JPH07507925A - Protein L and its production by recombinant DNA method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Protein L and its production method by recombinant DNA method This invention provides a novel immunoglobulin Bulrin-binding protein, its production method, recombinant DNA molecules encoding it, and The present invention relates to recombinant DNA molecules useful as probes for biochemistry.

More particularly, the invention provides substantially pure and/or complete and/or homogeneous A quality form of a protein called protein L and a group encoding protein L Concerning recombinant DNA molecules.

A surface protein that has affinity for mammalian immunoglobulins through interaction with heavy chains. A large number of Gram-positive bacterial species have been isolated that express the These immunoglobulins The best known binding protein is type 1 staphylococcus. Streptococcus protein A and type 2 Streptococcus protein G, which interact primarily with the C2-C5 boundary on the Fc region of human immunoglobulins. It has been shown that In addition, both proteins also have weak interactions with the Fab regions. Although it has been shown that this is the case, it is also due to the heavy chain of immunoglobulin.

Recently, Peptococcus magnus Protein L, a novel protein from Interaction with light chains in human, rabbit, pig, mouse, and rat immunoglobulins It was found that they can only be combined by In humans, this interaction It has been shown that this only occurs in the cold. Both chains of kappa and lambda are different Since protein L is commonly present in all human classes, Strongly binds to subunit form of IgM and also exhibits protein L chain binding. It is expected to bind to all classes in the species.

Peptococcus and Peptostreptococcus occus), both of which are proteins that bind to the kappa chain of human immunoglobulins. It has been reported that the protein L is produced. Protein L is known to be a virulence factor. It has been proposed that: non-virulent peptococcus and pebstrebutt Coccus neither expresses protein L nor has its structural gene. (Kastern et al., 1990).

Protein L is present in all classes and subclasses of immunoglobulins It is of particular interest because it has been reported to bind to kappa shichin. That's it As such, protein L is a useful protein that can be used in ELISA and RIA methods. It must be a diagnostic reagent. Current production methods for protein L generally involve cell surface production. Protein L is extracted and purified from bacteria that express protein L.

Such methods are inherently inefficient and produce impure proteins in low yields. It is produced at a high rate and takes a long time. In addition, the protease obtained in this way The link L is incomplete.

EP-A-0255497 describes the purification of protein L by standard protein purification methods. Attempts at production and characterization are described. Thereafter, EP-A-0255497 The authors have published a number of scientific papers that further explore the properties and structure of protein L. However, it has so far failed to fully characterize the protein. It's finished. Therefore, recently, Kastern (W) et al. “Protein L Bacterial Immunoglobulin-Binding Proteins and Possible Virulenes” A paper entitled “Infection and Immunit May 1990, pp. 1217-1222), protein L tripe The N-terminal amino acid sequence of the syn fragment was determined and the obtained sequence information was used to identify the gene. Gene encoding protein L by constructing a probe for isolation It has been stated that attempts to isolate the .

However, to date, the gene for protein L has not been isolated and characterized. problems remain unresolved and are therefore preventing significant improvements in Protein L production. ing.

Furthermore, sequence information on protein L is not available, and immunoglobulin kappa L chain It was not possible to identify the sequences involved in complex formation with.

This invention now allows the isolated cDNA insert encoding protein L to be fully isolated. is based on the cDNA sequence encompassing the Wear. This cDNA sequence and the amino acid sequence corresponding to its longest open reading frame are This is shown in FIG. The sequence of the longest open reading frame shown in Figure 1 is TTG (103). to AAA (3183), and the illustrated DNA is a non-mature protein. The code spanning from nucleotide 208 to nucleotide 3183 encoding Contains the code area.

Therefore, in one embodiment, the present invention provides immunoglobulin protein L, termed protein L. It provides a polypeptide capable of forming a complex with kappa light chain. , the polypeptide is in substantially homogeneous and/or complete and/or full-length form. It is characterized by being

The polypeptides of the invention preferably (i) are substantially homogeneous; (ii) ) be complete; and (iii) be in substantially full-length form. It has at least two, more preferably three, characteristics.

The above full-length polypeptide has a small length (both 900 amino acids, and preferably at least 950 amino acids in length, and most preferably at least 950 amino acids in length. At least 975 amino acids.

Full-length polypeptides provided by the invention include mature and non-mature sequences. Both are included. Triplet ATG (208) has 30 to 35 amino acids This appears to be the beginning of a signal sequence that spans. Therefore, the polypeptide of the present invention The immature polypeptides from Met (208) to Lys (3183) are included in the code. and the mature polypeptide with 30 to 35 amino acids omitted from the N-terminus. Included.

In summary, full length immature protein L (or preprotein L) is 992 The immature polypeptides of the present invention are believed to consist of amino acids and are at least 9 in length. Optimally, it consists of a polypeptide that is 90 amino acids.

The immature polypeptide according to the invention has the N-terminal two sequences Met　Lys　l1eAs n Lys Lys Leu Leu Met Ala Ala Leu Al at least 7 of acly Aha Ile Val Val Gly Gly, More preferably at least 10, most preferably at least 15 amino acids. It is preferred to have a corresponding N-terminal sequence.

These N-terminal sequences are as follows.

611111: Lys lla Asn LyS Lys LeuNet L ys Ile Asn Lys Lys Leu Leu See ^1aMe t Lys Ile Asn Lys Lys Lau Leu Met Al a Ala Leu^la Gly Ala The above-mentioned polypeptides are included. The signal sequence is 20-35 amino acids in length. acids, more specifically 23-27 amino acids in length. Therefore, the completeness of the present invention The long mature polypeptide begins with one of the following N-terminal sequences and begins with Leu The array shown in Figure 1 continues from (313) (.

cry^la Asn^la Tyr^1a^la Glu Glu Asp Asn Thr^sp^sn A311,,.

ALa Asn ALa Tyr Ala Ala Glu Glu Asp Asn ding hr Asp Asn Asn,,.

Hesn^1aTyr^1a^1aGLuGluAspAsnThrAspAsn Asn...

ALa Tyr ^1 & ALa Glu Glu Asp Asn Thc Asp Asn Asn,,.

”Tyr Ala Ala Glu GLu Asp Asn Thr Asp 　Asn　Asn　,　,　.

Ala Ala Glu Glu Asp Asn Thc Asp Asn Asn,,.

ALa Glu Glu Ag^in Thr Asp ASn Amn,, .

C+lu Asp Asn Thr Asp Asn Asn, .

Asp Asn Thr Asp Asr + Agn, 5゜Thr Asp Asn　Asn　,,.

The actual mature sequence of protein L is thought to start from the code 1 of the above sequence. i.e. in Figure 1 the mature sequence is marked by rMJ and the signal sequence is Columns are marked "SS".

Although the present invention also encompasses variants of the above polypeptides, all variants are Variants can form complexes with immunoglobulin kappa chains.

Desirably, the variant polypeptide of the invention has at least the amino acid sequence shown in FIG. Both have 75% sequence homology, preferably at least 90% sequence homology.

Most preferably, the variant polypeptide of the invention has the amino acid sequence shown in FIG. at least 95% sequence homology, preferably less (at least 98% sequence homology) Ru.

The full-length polypeptide shown in Figure 1 has the C-terminal sequence Leu Ala Ala Al a Ala　Leu　Ser　Thr　Ala　Ala　Gly　Ala　Ty It has rVat Ser Leu Lys Lys Arg Lys.

The polypeptide according to the invention has the C-terminal sequence Leu Ala Ala AlaA la Leu Ser Thr Ala Ala Gly Ala Tyr V At least 7 of alSer　Leu　Lys　Lys　Arg　Lys　 Preferably at least 10, most preferably fewer (both corresponding to 15 amino acids) It has a C-terminal sequence.

These C-terminal sequences are as follows.

Leu^la ALa Ala Ala A la　Gly^la　Tyr　Val　Se=The present invention further provides a second aspect. and provide a recombinant DNA molecule having an insert encoding the above polypeptide. Ru.

In one embodiment of the invention, a recombinant polypeptide encoding an immature polypeptide of the invention The DNA molecule begins with (a) ATG located at 208 and ATG located at 3183. The DNA coding sequence shown in Factor 1 extending to AA, and (b) the same amino acid sequence are selected from degenerate DNA sequences that code.

In yet another embodiment, recombinant DNA encoding a mature polypeptide of the invention (c) The molecule starts with a codon in the region from position 211 to position 313. The DNA coding sequence shown in Figure 1 extending to AAA at position 3183, and ( d) selected from degenerate DNA sequences encoding the same amino acid sequence.

In a third aspect, the invention provides an expression vector containing the above DNA coding sequence. immunoglobulin binding characterized by culturing a transformed host transformed with The present invention provides a method for producing a synthetic protein. The transformed host can be eukaryotic or prokaryotic. For example, bacterial or yeast or animal cells, cultured mammalian cells or It may be an insect cell.

In a fourth aspect of the present invention, at least 12 DNA sequences shown in the DNA sequence of FIG. 1, It preferably consists of a contiguous sequence of 15 bases, most preferably 17 bases, and is an immunoglobulin. As a probe to isolate other DNA sequences encoding binding proteins Useful recombinant DNA molecules are provided. Therefore, in the fourth aspect, as shown in FIG. A DNA sequence encoding a polypeptide having a sequence related to protein L Used to construct probes useful for probing gene banks of A sequences. .

In a fifth aspect, the present invention further provides a method for probing a gene bank with the above probe. culturing the expression vector containing the isolated DNA coding sequence by Provided is a method for producing an immunoglobulin-binding protein, which is characterized by:

The specific binding properties of protein C (its ability to bind to the kappa light chain of immunoglobulins) ) have recognizable repeated features within the amino acid sequence of the molecule. This seems to be due to the existence of a sequence. As used herein, "recognizably" The word "repeated features" means that the amino acid sequence has a length of 20 to 45 amino acids, respectively. consisting of at least two sequences of amino acids, which sequences are at least 759 amino acids from each other. 6, preferably at least 90% homology, most preferably at least 95% homology It means to have.

The polypeptide sequence shown in Figure 1 contains 10 sets of repeat sequences, of which a few It is thought that at least two of them are involved in immunoglobulin kappa L complex binding.

These 10 sets of repeat sequences are classified as follows at their N-termini.

(1) AI, A2 and A3; (2) Bl and B2: (3) C1, C2, C3 and C4: (4) DI, B2, B3 and B4: (5) El, B2 and B3; (6) Fl, F2, B3 and B4° (7) Gl and G2; (9) Hl and R2: and (10) R1, R2, R3, R4, R5, R6, R7 and R8 each repetition of these The sequence has a length of 25-45 amino acids.

The ability to bind to kappa L chain is determined by repeating sequences A, B, C, and DC (1) to It seems to be related to one or more of (4)).

Therefore, in Figure 1 at its N-terminus Al, A2 and A3; Bl and B 2; C1, C2, C3 and C4: and separated as Dl, B2, B3 and B4 a plurality of recognizably repeated binding domains selected from similar sequences; It is another feature of the invention to provide a synthetic immunoglobulin binding molecule consisting of Ru. Preferably, the synthetic immunoglobulin binding molecule contains 2 to 15 domains. stomach. These selected one or more domains are shown in FIG. At A1, A2 and A3; Bl and B2; C1, C2, C3 and C4; and sequences classified as Dl, B2, B3 and B4, or These and at least 75%, preferably at least 90%, most preferably less Variations may be made from the sequence provided they have at least 95% homology.

At its N-terminus El, B2 and B3: and Fl, F2, B3 and B4 Sequences classified as The synthetic binding molecules provided include sequences E1, B2 and B3; and Fl, F2, B3 and B4, or at least 75%, preferably less than at least 90% homology, most preferably at least 95% homology. and related sequences that vary from said sequence.

Characterization, isolation and labeling of the gene encoding pbutococcal protein L A comparison with isolation by quasi-protein purification methods is described in the Examples below.

Example 1 - Isolation and Characterization of Protein L by Standard Protein Purification Methods fruit A collection of 56 different clinical isolates of Gram-positive anaerobic cocci was collected from Luton Pub. ・Health Laboratory (Luton Public Health Labo ratory) Annaerobe Reference Unit (Anaerobe R efference Unit), Bass Public Health Laboratory ( Bath Public Health Laboratory) and sole Salisbury Public Health Laboratory ic Health Laboratory). pebtococcus and The concave forms of Peptostreptococcus and Peptostrectococcus are in the National Collection of Thailand. National Collection of Type Cultures), Central Public Health Laboratory (Ce ntral Public Health Laboratory), (Colin Dill, London).

1.1 Preparation of cell extract Todd-Huyts (Todd-Huyts) with the above strain added with 0.05% Tween 80 Hevitt's broth was cultured anaerobically in a conventional manner. Centrifuged at 8,000g Collect cells by separating and add to 50mM potassium phosphate buffer (pH 6,8). and then resuspended in the same buffer to achieve a cell density of 10% (wet weight/volume). did.

Mutanolysin (Sigma: per ml of bacterial suspension) 50 units) was added to perform dissolution. After incubation at 37°C for 2-3 hours, Cell extracts were centrifuged at 12,000 g for 15 min or aliquoted at -20°C. or boiled for 10 min in SDS PAGE loading buffer (buffer). -Harlot & Lane, 1988).

1.2 SDS PAGE and Western blotting of proteins Proteins from cell extracts were electrophoresed on a 7.5% polyacrylamide SDS gel. Western blotting (Barlow & Lane, 1988) electrophoretically using a nitrocellulose membrane (Hybond C); Transferred to Amarjam). - after overnight incubation, a lily containing 0.02% Tween 20; The membrane was washed several times in acid-buffered saline (PBS-T), followed by Protein binding sites are removed by incubation in gelatin (Sigma) for 1 hour. I blocked the position. Next, human IgG molecules conjugated with alkaline phosphatase were added. or phosphatase-conjugated light chain (reduction, alkylation and separation by FPLC). Prepared from human IgG by separation (Harlow and Lane, 1988) Immunoglobulin-binding proteins were detected by incubating. immunity Globulin-binding proteins were treated with nitro-blue tetrazolium and X-phos. (Barlow & Lane, 1988).

1.3 Protein L affinity purification 4-liter culture solution of strain 1018 Proliferated, harvested and lysed as described above. This cell extract is subjected to a centrifugation step. Heat treatment (80° C., 10 minutes) was performed before application. The supernatant was diluted with 250mM NaCl. and applied to a 5 ml column (diameter 15 mm) of IgG-Sepha0-se. This card Wash the lamb with 50mM Hepes-NaOH (pH 8,0) and remove the immunoglobulins. The protein-binding protein was eluted with 100mM glycine-HCl (pH 2,0). . The eluate containing this protein was analyzed by Western blotting. The solution was neutralized to pH 7.5 with Tris-HCl before washing.

immunoglobulin-binding proteins in Peptococcus magnus isolates. Sizes correspond to those reported for Protein L. in 1990 In contrast to the results made by Stern et al., we demonstrated that immunoglobulin Clinical isolates containing binding proteins include wound isolates or from infected surgical wounds. I found out that it is something.

The immunoglobulin-binding protein is alkaline phosphatide prepared from human IgG. was detected using a Tase-labeled strand preparation.

14 Conclusion These results indicate that immunoglobulin-binding proteins are present during the affinity purification process. is significantly degraded into smaller products that retain the ability to bind to IgGL chains. It is confirmed that it will be given.

Example 2 Determination of the complete nucleotide sequence of Protein L and other sequenced immunizations Comparison of translated amino acid sequences with globulin binding proteins 2.1 Materials Radiochemicals were obtained from Amerjam International. X-OmatS X-ray film was obtained from Kodak. Deoxynucleoside triphosphate and dideoxynucleoside triphosphate, DNA ligase, restriction endonuclease and and other DNA-modifying enzymes were obtained from Boehringer. agarose, acrylamide Bisacrylamide, bisacrylamide, and phenol were obtained from Bethesda Research Laboratory. I got it from The chromatography medium was Farmanar LKB (Uppsala, Sweden). Obtained from (Weden). Human immunoglobulin and serum albumin from Sigma Obtained. All other reagents were obtained from Sigma or BDH. Nitrocellulose is an Darman (Ander+can and Co,,) (Kickston-upon) -Thames, Surrey, UK).

2.2 Medium and culture conditions E. coli TGI in 2xYT broth (2% (w/v)) Ribton/1% (w/v) ) Cultured overnight at 37°C in yeast extract/1% (w/v) NaCl). medium It was solidified with 2% (w/v) Bacto agar (Difco). HT for M13 layer -Aga-Niha, 1% (w/v)) Ribton, 08% (w/v) N a Cl O and 0.8% (W/V) Bacto Agar (Difco). if necessary ampicillin at a concentration of 50 μg/ml for selection and propagation of transformants. was used.

Detection of functional β-galactosidase was performed using 5-bromo-4-chloroindolyl-β. - by adding D-galactone at a final concentration of 600 μg/ml, and If necessary, add isopropyl-β-D-thiogalactopyranone to a final concentration of 20 This was done by adding 0 μg/ml.

Purification of plasmid and phage RF DNA was carried out from E. coli by Brij lysis and This was done by CsCl/ethidium bromide density gradient centrifugation. Pebutokotka Isolation of chromosomal DNA was performed as described in both cases.

2.3 Gene manipulation procedure Use DNA-modifying enzymes in buffer and under conditions recommended by the supplier (Boehringer). used. Transformation of E. coli was performed essentially as previously described. DNA cut Electrophoresis of the pieces was performed in Tris-acetate buffer (40mM-Tris/20mM-sodium acetate). 1% (w/v) vertically in 1% (w/v) - Performed on an agarose slab gel. The size of the DNA fragment is limited by the front end Compare with fragments of lambda phage DNA digested with nuclease Hindll. It was evaluated based on the following. Purification of DNA fragments was carried out essentially as previously described. This was done by electroelution.

2.4 Nucleotide sequencing Checks on M13 molds using shotgun protocol to generate random molds The nucleotide sequence was determined by the terminator method. DNA5TARIn Multiple overlapping sequences were generated using the program provided by C (Madison, USA). collected. Using these same programs, sequenced genes and translated proteins The protein was analyzed. Synthesis of oligonucleotide primers in abrid biosynthesis It was performed using a Stemda 380B DNA synthesizer.

2.5 Ultrasonication of cells The cell suspension was subjected to sonication using an MSE ultrasonic tube (MSE sonicator). 150 sonicator (MS E5oniprep 1505onicato 3 x 30 s bursts at 18 MHz with 30 s intervals at 4°C).

2.61gG-affinity chromatography on Sepharose 4B ultrasound Affinity chromatography on IgG-Sepharose FF using the procedure Bacterial cells from small scale purification of PPL were disrupted. Culture solution (300ml ) was grown overnight and then centrifuged (15,000 g for 10 min at 4°C); 100mM Trix-HCL pH 7.5, in 250mM NaCl (3ml) resuspended in. This suspension was sonicated and centrifuged (4°C, 30,000 g for 10 min), and the supernatant was diluted with 100 mM) Lis-HCl, pH 7,5,250. 1 of IgG-Sephas FF equilibrated and washed with mM NaCl (5 ml). ml column (1,6 cm x Q, 9 Q cm internal diameter). 100mM glycine- Proteins were eluted with MCI, pH 2.0 and p I raised the H to 7.5.

2.7 PAGE Samples were solubilized under reducing conditions and electrolyzed on a 5DS-polyacrylamide slab gel. It was subjected to electrophoresis. acrylic in the LKB vertical electrophoresis unit using the Laemmli method. Amide (7.5%, w/v) slab gels were processed. kumasi protein - Stain with Brilliant Blue R-250 and ComoScan the protein bands -3 (Chomoscan -3) Laser optical densitometer (Joyce Rape) J oyce-Loebl), Gateshead, Tyne and Wear, UK). The apparent M was evaluated by scanning.

2.7 Western blotting Proteins were quantified by electrophoretic transfer from 5DS-polyacrylamide gels. cellulose membrane and probed with 1125-labeled protein.

2.8 N-terminal sequencing Abrid biostem by automated Edman phenylthiohydantoin degradation Determine the N-terminal amino acid sequence on a Da4fufu A-pulse liquid protein sequencer. Established. The PPL sample was dialyzed against 50mM-NaC+ and approximately 500 pmoles Applied to gas phase encoder. Operation of the equipment shall essentially be in accordance with the manufacturer's instructions. I went. By repeating Edman degradation, amino acids are continuously extracted from the peptide. This was identified using reverse phase HPLC.

2. Screening and identification of 93316 Part 5au of 3316 chromosomal DNA 6.0-8 from 3A digestion. Fragments of OKb were isolated by electroelution and dephosphorylated B Cloned into amH1 digested pMTL23. 1152 recombinant clones Place samples in 12 microtiter trays and incubate overnight at 37°C. did. By adding glycerin to each well at a final concentration of 10% (W/V) , this gene bank could be stored at -70°C. Possible immunoglobulin To rapidly identify binding proteins, these clones were isolated from 48 240 cells ( 2 microtiter trays) and incubated overnight at 37°C with shaking. I did it. Soluble cellular proteins released by sonication 11! l radioactive It was subjected to Western blotting using labeled human immunoglobulin L chain.

From the identified pools, repool the initial clone stocks into groups of 8 and probed. Finally, clones from the positive pool were individually rescreened for 3 We identified two human immunoglobulin light chain binding proteins.

2.10 Characterization The HiI-1gG affinity purified samples for all three clones were ゜When run on a 5% SDS polyacrylamide gel, after staining with Tumassie blue, The three main types are 104 kilodaltons, 96 kilodaltons and 90 kilodaltons. band was shown. This same band pattern was also observed using crude sonicated samples. The western blot was plotted using human IgG, IgA, IgM, IgD, IgE and This was also observed after the Lls probe was carried out with the light chain and kappa light chain, but it was not observed with the lambda chain. I couldn't. In addition, it was found to also bind human serum albumin.

Since proteins A and G were known to be extremely stable against heat, The present inventors developed the ultrasonic treatment by heating the crude ultrasonicated product at 80°C for 10 minutes. The stability of Tin L was tested. This treatment removes proteins precipitated from solution. It was discarded and the soluble fraction was analyzed by 5DS-PAGE.

In Cumann staining, most of the E. coli proteins were precipitated, but protein L was in solution. It was shown that it remained. Comparison with IgG-affinity purified protein L Heat treatment appears to be another rapid method of protein purification. . According to Western blot analysis, this protein remains isolated from kappa light chain and and retains activity against H8A. In contrast to what was observed with protein G In general, a smaller protein that appears to be a proteolytic fragment of protein L It is interesting that only protein binds H3A.

2.11 DNA sequencing DNA restriction mapping of the above three clones revealed that pPPI, 9 was 6.2 Kb. was found to contain the minimal insert of the ppl gene by subcloning. was limited to a 4.2 Kb Pstl fragment. Continue cutting this Pstl fragment. The sample was taken out and subjected to ultrasonication using a shotgun.

By sequencing these inserts, we obtained the complete DNA sequence and related components shown in Figure 1. A amino acid sequence was obtained.

2.12 Conclusion The pure and homogeneous protein L obtainable according to the present invention is characterized by the kappa light chain binding can be the basis for many systems in which a Therefore, protein L is useful for diagnostic tests. and in assays, e.g. for immobilizing antibodies on columns. Can be used as a reagent. Other uses include as pharmaceutical formulations and It is used as a reagent for preparing drugs.

Important properties of protein L according to the present invention include the following.

(1) Binding ability to L chain, i.e., not Fc-binding (Fab-binding (11) thermal stability) Qualitative, i.e. stable for at least 10 minutes at 80°C Binding ability to min (iv) pK of approximately 4.68 Synthetic immunoglobulin binding molecules according to the invention also include those in which kappa light chain binding is desired. can be the basis for many systems, such as test kits, biochemical reagents, and protocols. . In addition, these synthetic molecules are sequenced so that they have virtually no albumin binding ability. Sequences selected from E1, F2, F3, Fl, F2 and F3 may be omitted .

ττTGGAcAGτ　G(ACGAAACA　AGAACACTGA　TTτ Enter ATAAAT TGGTGAAATT CGkTTGTTf8 60 Lau　L@u　GLy　Asn CAA m CAT TTA AAT AGCATT Oiri A TGC AA AAA TTT 8AA AGG AGG AGA 162AAG ATT A AT AAG AAA TTA TTA AT CCT GCA CTT CA GGA GCA ATT GTA@2S8 GGA GAT GTT TCA GAT TCA GTA GAT CCT CTA GAA to AA Gkk ATA GACCAA S02 GCA TTA GCA AAA GCA TTA G(:A GAA GCT AAA GAA^CA GCA AAA AAA CAT S50 GAA AAA TTA GCA GCA GCA AAA GAA ACA GCA AAG AAA CAT ATA GAT CAA@642 Submission of translation of written amendment (Article 184-8 of the Patent Law) November 7, 1994

Claims (33)

[Claims] 1. be in substantially homogeneous and/or complete and/or full length form; It is characterized by the fact that it is called protein L and forms a complex with immunoglobulin kappa light chain. A polypeptide that can be 2. (i) substantially homogeneous; (ii) complete; and (iii) ) have at least two of the characteristics of being in substantially full length form. The polypeptide according to claim 1. 3. (i) substantially homogeneous; (ii) complete; and (iii) ) have at least three of the characteristics of being in substantially full-length form; The polypeptide according to claim 1. 4. according to any of the preceding claims, having an amino acid sequence substantially as shown in FIG. polypeptide. 5. A polyester according to any of the preceding claims, having a length of at least 900 amino acids. Ripeptide. 6. A polyester according to any of the preceding claims, having a length of at least 950 amino acids. Ripeptide. 7. A polypeptide according to any of the preceding claims, having a length of at least 975 amino acids. Ripeptide. 8. A polyester according to any of the preceding claims, having a length of at least 990 amino acids. Ripeptide. 9. A polyester according to any of the preceding claims, having a length of at least 992 amino acids. Ripeptide. 10. N-terminal sequence Met　Lys　Ile　Asn　Lys　Lys　Leu any of the preceding claims, having an N-terminal sequence corresponding to at least 7 amino acids of A polypeptide described in crab. 11. N-terminal sequence Met　Lys　Ile　Asn　Lys　Lys　Leu having an N-terminal sequence corresponding to at least 10 amino acids of LeuMetAla A polypeptide according to any of the preceding claims. 12. N-terminal sequence Met　Lys　Ile　Asn　Lys　Lys　Leu At least Leu Met Ala Ala Leu Ala Gly Ala according to any of the preceding claims, wherein the N-terminal sequence corresponds to 15 amino acids. polypeptide. 13. N-terminal sequence Met　Lys　Ile　Asn　Lys　Lys　Leu Leu Met Ala Ala Leu Ala Gly Ala Ile N-terminus corresponding to at least 20 amino acids of Val Val Gly Gly A polypeptide according to any of the preceding claims, having the sequence: 14. At least 75% sequence homology with the amino acid sequence shown in Figure 1, preferably less than Polypeptides according to any of the preceding claims, having a sequence homology of at least 90%. Chido. 15. At least 95% sequence homology with the amino acid sequence shown in FIG. Polypeptides according to any of the preceding claims, having a sequence homology of at least 98%. Chido. 16. C-terminal sequence Leu Ala Ala Ala Ala Leu Ser any of the preceding claims having a C-terminal sequence corresponding to at least 7 amino acids of The polypeptide described in. 17. C-terminal sequence Leu Ala Ala Ala Ala Leu Ser Thr　Ala　Has a C-terminal sequence corresponding to at least 10 amino acids of Ala A polypeptide according to any of the preceding claims. 18. C-terminal sequence Leu Ala Ala Ala Ala Leu Ser At least Thr Ala Ala Gly Ala Tyr Val Ser according to any of the preceding claims, also having a C-terminal sequence corresponding to 15 amino acids. polypeptide. 19. C-terminal sequence Leu Ala Ala Ala Ala Leu Ser Thr Ala Ala Gly Ala Tyr Val Ser Leu LysLys　Arg　C-terminal sequence corresponding to at least 20 amino acids of Lys A polypeptide according to any of the preceding claims having a sequence. 20. The signal sequence is omitted, and the signal sequence is 20 to 35 amino acids in length. acid, more particularly 23 to 27 amino acids in length, according to any of the preceding claims. polypeptides. 21. Starting from one of the following N-terminal sequences, shown in Figure 1 from Leu (315): 21. The polypeptide of claim 20, which is followed by a sequence of: [There is an array] 22. Starting from the N-terminal sequence below, from Phe (343) the sequence shown in Figure 1 21. The polypeptide of claim 20, followed by: [There is an array]. ．． ．． 23. having an insert encoding a polypeptide according to any of the above claims. Recombinant DNA molecule. 24. (a) D shown in Figure 1 from ATG at position 208 to AAA at position 3183 from an NA coding sequence, and (b) a degenerate DNA sequence encoding the same amino acid sequence. 24. A recombinant DNA molecule according to claim 23, which is selected. 25. Consisting of at least 12 bases adjacent to the DNA sequence shown in Figure 1, the immunoglobulin As a probe for the isolation of other DNA sequences encoding phosphorus-binding proteins. Recombinant DNA molecules useful in 26. Consisting of at least 15 bases adjacent to the DNA sequence in Figure 1, the immunoglobulin As a probe for the isolation of other DNA sequences encoding phosphorus-binding proteins. Recombinant DNA molecules useful in 27. Consisting of at least 17 bases adjacent to the DNA sequence shown in Figure 1, the immunoglobulin As a probe for the isolation of other DNA sequences encoding phosphorus-binding proteins. Recombinant DNA molecules useful in 28. (a) culturing a transformed host; is the DNA coding sequence according to claim 24 or (b) according to any one of claims 25 to 27. The DNA code isolated by probing the gene bank with the probes listed above. an immunoglobin, characterized in that it has been transformed with an expression vector containing the sequence Method for producing Robulin-binding protein. 29. In Figure 1 at its N-terminus A1, A2 and A3; B1 and B2; C1 , C2, C3 and C4; and classified as D1, D2, D3 and D4 Consisting of multiple recognizably repeated binding domains selected from the sequence , a synthetic immunoglobulin binding molecule. 30. The synthetic immunoglobulin binder according to claim 29, consisting of said domains from 30.2 to 15. Synthetic molecules. 31. The chosen domain or domains are A1 at its N-terminus in FIG. , A2 and A3; B1 and B2; C1, C2, C3 and C4; and D1 , D2, D3 and D4. at least 75%, preferably at least 90%, most preferably at least 9 Claims that vary from said sequence provided that they have 5% homology. 29 or 30. The synthetic immunoglobulin binding molecule according to 29 or 30. 32. sequences E1, E2 and E3; and F1, F2, F3 and F4, or at least 75%, preferably at least 90%, and preferably a small amount, of said sequence. Related sequences that have changed from the sequence provided that they have at least 95% homology. The synthetic immunosorbent according to any one of claims 29 to 31, further comprising a domain selected from epiglobulin-binding molecule. 33. Any of claims 29 to 32, wherein the domain has a length of 20 to 45 amino acids. A synthetic immunoglobulin-binding molecule as described in Crab.