasmids with various restriction enzymes. A plasmid was selected and designated pRH242 (restriction map: see Figure 11).

c) Preparation of the plasmid pRH243, which contains the yeast ADHI promoter, a gene coding for the yeast "mating" factor α (MFa) leader peptide (see J. Kurjan et al. in Cell 30, 933-943 [1982]), a multicloning site and the yeast ADHI terminator contains: The MFa leader peptide gene was chemically synthesized by yeast codon usage (see JL wetting et al., J.Biol. Chem. 2S7, 3026-3031 [1982]) ,

vector production

1 μρ pRH 242 was double digested with Xhol and XbaI. The vector fragment was purified by agarose gel electrophoresis, electroelution and precipitation, the DNA was dissolved in 30μΐ TE.

lnsertheretellung

Using a DNA synthesizer Applied Biosystems 381A, a set of 10 oligodeoxynucleotides was prepared:

Name sequence

MF1 TCGAGCCTCATATCAATGAGATTCCCATCTATTTTCACTGCTGTTTTGTr (60 mer)

MF2 AGCAGCGAACAAACAGCAGTGAAAATAGATGGGAATCTCATTGATATGAGGC (53 mer)

MF 3 CGCTGCTTCCTCCGCTTTGGCTGCTCCAGTCAACACTACTACTGAAGACGAAACTGCTCAAATTCCAGCT (70 mer)

MF4 CAGCTTCAGCTGGAATTTGAGCAGTTTCGTCTTCAGTAGTAGTGnGACTGGAGCAGCCAAAGCGGAGGA (70mer)

MF 5 GAAGCTGTCATCGGTTACTCTGACTTGGAAGGTGACTTCGACGTTGCT (48 mer)

MF6 GCAAAACAGCAACGTCGAA ₁ GTCACCTTCCAAGTCAGAGTAACCGATGa (48mer)

MF7 GTTTTGCCATTCTCCAACTCCACTAACAACGGTTTGTTGTTCATrAACACTACTATTGCATCGATTGCT'iegmer)

MF8 CCTTAGCAGCAATCGATGCAATAGTAGTGTTAATGAACAACAAACCGTrGTTAGTGGAGTTGGAGAATG ^ mer)

MF9 GCTAAGGAAGAAGGTGTTTCTrTGGACAAGAGGCCTCTGCAGGAATTCT (49mer)

MF10 CTAGAGAATTCCTGCAGAGGCCTCTTGTCCAAAGAAACACCTTCTT (46 mer)

60 pmoles of each oligonucleotide except MF1 and MF10 were phosphorylated in δμΙ solution by T4 polynucleotide kinase (PNK). The reaction were stopped by heating the samples at 100 ^° C. for ten minutes. 5μΙ MF1 (6OpMoI) and the MF-2 solution were taken together and also the solutions of MF3 with MF4; MF5 with MF6; MF7 with MF8 and 5μΙ of MF10 given to the solution of MF9. Taken together solutions turn wuiden to 100 ⁰ C. and slowly cooled to room temperature. After this "annealing" step the five solutions were taken together, 20 units of T4 ligase was added and ligation was conducted at '.4 ⁰ performed C16 hours.

Finally, 0.5 μΙ of the vector DNA and 7 μΙ of the above-mentioned ligation reaction were pooled and ligated with 5 units of T4 ligase. E. coli JM101 was transformed with 3μΙ of this ligation solution. The plasmids of several colonies were isolated with Xhol and XbaI double restriction followed by purification by agarose gel electrophoresis. Those piasmids containing an insert of the expected size (290 bp) were further characterized by subcloning of the insert in M 13mp8 and sequencing by the Sanger dideoxy chain termination method (see Sanger F. et al., Proc Natl Acad , 5463-5467 (1977)). A plasmid containing the expected insert (see Figure 12) was designated pRH243 (restriction map: see Figure 13).

d) Preparation of plasmid pRH 244, containing the ADHI promoter, the coding sequence for the FCeR-water-soluble fragment and the ADHI terminator (see FIG. 15):

insert manufacturing

20 μg of pGEM4-Fc _t R (plasmid pGEM [Promega Biotec, Madison WI53711, USA] containing the larger HindIII-EcoRI fragment of the FccR cDNA) was cut with 40 units each of EcoRI and HindIII and the sticky ends were blotted with Klenow®. Fragment of E. coli DNA polymerase I and the four deoxynucleoside triphosphates (dXTP's) filled. The DNA was dephosphorylated with 10 units of calf intestinal phosphatase (CIP, Boehringer Mannheim), deproteinized by two phenol / chloroform extractions and separated on an agarose gel. After electroelution and precipitation, the fragment containing the coding region of the FCeR-water-soluble fragment was dissolved in 10μΙ TE.

Since HindIII cuts the Fc _c R cDNA about 50 bp upstream of the first amino acid of the soluble fragment (MeM 50), the insert of pGEM 4-Fc _t R is again excised with Sau 3 A. This method not only removes the 5 But also the nucleotides encoding the first 18N-terminal amino acids of the soluble fragment Two linker oligodeoxynucleotides of the following formulas.

5 'TCGAGCTCATATACAATGG ATG GAA TTG CAA CTT TCC TCT GGT TTCGTT TGT AAC ACT TGT CCA GA /. AAG TG

3 'CGAGTATATGTTACC TAC CTT AAC GTT CAA AGG AG A CCA AAG CAA ACA TTG TGA ACA GGT CTT TTC ACC TAG

Sow 3 A

W / the synthesized to restore the complete gene by means of yeast codon usage of the following formula

mead XhoI CGAGTATATGT TAC Val 155 Ser Gly Phe Val 160 Asn Thi Cys Per 165 5 'TCGAGCTCATATACA ATG GTT Ser TCT GGT TTC GTT Cys AAC ACT TGT CCA Glu 3 ' Leu CAA TCC AGA CCA AAG CAA TGT TTG TGA ACA GGT GAA TTG Gin AGG ACA CTT AAC CAA Gil GTT GAA CTT

Lys Trp 3 ' AAG TG 5 ' TTC ACC DAY Sau3A

Ever 2BpMo! The oligonucleotides were annealed to each other and added to the 3μ Sau3A (B'PhoephaO-EcoRI-filled, dephosphorvUert) fragment and ligated in 20μΙ total by T4 DNA ligase. The resulting Xhol (EcoRI) fragment was cloned by Ag & rosegel Electrophoresis, electroelution and precipitation were purified and it was dissolved in 20μΙΤΕ

10μΙ of pRH 242 was cut with XbaI and the ends blunt-ended by Klenow-Auttull reaction. The DNA was excised again with Xhol and the large fragment was isolated by agarose gol electrophoresis, electroelution and precipitation. It was solved in 100μΙ TE. 1 μΙ vector and 1 μΙ insert were ligated in total ΙΟμΙ using T 4 ligase. (Ligation of a filled EcoRI site to a filled-in XbaI site restores both the EcoRI and XbaI sites.) 3μL of the ligation reaction was used to support E. coli JM101. Plasmids were isolated from several colonies and assayed for the presence of FceR-spar soluble fragment gene using several restriction enzymes. One of the plasmids was further sanctioned and the Xhol-XbaI fragment became J

partially sequenced to confirm the correct connection between ADHI terminator and the Hnman gene. This plasmid was designated pRH244 (see FIG. 14).

q) Preparation of the plasmid pRH245, which contains the yeast ADHI promoter, the yeast "mating" factor aLeader gene, the gene for the Fc _t R-water-soluble fragment and the yeast e-ADHI terminator (see FIG. A5V-vector production

10 μg of pRH 243 are digested twice with EcoRI and Stul. The large fragment was purified by agarose gel electrophoresis, electroelution and precipitation. Finally, it was dissolved in 100 μΙ TE. insert manufacturing

20μ were doubly digested with EcoRI and HindIII and dephosphorylated. The insert was excised with Sau3A again and the larger fragment was isolated. To recover the complete coding region for the soluble fragment of the following formula

mead

5 'ATG 3 ¹ TAC

GIu Leu Gin VaI Ser Ser GIy Phe VaI Cys Asn Thr C / s Pro GIu GAA TTG CAA GTT TCC TCT GGT TTC GTT TGT AAC ACT TGT CCA GAA CCT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT

Lys Trp

AAG TG 3 '

TTC ACCTAG 5 '

Sow 3 A

were two oligonucleotides of the following formula two oligodeoxynucleotides of the formula

3 'TACCTTAACGTTCAAAGGAGACCAAAG TAC CTT AAC GTT CAA AGG ACa (XA AAG CAA ACATTG TGAACA GGT CTTTTC ACCTAG 5'

synthesized.

Twenty-five pmoles of each oligodeoxynucleotide were annealed to each other and added to 3 μg of the Sau3A-EcoRI fragment .The ligation was carried out in 20μl solution by T4 DNA ligase The resulting DNA was purified by agarose gel electrophoresis, electroelution and precipitation DNA was dissolved in 20μΙ TE.

1μΙ vector fragment and 1μΙ insert fragment were ligated in 10μΙ total with T4 DNA ligase. 3μΙ of the ligation reaction were used to transform E. coli JM101. Plasmids from several colonies were isolated and examined for the presence of Fc _e R-water-soluble fragment gene by means of restriction enzymes. One of the plasmids was further selected and the Clal-XbaI fragment was partially sequenced to confirm the correct linkage between the mating factor a part and the FccR-water-soluble Friigmont gene This plasmid was named pRH245 (see Fig. 15).

f) Preparation of yeast vectors

pRH244 and pRH245 were constructed using an E. coli vector (Bluescribe M13 +), which allows for the sequencing of the inserts. In order to express both versions of the FceR-soluble Fragmont gene in yeast, both recombinant plasmids must be cut with HindIII and BamHI, resulting in the expression cassette consisting of ADHI promoter, MFa leader gene (in the case of pRH245), Fc _£ R-iscerol soluble fragment gene and ADHI terminator can be ligated These DNAs can be ligated into almost any yeast vector having suitable restriction enzyme sites For example, plasmid pJDB207 was chosen (see VD Beggs in "Gene cloning in yeast" [Genocloning in yeast], Herg. R.Williamson: Genetic

Engineering 2,175-203 [1982], deposited at the German Collection of Microorganisms under DSM3) 81) pJDB207 contains a 2 ^ origin of replication and an Ieu2 selection marker. This plasmid replicates extrachromosomally in

vector production

10pg pJDB207 were double digested with HindIII and BamHI. The large fragment was isolated by agarose gel electrophoresis, electroelution and precipitation. The DNA was dissolved in 50μΙ TE.

Inserthersteltung

10 \ iq pRH244 and pRH245 were digested twice with HindIII and BamHI. The expression cassettes were isolated by agarose gel electrophoresis. The inserts were dissolved in 10 μl TE.

1 μΙ vector and 2 μΙ inserts were mixed and ligated in ΙΟμΙ total with T4 DNA gaseous. E. coli JM101 was transformed with 3μΙ of the ligation solution. The plasmids of some of the resulting colonies were analyzed for the accuracy of the construction by restriction analysis.

Each of the plasmids was selected and designated as follows:

pJDB-244 containing the expression cassette without the MFa leader sequence and pJDB-245 containing the expression cassette with the MFa leader sequence.

Both plasmids were isolated on a larger scale and used for transformation (see Beggs JD in Nature 275, 104 (1978)).

of yeast strain WS21-3 (a, leu2, his3, ura3, pep4). »

Part II:

Preparation of Plasmids 289a 1 and 289b3

1 \ iq of the yeast plasmid YEpl3 was digested with 3 units of HindIII and BamHI at 37 ° C within 3 hours in Core-buffer (Tris-HCI SOmMoI, pH8,0,1OmMoI MgCl _2, 5OmMoI NaCl). The linearized vector was isolated by agarose gel electrophoresis using 0.7% agarose gel (see Dretzen et al., Anal. Biochem 112,295).

1 μg of the plasmids pRH244 and pRH 245 were also digested with HindIII and BamHI. The 1650 bp fragment from pRH 244 and the 1900 bp fragment from pRH 245 was isolated by the method described above.

50 ng of the linearized vector and 200 ng each of the expression cassettes were incubated overnight at 14 ⁿ C in 20 μM gas buffer (66 mM Tris-HCl, pH 7.6.6.6 mM MgCl ₂ , 10 mM MoD DDT, 1 mM rATP, 0.1 mg / ml BSA ) in the presence of 1 unit T4 DNA ligase.

ΙΟμΙ of the ligation mixture were used to transform E. coli HB101 (see Maniatis et al., Molecular Cloning - A Laboratory Manual, [Molecular Cloning - A Handbook for the Laboratory], p. 250) and selected for ampicillin resistance.

The plasmids of some of the resulting colonies were checked by restriction analysis for the accuracy of their construction. Two plasmids were derived from pRH 244 and pRH 245, respectively (designated as plasmid 289a 1 [derived from pRH 244] and designated as plasmid 289b 3 (derived from pRH245).

Yeast WS21-1 (aleu2, his3, Up 1 pep4) was transformed with plasmids 289a 1 and 289b3 (see Nature 275, 104 [1978]).

Example 12

Expression of the Fc _t R-soluble fragment in E. coli

vector production

10pg pRH100 (see Example B and Himmler A., Captain R., Adolf G. and Swetly P. in J. Interferon Res. (1986), in print were digested with SstI.) The 3 'protruding ends became xh addition 5 units of E. coli DNA polymerase I and DGPT removed. The linearized plasmid was prepared by adding 10 units CIP linearized plasmid was dephosphorylated by adding 10 units CIP and by incubating for 30 minutes at 37 ⁰ C. After two phenol / chloroform Extractions were further purified by agarose gel electrophoresis, electroelution and precipitation, and the linearized vector was dissolved in ΙΟμΙ TE.

insert manufacturing

20pg pGEM4-Fc _c R (plasmid GEM4 (Promega Biotec, Madison WI53711, U.S.A.) containing the larger HindIII EcoHI fragment of the Fc _t R cDNA was double digested with Eco RI and Hind III The 5 'protruding ends were blunt-ended by addition of Klenow fragment of DNA polymerase I and all four dXTPs The 5'-phosphate groups were removed with O. After agarose gel purification, the fragment containing the Fc _t R-soluble Fragmenl gene was included Sau3A and the larger fragment was isolated 50 pmoles of each oligodeoxynucleotide

5 'GAACTGCAGGTGAGCTCTGGTTTCGTTTGCAACACTTGCCCGGAAAAATG 3'

3 'CTTGACGTCCACTCGAGACCAAAGCAAACGTTGTGAACGGGCCTTTTTACCTAG 5,

Sau3A

which restore the reader's host, starting with the second codon (GIu) of the FcJ-soluble fragment gene, with the E. cüli codons preferably being used (Sharp PM, Li W.-H. Nucl. Acids Res., 14, 7737-7749 [ 1986]), were "annealed" in 10μ1 solution by heating to 100 ⁰ C and slowly cooling. the Oligodesoxynucleotido and the insert DNA were ligated with T4 DNA ligase. After heat denaturation of the enzyme T4 polynucleotide kinase and ATP were added to ^d:.. to phosphorylate e 5 'ends of the insert After agarose gel purification the DNA was belöst in 10μΙ TE 1 μΙ linearized vector DNA and 5μΙ insert DNA were pooled and ligated half of the material was used to transform. E. coli HB101 (F-, hsdS20 (rb-, mb-), recA13, ara-14, proA2, lacY1, galK2, rpsL20 (Sm-resistant), xyl-5, mitl-1, supE44, lambda minus) The plasmids of some of the ampicillin-resistant colonies were isolated and restricted to Rich by restriction enzyme analysis The construction was checked. A plasmid was selected and further analyzed by DNA sequencing of the compound Trp promoter and FCcR-soluble fragment gene. After this last proof, plasmid was named pRH246 (see Figure 16).

Example 13

Construction of the plasmid psFc _e R-1

a) 350pg pBSF2-38 (see Nature 324, 73-76 (1986) containing the BSF-2 cDNA in the SmaI site of pGEM4 were digested with 700 units of EcoRI and BamHI in 500μl of a high saline buffer (10 oMM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1 mM MgCl ₂ ,

1 mM OTT) for 2 hours at 37 ⁰ C digested. The digested DNA was applied to a preparative 1% agarose gel electrophoresis and the EcoRI-BamHI fragment containing the full length of the 1.2 kbp BSF-2 cDNA was electroeluted from the gel, precipitated with 70% ethanol and added a construction of 1 μg / μΙ dissolved in TE buffer. 20 μg of this fragment were digested for 1 hour with 40 units of Hinfl in 50μΙ of a high saline buffer, extracted with phenol and precipitated with ethanol. The digested DNAs were dissolved in 25 μl of a 1 μl nick translation buffer (50 mM Tris-HCl, pH 7.2, 1 mM MgSO ₄ , 0.1 mM DTT, 50 μg / ml BSA) and 1 ml at 20 ° C. for 30 minutes 8.2 units / μΙ Klenow fragment and 1 mM dNTP incubated. The AuffOilreaktion was terminated by five minute incubation at 7O ⁰ C. The resulting 127-bp, blunt-ended fragment was extracted with phenol, with 40 units KpnI for 1 hour at 37 ⁰ C in 50μΙ a little salt-containing buffer (1OmM Tris-HCl, pH 7.5,1OmM MgCl ₂ , 1 mM DTT) digested, incubated for 30 minutes at 65 ⁰ C with 2.5 units of bacterial alkaline phosphatase and then on 1% preparative agarose gel The 110bp fragment containing the BSF-2 leader sequence was electroeluted and ethanol precipitated.The 110bp fragment was ligated into 10μl of ligation buffer (50mM Tris-HCl, pH 7.4, 0.1mM MgCl ₂ .10mM DTT, 1 mM spermidine, 1 mM ATP, 0.1 mg / ml BSA) and untreated d ligated with 1 \ ig digested by Kpnl and Smal pGEM4 by incubating at 4 ⁰ C for 16 hours with 200 units of T4 ligase, and transfected into E.coii (MC 1065). From colonies collected, four colonies were picked, one clone was selected, propagated, and upon confirmation that the plasmid of this selected clone contains only one leader sequence, it was designated pBSF2-L8 (see Figure 20).

b) 80μρ of plasmid LE-392 or pGEM4 (pFcJM) (1OmM Tris-HCI, pH 7,5,1OmM MgCl _2, 1 mM DTT) were digested at 37 ⁰ C for 1 hour with 150 units of HindIII in 200μΙ einos little saline buffer and subjected to 1% preparative agarose gel electrophoresis. The HindIII fragment containing the soluble Fc _e R region was electroeluted from the gel, precipitated with ethanol and dissolved in TE buffer at a concentration of 1 pg / μΙ. 1 \ ig of HindIII fragment were to fill at 20 ° C for 30 minutes with 8.2 units Klenow fragment and 1 mM dNTP in 10μΙ of 1x "nick" -Translationspuffer inkubiort the recessed 3 'ends, extraction with phenol followed by and ethanol precipitation The HindIII fragments whose 3 'ends were filled in were washed with 2 units of PstI in 10 μM of the medium salt buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM DTF) for 1 hour at 37 ⁰ C is digested, incubated for 30 minutes with 0.25 units bacterial alkaline phosphatase at 65X and precipitated with ethanol. Separately, 1 pg pBSF 2-L8 with

2 units of BamHI long ⁰ C for 1 hour digested in 20μΙ of a high salt buffer at 37, extracted with phenol and precipitated with ethanol. The digested by BamHI pBSF-2-L8 was inV.ubiert in 10μΙ Ix "nick" -Translationspufferg9löstund with 8.2 units Klenow fragment and 1 mM dNTP at 20 ⁰ C for 30 minutes to fill in recessed 3 'ends with The precipitate was dissolved in 20 μl of a high saline buffer, digested with 2 units of PstI at 37 ° C. for 1 hour, extracted with phenol and precipitated with ethanol. containing the soluble FCcR coding region and PstI-digested pBSF2-L8 was ligated by incubation with 200 units of T4 ligase in 10μΙ ligation buffer at 4 ° C for 16 hours and transfected into E. coli (MC 1065) One clone was selected, propagated, and designated as psFc _t R-1 after confirmation of plasmid construction The augmented psFc _c R-1 contains seven bases from multiple cloning in pGEM4 between B SF-2 leader and Fc _c R sequences in frame (see Figure 17: nucleotides 137-143).

Example 14

Expression of Fc _e R cDNA

The plasmid pSFc _e R-1 containing the modified Fc _t R cDNA was linearized by digestion with the restriction enzyme BamHI. mRNA was synthesized with SP6 RNA polymerase using linearized plasmid DNA as a template according to Melton et al.

About mRNA per 10 ng were injected into oocytes of Xenopus leavis and the oocytes were incubated at 20 ⁰ C in Barth's modified medium supplemented with 100 pg / ml penicillin and 1 ug / ml streptomycin. After the two-day incubation, the culture supernatant was collected and the oocytes were homogenized in Dulbecco's PBS containing 1 mM PMSF. The PBS lysate was separated by high performance centrifugation at 15,000 rpm for 10 minutes, and the pellet was again extracted with NP-40 to eolubilize the membrane-bound receptor (0.5% NP-40.0, 1 M NaCl , 0.05 M Tris-HCl,

Example 15

SDS-PAGE analysis of Fc _c R in oocytes

Ten injected with mRNA, oocytes were contained with 10ΟμΙ modified Barth medium containing 150μ Ci ³⁵ S-methionine, incubated for 24 hours at 2O ⁰ C. Selected oocytes were incubated in 1 ml lysis buffer (0.5% NP-40,0,1 M CaCl, 0.05 M Tris-HCI, pH 7.5) and 10 minutes at 15000 rpm ^'1 The clarified lysate and centrifuged The culture supernatant was precleared with normal mouse Ig-coupled Sepharose 4B Perleri and then incubated for 60 minutes with frequent shaking on ice with 3-5 (IgGI) antibody-coupled Sepharose 4B beads. The beads were washed twice with lysis buffer and twice with high salt buffer (0.5% NP-40, 0.5 M NaCl, 0.05 M Tris-HCl, pH 8.0) and finally washed once more with lysis buffer. The immunoprecipitates were eluted by boiling with SDS sample buffer for two minutes. The samples were analyzed on 9% SDS Page (see Figure 24).

Example 16 Detection of FC _e R

FccR activity was measured with an enzyme-linked immunosorbent assay (ELISA) with two antibodies using two different monoclonal anti-FCeR antibodies, 3-5 (IgGI) and 8-30 (IgM), which had two different epitopes respond to Fc _e R 96 well microtiter plates (Nunc, Roskilde, Denmark) were pre-coated with ΙΟΟμΙ / well 3-5 antibody (10pg / ml) in coating buffer (0.1M carbonate buffer, pH 9.6,0,02%) and overnight 4 = C incubated. The plates were then washed four times with rinse buffer (Dulbecco's phosphate buffer, pH 7.4, containing 0.05% (v / v) Tween 20) and then 100μl samples diluted with fertilization buffer (0.05% Tris-HCl, pH 8 , 1, diluted with 1 mM MgCl ₂ , 0.15 M NaCl, 0.05% Tween 20.1% BSA and 0.02% NaN ₃ ). The plates were incubated for 2 hours at room temperature, washed four times with rinse buffer, and then 100 ml of 8-248 antibody-alkaline phosphatase conjugate (0.75 mg / ml) was added. After a four hour incubation period, the plates were washed four times and the Enzyme ReaK.ion was prepared by adding 100 μl / well of 1 mg / ml p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, MO) in substrate buffer (0.05M carbonate buffer, pH 9 , 8.1 μm MgCl ₂ ). After an appropriate incubation period (60-90 minutes), the optical density was read using 405 and 620 nm wavelengths by means of an automatic micro ELISA display device (Nippon Inter. Med., Tokyo, Japan) (see Figures 21 and 22). ,

Example 17

Determination of the binding of Fc _e R to IgE

The antibody 3-5 coated plates were coated with 100 μΙ samples for 2 hours, washed four times with rinse buffer, and then 10 μg / ml of human monoclonal IgE (PS myeloma) was added. After two hours incubation at room temperature, the plates were washed four times and further incubated with alkaline phosphatase-conjugated monoclonal anti-human IgE and then described by addition of substrate p-nitrophenyl phosphate as described above.

Example 18 IgE rosetting

Fc ₁ R on lymphocytes are detected by a test using human IgE coated fixed ox RBC (ORBC) (Gonzalez-Molina, A. and Spiegelberg, HLJ Clin. Invest 59,616 [19771]. The number of IgE rosetting cells is determined by subtracting the number of nonspecific binding with fixed ORBC coated with bovine serum albumin. For IgE-Rossette inhibition, 25μΙ Fc _c R-bearing cells (5 x 10 ⁶ / ml) are mixed with a specific volume (e.g., 100μΙ) of the test sample or control medium and incubated for 1 hour at 4 ° C. The number of rosettes with 3 or more ORBC is counted. In experiments I and III, the Fc _e R-bearing cells are RPMI8866 cells and in experiment I) they are SKW6-CL4 cells. The control medium is the supernatant of the control cells, ie non-transformed oocytes

 It can be seen from the previous test results

a) The SDS-PAGE-Analyso shows the product of psFc _c R-1 from the oocytes, which is recognized by both antibodies 3-5 and 8-30 and has the IgE-binding activity, and which produced broad protein bands (see Fig. 24) and b) that the product of pANFc _c R-1 from the oocyte, which lacks the N-terminal transmembrane region in comparison, can be recognized by the 3-5 antibody but not by the 8-30 antibody and has no IgE-binding activity. These results show that the product of pANFc & R-1, which has no signal sequence is not processed properly and thus that a proper processing as in clone ps ^ c _e R-1 creates an epitope which is recognized by the antibody 8-30 and has IgE-binding activity.

Expression of the water-soluble portion of the Fc _c receptor may be obtained by culturing the corresponding E. coli or yeast or other organisms by standard fermentation techniques followed by concentration and purification of the desired water-soluble fragment by the means described in section a) "Isolation and Purification of the Water Soluble Part of Fc _t R ".

For example, yeast WS 21-1 transformed with plasmid 289b3 (WS 21-1 / 289 be) was cultured in YHK8 medium in a fermentor for about 40 hours until an optical density (546nm) of about 45 (dry cell mass: 13g / l) was achieved was. After centrifugation of the cells, the yield of Fc _c R in the recovered supernatant was determined by a specific EISA method

Yield: 2.5 E / ml Fc ₆ R (1 U / ml Fc _E R corresponds to the activity of a supernatant of 1 × 10 ⁵ RPMI / cells / ml).

In 11 YHK8 medium are included:

8.0Og (NH ₄ I ₂ SO ₄ ,

2.56 g (NH ₄ I ₂ HPO ₄ ,

0.60 g of MgSO ₄ .7H ₂ O,

0.56 g CaCl ₂ x 2H ₂ O,

0.04 g of biotin, 80.0 mg of m-inositol, 40.0 mg of Ca-pantothenate,

8.0mg of thiamine,

2.0 mg pyridoxine,

3.1 mg CuSO ₄ × 5H ₂ O, 19.0 mg FeCl ₃ × 6H ₂ O, 12.0 mg ZnSO ₄ × 7H ₂ O, 14.0 mg MnSO ₄ × H ₂ O,

5.0 mg boric acid,

2.0 mg NaMoO ₄ χ 2H ₂ O,

1.0 g of yeast extract,

0.5g citric acid, 0.2g uracil, 0/1g adenine, 15.0g glutamic acid, 0.5g tryptophan, 0.2g histidine, 100.0g glucose

Although in connection with the water-soluble fragment of the Fc _e receptor, the construction of vectors, the transformation of host organisms with them and the expression of the fragment in the foregoing are described in detail for the water-soluble fragment starting with amino acid 150 of the full Fc _e receptor it has been found that the processes of construction, transformation and expression can be carried out in a similar manner to recover other water-soluble fragments starting, for example, with amino acids 50 to 149 of the entire Fc _t receptor.

Table 1: Purification of Fc _e R from RPMI-8866 cells

material

Total-

protein

(MO)

Total activity (units *)

Specific activity (E / pg)

recovery

cleaning

Cell culture supernatant 180000 78,8Ou 0.44 100 1 Concentrated supernatant 172000 75,000 12:44 95.2 1 (19Of) NMIg sequence 132000 55,000 0.42 70 1 3-5-Sepharose eluate 441 36,800 83.4 47 190 HPLC 16 26,000 1,630 33 3,710

Crude cell lysate NMIg effluent 3-0 Sepharose eluate HPLC purified 117,000 99,000 306 6,160 2,475 3,795,649 0.05 0.02 12.4 649 3-5 Sepharose eluate procedure NMIg-Sepharose eluate drain 100 40.2 61.6 10.5 1 0.47 236 12,400 * 1 unit is the activity of 1 χ 10 ^s cells and corresponds to a 190x concentration: ism supernatant. Table 2 2470 4.5 85.5 1170 material IgE-Sepharose eluate procedure HPLC-purified

Table 3 CTCCT 5 GCTTAAACCT CTGTCTCTGA CGGTCCCTGC Leu Per bad 15 35 GAGGA GIu GIy GIn CGAATTTGGA GACAGAGACT GCCAGGGACG CTT CCC AGG bad GGTCGACCCC GAA GGT CAA AACACACTAG GAGGACAGAC ACAGGCTCCA GAA GGG TCC AGG 85 CAATCGCTCT CCAGCTGGGG CTT CCA GTT TTGTGTGATC CTCCTGTCTG TGTCCGAGGT TCC GTTAGCGAGA AGTGACCAGA 20 GCTGTGATTG TGCCCGCTGA GTGGACTGCG Leu Gly Leu 30 135 AACTCCACTA TCACTGGTCT Cys Arg Arg CGACACTAAC ACGGGCGACT CACCTGACGC CTG GGG CTG Val TTGAGGTGAT GTGAGTGCTC TGC AGG CGT CATCATCGGG AGAATCCAAG CAGGACCGCC GAC CCC GAC GTG 185 TTGTCAGGGA CACTCACGAG ACG TCC GCA GTAGTAGCCC TCTTAGGTTC GTCCTGGCGG CAC AACAGTCCCT 35 10 Leu Leu Leu 45 AIa Leu Trp Tyr Ser GIu He GIu GIu CTT CTC CTG Trp Mot GIu GCT CTG TGG ACT TCA GAG ATC GAG GAG GAA GAG GAC TGG 230 ATG GAG CGA GAC ACC ATA AGT CTC DAY CTC CTC ACC TAC CTC 25 GIy Thr Gin Hey VaI Leu Arg Cys GGG ACT CAG ATC GTG CTG 275 CGG TGT CCC TGA GTC DAY CAC GAC GCC ACA 40 Ala GIy Leu Leu Thr Leu Thr Ala GCT GGG CTG CTG ACT CTG 320 ACC GCC CGA CCC GAC GAC TGA GAC TGG CGG

Trp Asp Thr 50 Gin Ser Leu Lys 55 Leu Glu Glu -29 - 263 538 1: S i I t * TGG GAC ACC Thr CAG AGT CTA AAA Gin CTG GAA GAG 60 i His ACC CTG TGG ACA GTC TCA GAT TTT CAG GAC CTT CTC bad Ala CAC TGT GTC AGG GCT 365 ''] GTG bad Asn Val 65 Gin Val Ser Lys 70 Leu Glu Ser TCC CGA Ί CGG AAC GTC Ser CAA GTT TCC AAG Asn TTG GAA AGC 75 Ala GCC TTG CAG TCT GTT CAA AGG TTC AAC AAC CTT TCG His His ^: J GCC AGA TTG CAC CAC 410 CGG Asp Gin mead 80 Gin Lys Ser Gin 85 Thr Gin He GTG GTG GAC CAG ATG Ala CAG AAA TCC CAG Ser ACG CAG Old 90 • Gly CTG GTC TAC GCG GTC TTT AGG GTC TCC TGC GTC TAA Ser gin GGT CGC AGG TCA CAG 455 CCA Leu Glu Glu 95 bad Ala Glu Gin 100 bad Leu Lys AGT GTC j CTG GAG GAA Leu CGA GCT GAA CAG Gin AGA TTG AAA 105 Glu GAC CTC CIT CTT GCT CGA CTT GTC CAG TCT AAC TTT Ser gin GAA GAA GTC TCT CAG 500 CTT Leu Glu Leu 110 Trp Asn Leu Asn 115 Leu Gin Ala AGA GTC TTG GAG CTG Ser TGG AAC CTG AAC Gly CTT CAA GCA 120 Asp AAC CTC GAC TCC ACC TTG GAC TTG GGG GAA GTT CGT Asp Leu GAC AGG CCC GAT CTG 545 CTG Ser Phe Lys 125 Gin Glu Leu Asn 130 bad Asn Glu CTA GAC AGC TTC AAG Ser CAG GAA TTG AAC Glu AGG AAC GAA 135 Ser TCG AAG TTC TCC GTC CTT AAC TTG districts TCC TTG CTT Ala Ser AGC AGG CTC GCT TCA 590 TCG Leu Leu Glu 140 Leu bad Glu Glu 145 Thr Lys Leu CGA AGT TTG CTG GAA bad CTC CGG GAG GAG Val ACA AAG CTA 150 Asp AAC GAC CTT AGA GAG GCC CTC CTC GTG TGT TTC GAT bad mead GAT TCT CAC AGG ATG 635 CTA Leu Gin Val 155 Ser Gly , Phe Val 160 Asn Thr Cys TCC TAC TTG CAG GTG Ser AGC GGC TTT GTG Cys AAC ACG TGC 165 Glu AAC GTC CAC TCC TCG CCG AAA CAC TGC TTG TGC ACG Per Giu GAG AGG ACG CCT GAA 680 CTC Trp He Asn 170 Gin bad Lys Cys 175 Tyr Phe Gly GGA CTT TGG ATC AAT Phe CAA CGG AAG TGC Tyr TAC TTC GGC 180 Lys ACC DAY TTA TTC GTT GCC TTC ACG TAC ATG AAG CCG Lys Gly AAG AAG ATG AAG GGC 725 TTC Lys Gin Trp 185 His Ala bad Tyr 190 Cys Asp Asp TTC CCG AAG CAG TGG Val CAC GCC CGG DID Ala TGT GAC GAC 195 Thr TTC GTC ACC GTC GTG CGG GCC ATA GCC ACA CTG CTG mead Glu ACC CAG CGG ATG GAA 770 TGG Gin Leu Val 200 He His Ser Per 205 Glu Gin Asp TAC CTT CAG CTG GTC Ser ATC CAC AGC CCG Glu GAG CAG GAC 210 Gly GTC GAC CAG AGC DAY GTG TCG GGC GAG CTC GTC CTG Phe Leu GGG TCG CTC TTC CTG 815 CCC Lys His Ala 215 His Thr Gly Ser 220 He Gly Leu AAG GAC AAG CAT GCC Ser CAC ACC GGC TCC Trp ATT GGC CTT 225 Thr TTC GLA CGG AGC GTG TGG CCG AGG TGG TAA CCG GAA bad Asn ACC TCG ACC CGG AAC 860 TGG Asp Leu Lys ¹ 230 Glu Phe He Trp 235 Asp Gly Ser GCC TTG GAC CTG AAG Gly GAG TTT ATC TGG Val GAT GGG AGC 240 Leu CTG GAC TTC GGA CTC AAA DAY ACC GTG CTA CCC TCG IHs Val TTG CCT CAC CAT GTG 905 AAC Tyr Ser Asn 245 Ala Per Gly Glu 250 Thr Ser bad GTA CAC TAC AGC AAC Trp GCT CCA GGG GAG Per ACC AGC CGG 255 Asp ATG TCG TTG TGG CGA GGT CCC CTC CCC TGG TCG GCC Ser Gin GAC ACC GGG AGC CAG 950 CTG TCG GTC

Glu Asp Cys 260 mead mead bad Gly 265 Gly bad Trp -30 - 263 538 I 1 I i; GAG GAC TGC Val ATG ATG CGG GGC Ser GGT CGC TGG 270 Gly CTC CTG ACG GTG TAC TAC GCC CCG TCC CCA GCG ACC Asn Asp 1 GGC CAC AGG AAC GAC 995 CCG Phe Cys Asp 275 Lys Leu Gly Ala 280 Val Cys • Asp TTG CTG TTC TGC GAC bad AAG CTG GGC GCC Trp GTG TGC GAC 285 j Ala AAG ACG CTG CGT TTC GAC CCG CGG TGG CAC ACG CTG bad Leu GCC GCA ACC CGG CTG 1040 CGG Thr Cys Thr 290 Per Ala Ser Glu 295 Ser Aia Glu GCC GAC ACA TGC ACG Per CCA GCC AGC GAA Gly TCC GCG GAG 300 Ala TGT ACG TGC CCG GGT CGG TCG CTT GGT AGG CGC CTC Ser mead GCC GGC CCA TCC ATG 1085 CGG Per Asp Ser 305 Per Asp Per Asp 310 bad Leu Per AGG TAC CCT GAT TCA bad CCA GAC CCT GAC Gly CGC CTG CCC 31b Gly GGA CTA AGT AGA GGT CTG GGA CTG GGC GCG GAC GGG Thr Per GGA TCT CCG ACC CCC 1130 CCT TGG GGG

Ser AIa Pro Leu His Ser · TCT GCC CCT CTC CAC TCT TGA AGA CGG GGA GAG GTG AGA ACT

GCATGGATA CAGCCAGGCC CAGAGCAAGA CGTACCTAT GTCGGTCCGG GTCTCGTTCT

CCCTGAAGAC CCCCAACCAC GGCCTAAAAG CCTCTTTGTG GCTGAAAGGT GGGACTTCTG GGGGTTGGTG CCGGATTTTC GGAGAACACAC CGACTTTCCA CCCTGTGACA TTTTCTGCCA CCCAAACGGA GGCAGCTGAC ACATCTCCCG GGGACACTGT AAAAGACGGT GGGTTTGCCT CCGTCGACTG TGTAGAGGGC CTCCTCTATG. GCCCCTGCCT TCCCAGGAGT ACACCCCAAO AGCACCCTCT GAGGAGATAC CGGGGACGGA AGGGTCCTCA TGTGGGGTTG TCGTGGGAGA CCAGATGGGA GTGCCCCCAA CAGCACCCTC TCCAGATGAG AGTACACCCC GGTCTACCCT CACGGGGGTT GTCGTGGGAG AGGTCTACTC TCATGTGGGG AACAGCACCC TCTCCAGATG CAGCCCCATC TCCTCAGCAC CCCAGGACCT TTGTCGTGGG AGAGGTCTAC GTCGGGGTAG AGGAGTCGTG GGGTCCTGGA GAGTATCCCC AGCTCAGGTG GTGAGTCCTC CTGTCCfCC TGCATCAATA CTCATAGGGG TCGAGTCCAC CACTCAGGAG GACAGC. OG ACGTAGTTAT

AAATGGGGCA GTGATGGCCT TTTACCCCGT CACTACCGGA

CCCA GGGT

Claims (19)

claims: An ancestor for producing a human Fce receptor with low affinity or a water-soluble part thereof having an N-terminal cytoplasmic domain, a C-terminal extracellular domain and a molecular mass of about 46 kd and its glycosylated derivatives, characterized in that a suitable host organism with an expression vector containing as a coding sequence replicon and control sequences for expression in prokaryotes and eukaryotes, E. coli or in yeast for the desired polypeptide at an appropriate site, and the desired polypeptide is isolated from the resulting transformants. 2. The method according to claim 1, characterized in that as replicon and Kontiollsequenzen those of the plasmid pER 103 or plasmid pGEM 4 are used or as a replicon 2-p-origin and as control sequences contains the ADHI promoter and the ADHI-Terrninator and additionally contains the "mating" factor a-leader sequence (MFa). 3. The method according to claim 1, characterized in that as expression vector, a plasmid vector with the coding sequences mentioned in claim 2 and a recombined DNA molecule can be used. 4. The method according to claims 1 to 3, characterized in that as plasmid plasmid LE392 deposited in E. coli HB101 under FERm BP-1116 is used, j and the DNA sequence of the formula j ATG GAG GAA GGT CAA ACT TCA GAG ATC GAG GAG CTT CCC AGG AGG] TAC CTC CTT CCA GTT ATA AGT CTC TAG CTC CFJ GAA GGG TCC TCC | CGG TGT TGC AGG CGT GGG ACT CAG ATC ClG CTG CTG GGG CTG GTG | GCC AGA ACG TCC GCA CCC TGA GTC TAG CAC. GAC GAC CCC GAC CAC! ACC GCC GCT CTG TGG GCT GGG CTG CTG ACY CTG CTT CTC TGG TGG CGG CGA GAC ACC CGA CCC GAC GAC TGA GAC GAA GAG GAC ACC CAC TGG G, * C ACC ACA CAG AGT CTA AAA CAG CTG GAA GAG AGG GCT GTG ACC CTG TGG TGT GTC TCA GAT TTT GTC GAC CTT CTC TCC CGA GCC CGG AAC GTC TCT CAA GTT TCC AAG AAC TTG GAA AGC CAC CAC CGG GCC TTG CAG AGA GTT CAA AGG TTC TTG AAC CTT TCG GTG GTG GGT GAC CAG ATG GCG CAG AAA TCC CAG TCC ACG CAG ATT TCA CAG CCA CTG GTC TAC CGC GTC TfT AGG GTC AGG TGC GTC TAA AGT GTC GAA CTG GAG GAA CTT CGA GCT GAA CAG CAG AGA TTG AAA TCT CAG CTT GAC CTC CTT GAA GCT CGA CTT GTC GTC TCT AAC TTT AGA GTC GAC TTG GAG CTG TCC TGG AAC CTG AAC GGG OTT CAA GCA CT CTG CTG; AAC CTC GAC AGG ACC TTG GAC TTG CCC GAA GTT CGT CTA GAC; AGC AGC TTC AAG TCC CAG GAA TTG AAC GAG AGG AAC GAA GCT TCA TCG TCG AAG TTC AGG GTC CTT AAC TTG CTC TCC TTG CTT CGA AGT GAT TIG CTG GAA AGA CTC CGG GAG GAG GTG ACA AAG CTA AGG ATG CTA AAC GAC CTr TCT GAG GCC CTC CTC CAC TGT TTC GAT TCC TAC GAG TTG CAG GTG TCC AGC GGC TTT GTG TGC AAC ACG TGC CCT GAA CTC AAC GTC CAC AGG TCG CCG AAA CAC ACG TTG TGC ACG GGA CTT AAG TGG ATC AAT TTC CAA CGG AAG TGC TAC TAC TTC GGC AAG GGC TTC ACC TAG TTA AAG GTT GCC TTC ACG ATG ATG AAG CCG TTC CCG ACC AAG CAG TGG GTC CAC GCC CGG TAT GCC TGT GAC GAC ATG GAA TGG TTC GTC ACC CAG GTG CGG GCC ATA CGG ACA CTG CTG TAC CTT GGG CAG CTG GTC AGC ATC CAC AGC CCG GAG GAG CAG GAC TTO CTG CCC CTG GAC CAG TCG TAG GTG TCG GGC CTC CTC CTC AAG GAC ACC AAG CAT CGG AGC CAC ACC GGC TCC TGG ATT GGC CTT CGG AAC TGG TTC GTA CGG TCG GTG TGG CCG AGG ACC TAA CCG GAA GCC TTG TTG GAC CTG AAG GGA GAT TTT ATC TGG GTG GAT GGG AGC CAT GTG AAC CTG GAC TTC CCT CTC AAA TAG ACC CAC CTA CCC TCG GTA CAC GAC TAC AGC AAC TGG GCT CCA GGG GAG CCC ACC AUC CGG AGC CAG CTG ATG TCG TTG ACC CHA GGT CCC CTC TGG TGG TCG GCC TCG GTC GGC GAG GAC TGC GTG ATG ATG CGG GGC TCC GGT CGC TGG AAC GAC CCG CTC CTG ACG CAC TAC TAC GCC CCG AGG CCA GCG ACC TTG CTG GCC TTC TGC GAC CGT AAG CTG GGC GCC TGG GTG TGC GAC CGG CTG CGG AAG ACG CTG GCA TTC GAC CCG CGG ACC CAC ACG CTG GCC GAC GCC ACA TGC GGC GGT CGG TCG CTT CCA AGG CGC CTC AGG TAC GGA CCT GAT TCA AGA CCA GAC CCG GAC GGC CGC CTG CCC ACC CCC CCT GGA CTA AGT TCT GGT CTG GGA CTG GGA CTG CCG GCG GAC GGG TGG GGG TCT GCC CCT CTC CAC TCT TGA
AGA CGG GGA GAG GTG AGA ACT or a degenerative derivative thereof and its glycosylated derivatives within the plasmid pGEM ™ 4 as EcoRI insert. 5. Process according to claims 1 to 3, characterized in that plasmid pDE2-FceR-1 is used as the plasmid and contains the DNA sequence according to claim 4. 6. The method according to claims 1 to 3, characterized in that as plasmid Plasrnid pRH 246 is used, which is the DNA sequence of the formula ATG TAC GAG TTG CAG GTG TCC AGC GGC TTT GTG TGC AAC ACG TGC CCT GAA CTC AAC GTC CAC AGG TCG CCG AAA CAC ACG TTG TGC ACG GGA CTT AAG TGG ATC AAT TTC CAA CGG AAG TGC TAC TAC TTC GGC AAG GGC TTC ACC TAG TTA AAG GlT GCC TTC ACG ATG ATG AAG CCG TTC CCG ACC AAG CAG 1 3 GTC CAC GCC CGG TAT GCC TGT GAC GAC ATG GAA TGG TTC GTC ACC CAG GTG CGG GCC ATA CGG ACA CTG CTG TAC CTT GGG CAG CTG GTC AGC ATC CAC AGC CCG GAG GAG CAG GAC TTC CTG CCC GTC GAC CAG TCG TAG GTG TCG GGC CTC CTC GTC CTG AAG GAC ACC AAG CAT GCC AGC CAC ACC GGC TCC TGG ATT GGC CTT CGG AAC TGG TTC GTA CGG TCG GTG TGG CCG AGG ACC TAA CCG GAA GCC TTG TTG GAC CTG AAG GGA GAT TTT ATC TGG GTG GAT GGG AGC CAT GTG AAC CTG GAC TTC CCT CTC AAA TAG ACC CAC CTA CCC TCG GTA CAC GAC TAC AGC AAC TGG GCT CCA GGG GAG CCC ACC AGC CGG AGC CAG '' CTG ATG TCG TTG ACC CGA GGT CCC CTC TGG TGG TCG GCC TCG GTC GGC GAG GAC TGC GTG ATG ATG CGG GGC TCC GGT CGC TGG AAC GAC CCG CTC CTG ACG CAC TAC TAC GCC CCG AGG CCA GCG ACC TTG CTG CGG TTC TGC GAC CGT AAG CTG GGC GCC TGG GTG TGC GAC CGG CTG CGG AAG ACG CTG GCA TTC GAC CCG CGG ACC CAC ACG CTG GCC GAC GCC ACA TGC ACG CCG CCA GCC AGC GAA GGT TCC GCG GAG TCC ATG CGG TGT ACG TGC GGC CGC CGG TCG CTT CCA AGG CGC CTC AGG TAL GGA CCT GAT TCA AGA CCA GAC CCT GAC GGC CGC CTG CCC ACC CCC CCT GGA CTA AGT TCT GGT CTG GGA CTG CCG GCG GAC GGG TGG GGG TCT GCC CCT CTC CAC TCT TGA
AGA CGG GGA GAG GTG AGA ACT or a degenerative derivative thereof, as well as its O-glycosylated derivatives and those of the plasmid pER103 within the plasmid pBR322a.s EcoRI insert with the restriction map shown in FIG. 7. Process according to claims 1 to 3, characterized gekonnzeichnet that plasmid as plasmid 'pRH 244 is used, the DNA sequence according to claim 6 and the replicon sequence of the 2 pm origin and contains as control sequence the ADHI promoter and the ADHI promoter within the plasmid Blueocribe M13 + as Barn HI / Hind III insert with the restriction map shown in FIG. 8. Process according to claims 1 to 3, characterized in that plasmid pRH 245 is used as the plasmid containing the DNA sequence of claim 7 with the restriction map shown in Figure 15. 9. A method: ^ rh claims 1 to 3, characterized in that as the plasmid plasmid pSFc _E R-1 is used which contains the DNA sequence according to claim 6 with the restriction map shown in Figure 19. 10. The method according to claim 1, characterized gekenr characterized in that as expression vector, a yeast vector containing the DNA Sequsnz according to claim 8 or 9, is used. 11. The method according to claim 1,2 and 10, characterized in that the yeast vector of the yeast vector pJHDB-244, which contains the DNA sequence according to claim 7 as BamHI / Hind III insert within the plasmid pJDB207, is used. 12. The method according to claim 1,2 and 10, characterized in that the yeast vector of the yeast vector pJDB-245, which contains the DNA sequence according to claim 8 as BamHI / Hind III insert within the plasmid pJDB207, is used. 13. The method according to claim 1,2 and 10, characterized in that the yeast vector of the yeast vector 289a 1, which contains the DNA sequence according to claim 8 as BamHI / Hind III insert within the plasmid YEp 13, is used. 14. The method according to claim 1,2 and 10, characterized in that as yeast vector of the yeast vector 289b3, which contains the DNA sequence according to claim 7 as BamHI / Hind III insert within the plasmid YEp 13, is used. 15. The method according to claims 1 to 14, characterized in that the cultivation of lymphoblast-like B cells and separation of the Fc _£ R from the supernatant or from the lysed cells is carried out by sequential immunoaffinity purification. 16. The method according to claims 1 to 15, characterized in that RPMI-8866 cells are used as lymphoblastic cells. 17. The method according to claims 1 to 16, characterized in that a solution containing Fc _E R is absorbed sequentially on BSA-Sepharose, human transferrin-Sepharose and normal mouse Ig Sepharose, the sequence on an anti-FceR Affinity column is applied and the process is further purified by HPLC. 18. The method according to claims 1 to 17, characterized in that an immunoaffinity column is prepared by coupling a corresponding antibody to Sepharose. 19. The method according to claim 1, characterized in that for the production of partial amino acid sequences of the formula Met-Glu-Leu-Gln-Val-Ser-Ser-Gly-Phe-Val, Gly-Glu-Phe-Ile-Trp-Val-Asp-Gly-Ser-His-Val-Asp-Tyr-Ser-Asn-Trp-Ala-Pro-Gly-Glu-Pro-Thr, Lys-His-Ala -Ser-His-Thr-Gly-Ser-Trp-Ile-Gly-Leu-Arg-Asn-Leu-Asp-Leu-Lys and Lys-Trp-Ile-Asn-Phe-Gln-.