JPH05500005A - Plasma glycoprotein expression - 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] Plasma ring protein expression Technical field The present invention relates to the expression of α-macroglobulin and its derivatives and variants, particularly in mammalian cells. Expression of human α2-macroglobulin (C2M) in its active form and its genetic engineering Regarding the expression of mutants. Such recombinant α-macroglobulin, especially recombinant αz M(rαZM) and its mutants are used for therapeutic purposes and for growth in mammalian cell culture. examples from the medical field for the development of newly defined growth media for It is listed.

Background technology Anal-macrologoblin') 1'03 Goshu Chemical Proteinase protein synthesized in the liver Synthetic glycoprotein α2M, along with coproteins C3, C4, and C5, are structurally and functionally related. It constitutes a separate class from the larger plasma proteins. For recent research, 5ottrup-Jensen, ll, (1987) in: The P lasmaProteins (Putnam, F, W, li), 2nd edition, 5: 191 291. Academic Press, 0rlando, Fl) Please refer to

Apart from C5, these proteins contain internal β-cysteinyl-T-glutamylthiol α, M, C3, Enables the proteolytically activated form of C4. (Sottrup-Je nsen, t, et al. (1980) FEBS Lett, 121: 27 5-280; 5alvesen, C.S., and Barrett, A.J. (1981) Old ochem, J, 187: 695-701) This thiol-S In the active protein, the ter is slowly cleaved with many small nitrogen nucleophiles. α2M biology constitutes a unique post-synthetic modification of proteins. It plays a major role in the characteristics of The presence of active thiol esters in α2M is revealed by a specific pattern of thermal decomposition.

(Harpel, P. C., et al. (1979) J. Biol. Chem. 25 4: 8869 8878) Traditionally, α2M inhibits plasma brotinase. Although studied within the context of agents, it is unique according to several criteria.

Most plasma brotinase inhibitors are monomeric pallors of approximately similar size, but α 2M is a trimer whose 180kl) subunit contains 1451 residues. ( Sottrup-Jensen et al. (1984) J. Biol. Chem. 2 59:8318-8327) Furthermore, most other proteinase inhibitors Phosphoproteinase and the active site of the proteinase and the reactive site of the inhibitor engage. α2M, in contrast, forms a 1:1 complex with substrate specificity and catalytic A wide range of proteinases with different mechanisms, e.g. trypsin, leucocytes, Lastase, chymotrypsin, bankretic elastase, canapsin G 1 forms a complex with plasmin, plasma kallikrein, and thrombin.

The second-order reaction constants for the association of these blotinases with α2M are several orders of magnitude larger. 1:1 and A 2:1 blockinase α2M complex can be formed together, disulfide-bridged dimer (360ko) is considered to be the functional unit of αzM. (Sottrup-Jen sen+ L, (1987) in: The Plasma Protei ns (Putnam, F.W., ed.), 2nd edition, 5:191-291+Ac academic Press, 0rlando, Fl) “Classic” blog In contrast to the proteinase inhibitor complex, the α2M-linked proteinase remains active. Yes, especially for small synthetic substrates. (Sottrup-Jens en, L. (1987) in: “The Plasma Proteins’ (Putnam, F.W.i), 2nd edition, 5: 191-29L Acade -mic Press, Orlando, Fl) Proteinase binding by α2M The mechanism of this is described by the ``trap'' (Barrett, A., J. and 5tarkey, P. M. (1973) Biochem, J., 133: 709, 724), where the 180-kD subunit (“bait” by proteolytic cleavage of a particularly exposed peptide stretch near the center of the The proteinase is captured by changing the conformation of the α2M tetramer.

The essentially irreversible nature of the formation of the blotinase complex with α2M after a long period of time It's not clear either. However, recent studies have shown that the main fraction (typically More than 80 to 90% of the proteinases produced were further expressed by nibsilone-lysyl ( (brotinase)-γ-glutamyl (α2M) bond. It is shown. (Sottrup-Jensen, L.

et al., (1981) FEBS Lett, 128: 127 132; 5a nd, O, et al. (1985) J, Biol, Chem, 260: 15723- 15735; Pochon, F. et al., (1987) FEBS L ett, 217: 101-105) of brotinase α2M, The α2M-brotinase complex is rapidly being elucidated in journals (Ohlsson +, (1971) Acta Pbysiol, 5cand, 81: 269-272; Imber, l'1. J, and Pjzzo, S, V, (1 981) J. Biol. Chem. 256:8134-8139). The general role of proteinases as ``clean vehicles'' has not been investigated. Ru.

The main physiological targets are coagulation, fibrinolytic brotinase and plasma kallikrein, and and possibly even brotinase-like leucosites, elastase, and canapycetes. G1 collagenase and other proteinases released during cell rotation. . (Sottrup-Jensen, L., and Birkedal-Hanse r++H, (1989) J.

Biol, Chem, 264:393 401) α2M causes healthy inflammation. In unstrained tissue, the inhibitor and its block may be mostly confined to the vasculature. The rotinase complex is present in plasma in rheumatoid joint inflammation and exudate and gingival crevicular fluid. It has been found at values close to the value. (Tollefsen, T., and 5altve d, E. (1980) J, Periodont, Res, 15: 96- 106; Borth + What, 1st Prize, (1983) Ann, N, Y, Acad, Sci, 421: 377-381) Plasma α2M is synthesized in the liver. It seems that (Schre-iber+G, (1987) in: “The Plasma Proteins'' (Putnam, F, W.

), 2nd edition, 5: 294 363 + Academic Press, 0r lando, Pi), other synthesis sites also exist. Several strains produced α2M during culture. fibroblasts (Mosher, D, F, et al. (1977) J, Cl1n , Invest, 60:1036-1045), Monocytes/Macrophages (H ovi+T-+ et al. (1977) J, Exp, Med, 145:1580 15 89).

Liver hepatocytes and clohost cells are present in plasma (Davi-dsen, O., et al. , (1985) Biochem, Biophys, Acta 846: 8 592), fibroblasts (Van Leuven, F., et al. (1979) J. Biol, Chem, 254:5155-5160; Gate Osher, D. F., and Vaheri, A. (193Q) Biochem.

Biophys, Acta 627: 113 122), macrophages (D ebanne.

Mon, To et al. (1975) Biochem, Biophys, Acta 41 1: 295 304; Kaplan, J., and Nelsen, M. Le, ( 1979) J, Biol, Chem, 254 Near 323-7328) Although important for elucidating the α2M-proteinase complex, α2M-proteinase It also has receptors for the enzyme complex.

These observations indicate that there is considerable extravascular rotation of α2M, probably primarily due to We propose that the brotinase function is transported into the cell microenvironment. (Sot trup-Jensen+ L, and Bfrkedal-Hansen, H, ( 1989) J, Biol, Chem, 264: 393 401) Development of the invention. Show Briefly, the present invention provides recombinant α-macroglobulin, particularly human α2M; Discloses a method for producing mutants in active form.

In a preferred embodiment, the host cells to be cultured are mammalian cells, insects, plants, etc. cells obtained from organisms such as yeast and other fungi, such as Aspergillus. It is a eukaryotic cell.

The present invention further provides a DNA comprising a gene encoding the expression of human α2M and its mutants. sequence, a vector containing the DNA sequence, and a suitable host transformed with such a vector. Concerning the Lord.

Yet another feature of the invention is the use of recombinant α2M as a protein carrier in enzyme exchange therapy. and the use of its variants.

Yet another feature of the invention is the use of recombinant α2M as a DNA carrier in gene therapy. and the use of its variants.

Yet another feature of the invention is that the Recombinant α-macroglobulin, especially human, as a component of the growth medium, as expressed α2M and its mutants.

definition Prior to describing the present invention, it is important to provide definitions of certain terms used hereinafter. , will help you understand.

DNA or cDNA A DNA molecule or sequence that is enzymatically synthesized from a sequence that was present in an mRNA template.

Yun person Tsukio Isolated in a partial form from a natural gene or linked or proximate in a way that does not occur in nature a single-stranded or double-stranded DNA molecule that has been modified to contain a DNA fragment that has been Genetic information that allows such a molecule to be replicated when integrated into a host cell A DNA construct containing. Plasmids generally contain at least one component to be expressed in the host cell. a promoter that contains one gene sequence and further assists in the expression of such genetic information; , sequences encoding functions such as the transcription start position.

DNA sequences are said to be linked when they are joined by a phosphodiester bond to the 5' end. say. The ligation may include methods such as ligation of paired or complementary ends, cDNA cloning, etc. synthesis of concatenated sequences through This can be achieved by removing existing sequences.

mutant It is related to the original peptide, but due to mutations in the gene encoding the original peptide, Peptides with altered amino acid sequences.

A=A 1 a=alanine V=Va　1=valine L = Leu = Leucine 1=11e=isoleucine P=Pro=proline P=Phe=phenylalanine W=Trp=)ributofan M=Met=methionine G=G], y=Nibrisi N=Ser=Saison T=Thr=threonine N=Asn=asparagine Q=G l n-glutamine D=Asp=aspartic acid E=G　u　u=glutamic acid ni=Lys=lysine R=Arg-arginine H=His=histidine Bukjo Shiodama T = Thymine (DNA only) U = uracil (RNA only) Brief description of the drawing Figure 1a shows the construction of plasmid pH36.

Figure 1b shows the construction of plasmid pH67.

Figure 2 shows the structure of plasmid pL136.

Figure 3 shows gel electrophoresis of thermal fragmentation products generated from α2M and 172M (10~ 20% 5DS-PAGE).

Figure 4 shows the thermal fragmentation products generated from methylamine-treated α2M and 172M. Gel electrophoresis is shown.

Figure 5 shows the gel voltage of the reaction products generated from trypsinized α2M and 172M. Showing pneumophoresis.

Figure 6 shows α2M and 172M treated with methylamine and treated with trypsin. Gel electrophoresis of the generated reaction products is shown.

Figure 7 shows unreacted natural α2M and 172M and trypsinized α2M and 172M. Showing rate gel electrophoresis.

Figure 8 shows unreacted natural α2M and 172M and those treated with methylamine. Showing "rate gel" electrophoresis.

Figure 9 shows the chromatograms of α2M and 172M on a Superose 6 column.

Figure 10 shows chymotrypsin-treated human α2M, human PZP, rαzM-PZP. Figure 2 shows gel electrophoresis (10-20% denaturing 5DS-PAGE) of the reaction product.

FIG. 11 shows human α2M and human PZP treated with elastase.

Gel electrophoresis of the reaction product of 172M-PZP (10-20% denatured 5DS-PA GE).

Figure 12 shows human α2M1 human PZP treated with trypsin.

Gel electrophoresis of the reaction product of 172M-PZP (10-20% denatured 5DS-PA GE).

Figure 13 shows human α treated with Glu-specific proteinase derived from Staphylococcus aureus. Gel electrophoresis of reaction products of zM, human PZP, rcxzMPZP ( 10-20% denaturing 5DS-PAGE).

Light Kaidan Pressed Gasa Goyu According to the present invention, α-macroglobulin, in particular human α2-macroglobulin, or Provided are methods for producing fragments and derivatives thereof (including variants thereof). This person In this method, α-macroglobulin, especially human α2-macroglobulin, or its fragments, derivatives (including variants thereof), or alleles of such genes. A functional expression vector containing a gene encoding the expression can be used to express the gene. Introducing into a suitable host, the host is enriched with assimilable carbon, nitrogen sources and other essential nutrients. The expressed α-macroglobulin, especially human α2-macroglobulin, is cultured in a suitable nutrient medium. Recover cloglobulin or its fragments and derivatives.

In particular, proteins synthesized in mammalian cells may undergo one or more post-translational modifications. Receive decoration (processing). Final purpose and specific function of newly synthesized protein Depending on the T-carboxylation, β-hydroxylation, sulfecigone, amidation , thiol ester formation, phosphorylation, and fluorescence at precursor processing sites. undergoes white degradative cleavage fatty acid acylation. (Rosner, M. R. (1986 ), in: Mammal-ian Ce1l Technology”+( Tilly, W.G.M), ButterivorthPublisher S., Stoneham, MA: 63-89) and many different sizes. A protein that undergoes post-translational modification is transformed into a heterologous protein transformed using recombinant DNA technology. is successively expressed in mammalian host cells. To give an example of 2.3, human aggregation factor Child ■a, IX undergoes corrections such as γ-carboxylation and glycosylation. ) BHK transformed with post-translational modification (Syrian baby hamsta kidney 11i & ) is expressed in cells (THirm + L- et al. (1988) Bioc hemistry 27: 7785; 7793; busby, S, et al. (1985) Nature 316: 271-273) Human platelet-derived The growth factor AB heterosimer was introduced into transformed CHO (Chinese hamster). A and B chain precursors undergo corrective processing. The AB heterosimmer undergoes correction assembly. Human aggregation factor ■Trait Expressed in transformed CHO cells, the precursor undergoes corrective processing and becomes trophic. It is derived from a double-stranded molecule that can be activated with protein and factor Xa.

(Kaufman, R, J, et al. (1988) J, Biol, Chem, 26 3: 6352 6362; Pittman, D, D, and Kaufma n, R, J, (1988) Proc, Natl, Acad, Sci.

USA 85: 2429-2433) So far, active thiol esters There are no reports on the heterologous expression of proteins in which the formation of a protein is a major post-translational modification.

The biosynthesis of the internal all ester of the third complement component (C3) in rabbits has been studied. There is. (I ij ima, M, et al. (1984) J, Bio-chem , 96: 1539-1546) Rabbit liver mRNA was lysed from rabbit reticulocytes. The C3-specific product synthesized by in vitro translation in the system is radiolabeled methane. Luamine was not included. On the other hand, radiolabeled iodoacetamide was synthesized reacted with a C3-specific product. These results indicate that free thiol groups are It is shown that it is present in the primary 03-specific translation product rather than the all ester. Ta. Contains liver homogenate supernatant (S-13) containing sidesils and microsomes. O3-specific products could incorporate methylamine if It was supposed to be. By increasing the concentration of the 5-13 component, the concentration of C3-specific products can be increased. Methylamine uptake increases and at the same time iodoacetamide uptake decreases. It was a little. When the 5-13 fraction was treated at 65°C for 5 minutes, its activity was completely lost. .

The results of this study indicate that the post-translational formation of active thiol esters in rabbit C3 requires It is strongly suggested that glutaminase-like or other types of enzymes be included. Ru. formation of thiol esters with other α-macroglobulins, e.g. α2M. Although there is no similar research aimed at Formation of thiol esters with other α-macroglobulins synthesized in the liver of animals It is expected that this is the cause.

Through this research, many developments were made that are considered to be within the scope of the present invention.

These include α-macroglobulin, especially α2-macroglobulin, fragments thereof, A DNA sequence containing a gene encoding the expression of a fragment, derivative, or variant, such as SEQ ID NO:1 and SEQ ID N0=3 are included.

Another feature of the invention is that at least one α-macroglobulin, in particular human α2- macroglobulin, or a fragment, derivative, or variant thereof, or any such gene. It relates to functional expression vectors containing genes encoding the expression of alleles.

Such vectors are further provided with the necessary controls for stable maintenance of the vector in mammalian cells. Preferably, it includes the element.

In addition, such vectors also contain sequences corresponding to the processing and secretion of the expression product. can be included.

Regarding the use of recombinant α-macroglobulin, especially 172M, in growth media, Other desired gene products may be co-expressed and, as a result, the vectors of the invention The gene can also contain one or more other genes encoding the desired gene product.

The present invention further provides human α2-macroglobulin or a fragment, derivative, variant, Alternatively, the machine of the present invention comprises a gene encoding the expression of an allele of such a gene. Transformed hosts containing a functional expression vector.

The host can be from the group consisting of bacterial strains, fungal strains, mammalian cell lines, and mammalian animals. can be selected, especially fungi, such as those belonging to the genus Aspergillus, or yeasts. strain, preferably one belonging to the genus Satucharomysis.

Other preferred types of hosts are mammalian cell lines, preferably Syrian vectors. Ivy Hamster Kidney (BHK) cell line, specifically N o, which can be obtained by specifying CRL1632.

The present invention further provides recombinant human α2-macroglobulin, SEQ ID NO: 2 and their active form with the amino acid sequence specified by SEQ ID No: 4. Concerning mutants.

α-macroglobulin, rα2-macroglobulin heart, use one The present invention discloses the application of α-macroglobulin, especially 172M. These are limited Recombinantly derived α-macroglobulin should not be considered as a It should be considered as one example of many uses.

α-macroglobulin as a stand-alone transformed or not Degradation of specific heterogeneous products produced in mammalian cell lines This is a big problem in generation. This is because many host cells contain one or more different proteins. This is due to the fact that it secretes rotinase.

When the production cell line is grown in the presence of, for example, 10% fetal calf serum, secretion Such proteolytic enzymatic degradation of recombinant or natural protein products This is only a minor problem due to the buffering effect of the serum proteins present.

However, the use of fetal bovine serum in large-scale propagation (fermentation) of mammalian cell lines is This is not desirable for many reasons.

First, fetal bovine serum is a very expensive component of complex growth media. Second, increase The demand for fetal bovine serum from the emerging biopharmaceutical industry will easily be met in the future. Thirdly, the use of fetal bovine serum is prohibited in the production of human drugs. with the potential problems of quality control required in production.

To overcome these problems, there are limited growth media for use with mammalian cells. We look forward to your efforts in the development field.

Upon addition of various proteinase inhibitors to such newly defined growth media, It is necessary to ensure integrity with secreted products. Alternatively, through genetic engineering, The proteinase inhibitor is produced and distributed along with the desired product in the cell line as a producer. It is also possible to give the ability to secrete secretions.

α-Macroglobulin, especially rαzM, inhibits blockinase with broad specificity. agent and therefore the product produced as a media additive or in conjunction with the desired product. As a composition, it may be used according to the invention as a component of growth media specifically for mammalian cells. used.

A number of different brotinases, e.g. bovine trypsin, streptomycin Liceus trypsin, papain, porcine elastase, bovine chymosin, ushikimoto Lipsin, Staphylococcus aureus strain ■8 brotinase, human plasmin, bovine toro Thermolysin, Subtilisin Novo, Strebtomysis griseu The attack target site of subrotinase B is human tBM (Mortensen, S, B. et al. (1981) FEBS Lett, 135:295-300) and other α-macroglobulins (Sottrup-Jensen).

L,, 5and+0. + Kr1stensen+L, and Fey, G, H, J, Biol, Chem, 264°15781-15789.1989) depression The area is mapped. α2M and other α-mas as proteinase inhibitors It is clear that cloglobulin has wide application specifications.

Blotinase inhibition spectrum of α-macroglobulin, e.g. α2M, is generated. In situations where prevention of degradation is not sufficient, site-specific mutagenesis, protein engineering, etc. et al., α-macroglobulin, such as α2M, was used as a blockinase inhibitor. It is possible to change the specificity of each. Desired specificity to the concave region of recombinant α2M Blocking mutated α2M by incorporating target proteinase attack sites The specificity as an agent changes. Furthermore, it has a specific or broad inhibition spectrum. To increase its versatility as a proteinase inhibitor, α- Novel specific or general proteinase attacks on the concave region of macroglobulins It is also possible to construct a target site. Furthermore, due to the action of natural proteinase inhibitors, Avoiding degradation of the variant in question by specific brotinases in more inhibited situations Therefore, it is also possible to remove the specific attack target site of α-macroglobulin. Ru.

Recombinant products in fungi, e.g. Aspergillus or Satucalomysis species or strains The production of will all be met with the potential problem of product decomposition. depending on the case It is also possible to isolate proteinase-negative mutants of the desired production strain. be. This is not always the case; α-macroglobulin, e.g. α2M or α ! M-mutant, by co-expression with the desired product, the protein of the product in question Degradability can be prevented.

The amino acid sequence of the concave region of α-macroglobulin is sensitive to various proteinases. determine the specificity of α-macroglobulin. Cutting patterns of various proteinases A comparison of the concave region sequences of five mammalian α-macroglobulins was recently published. It was done. (Sottrup-Jensen, L, +5and,, Kr1s tense, L, and Fey+G, H, The α-macroglobuli n bait region, 5 sequence diversity and 1 localization of cleavage 5ites for prote-inase in five n+ammalian α-mac roglobulins, J. Biol. Chem.

264, 15781-15789.1989) Recesses of each α-macroglobulin It has previously been shown that the has been done. The present invention provides human We demonstrate that the specificity of α2M as an inhibitor can be modulated.

In the present invention, the concave region of human α2M (residues 690 to 7 in SED ID NO: 2) 30) can be arbitrarily mutated to form a proteinase inhibitor of this macroglobulin. It has been demonstrated that it is possible to obtain new profiles as follows. The present invention provides The examples shown describe the construction of hybrid macroglobulins. This ha In hybrids, the concave region of the human pregnancy zone fluorophore (PZP) is M and then removed the natural concave region. The hybrid molecule is a recombinant D Although constructed using NA technology, the inhibitor profile is similar to that of PZP. revealed the brotinase inhibitor profile.

Thus, the present invention provides a blockinase inhibitor with modified or new inhibitor specifications. This demonstrates the possibility of arbitrarily designing and producing inhibitors.

This discovery is important for the design of new proteinase inhibitors. macrogro The concave region of brin has low antigenicity (VanLeaven, F., l'1aryn er++P,, Cassimam, J, -J, and Van den Ber- ghe+H, Mapping of st, structure-function relationships 1nproterns with a pan el of monoclonal antibodies, A, 5tud yon human alpha-2-macroglobulin, J, T mmunol, Methods ITT.

39-49.1988. and Delain, E., Barry+M,+Ta pon-Breaudiere+J,, Pochon, P, +Maryne n+P, , Cassiman, J, -J, , Van den Ber-gh e, H., and Van Leuven, F., The Molecular Or. ganization of Human alpha-2-Macroglob ulin, AnImmunoelectron m1cros-copic 5tudy with monoclonal antibodies, J. Biol, Chem.

263, 2981-2989.2981-2989.1988), so now the book The use of the technology described in the invention allows for the creation of new, non-immunogenic drugs that can be used, for example, in the treatment of diseases. It is possible to design new blockinase inhibitors. aggressive proteiner ze becomes a fear for human health.

Herein, we demonstrate the generation of α2M mutants by the construction of hybrid macroglobulins. This is a statement. However, those modified methods are also available, for example, International Publication No. HQ891 As stated in 06279 (NOVOINDLISTRI A/S), It is clear to those skilled in the art that it can be obtained by other genetic engineering methods. human α Instead of 2M, 5ottrup-Jensen, L., et al. (1989), supra. It is clear that other α-macroglobulins can also be used, such as those described in .

, 62, Another application of rα2Mα2M as a white carrier in the method is fluorescent white , as a carrier for macromolecules such as nucleic acids. α2M in solution When it reacts with α2M to form a complex, α2M is present in the solution. may bind to other operculum (which may be a non-proteinase protein). (Sal vesen, G. S., et al. (1981) Biochem, J.

195:453-461) X-chromoseme-related differences in glycosphingolipid metabolism In the case of Fabry's disease, which is common, α2M is Recently demonstrated to work as a carrier of enzyme exchange therapy (ERT) in vitro It was. (Osada, T. + et al., (1987) Biochen+, Biop Hys, f? es, Ccmw, 142: 100 106) αg M is tri Due to the action of psin, it is conjugated to coffee bean α-galactosidase and formed. The complex has α2M specificity (Van Leuven+F, 1 et al., (1981) 1 ．． Biol, Chem, 256:9016, 9022) to endocytox taken up and sent to lysosomes. Lysosomes contain α2M-protein is the target organelle for α2M-receptor-mediated uptake of the enzyme complex. (Willin gham, M.C., and Pa5tan, 1. , (1980) Cetl 2 1:6hu77) Such a plan in ERT allows the lysosotropy of the enzyme in question At the same time, the use of heterozygotes in therapy is provided. Potential antigenic problems arising from this are eliminated. One limitation of this ERT type (O Sada, T., et al. (1987).

Biochei, Biophys, Res, Commu, 142: 100 -106) are potential target cell types that can be treated with this regimen. clearly , they will express α2M-receptors. In future development of the system α2M by replacing the receptor binding region of α2M with other receptor ligands. The possibility exists to redesign the cell specificity of M uptake. As a result, α2M is Designed to enter all cells known to express specific uptake receptors. It can be measured.

This type of development would of course require a recombinant production system derived from α2M. ERT　 The use of natural human α2M as a carrier in (above) is not preferred. this is a well-known example of using blood derived from products in the treatment of human diseases. Because of the danger.

The production of recombinant α2M according to the present invention is accomplished by providing a method for mass production of 172M. to alleviate this problem.

Gene transfer into rα2M mammalian cells as a DNA carrier in gene therapy Advances are paving the way for the potential treatment of various genetic abnormalities through gene therapy. It was. The main problem in gene therapy is identifying the appropriate gene in the body. (Williamson, B. +1982, N. ature 298:416-418; Anderson, W. F., 19 84.5science226: 401-409; Parkman, R. 1986, 5science 232: 1373-In recent years, bacteria plus A structure containing chloramphenicol acetyltransferase (CAT) on the A synthetic foreign gene can attack the murine liver by internalizing it to specific receptors. It was announced that (Wu, G. Y., & Wu, C. H., 1988 ol, Chem, 263: 14621 14624). This DNA carrier , galactose-terminated (asialo)glycoprocovalently linked to poly-L-lysine Consists of Ting and Asialoorosomucoid.

This polylated L-lysine converts DNA into a strong non-covalent, non-damaging phase. Can be combined by interaction. Complexly bound DNA is unique to hepatocytes. can be internalized by cell surface asialoglycoprotein receptors. Ru. This complex was injected intravenously and analysis showed that only the liver showed CAT activity.

The present invention proposes the use of 172M as a DNA carrier in gene therapy. proposed. In the presence of covalently closed circular plasmid DNA, trypsin, etc. By reacting proteinase or methylamine with 172M, complex DNA is likely to be partially or completely captured in the α2M molecule. like this After intravenous injection of the conjugate with exposed receptor binding areas, the conjugate is rapidly removed from the blood. purified and specific target cells, such as hepatocytes or Kupffe. r) Internalized by cells etc. Protein technology for the 172M receptor binding region Through this process, it is possible to create DNA carriers specific to other cell types. Asia The advantage of this system compared to the above-mentioned systems that use glycoprotein receptors is that There is no need to identify different DNA carrier systems for each new cell type. .

Blood loss■ Hiroshi Hayashi Microorganisms and cell lineages E. coli K12 (MC1061), for example, is located in California, California, 92037. Stratagene, 3511099 North Tray Pines Road, Jola Available from Inc.

HepG2 (human liver tumor cell line) is freely available from ATCC (No. , HB8065).

BHK (Syrian hamster kidney cell line, thymidine kinase mutant line t) k’s 13) (Waechter and Baserga (1982) Pro c, Natl, Acad, Sci, US^79: 1106-1110) , freely available from ATCC (No. CRL 1632).

Plasmids and vectors 7” Las-; FpCDVI-PL and pSP62-2 are from 606 in Kyoto, Japan. Available from Dr. Tusk Hongjo of Kyoto University School of Medicine. psP62-2 is , plasmid pSP62-PL (Wedgwood Way, Steve nage, HerfordShire, 5G14ON's New Engla nd Nuclear/DuPont (UK) Inc.) was induced. pCDVUI-PL is pcDVI (Okayama+H, and Berg, P. (1983) Molec, Ce11. Biol,s: 2 80-2890 KAYAMA) from (Noma et al. (1986) Natur e, 319: 640-646) was induced as described.

Ml 3mp 18 is Pharmacia LKB Biotechnolo gy (catalog #27-1552-01). (Norrand Er.

J., Kempe, T., & Messing, J., Gene 26: 1 0101-106) 3mp 19 is, for example, International Biotechnolo-gies, Inc. P, O, Box 9558+ 275 Winchester Avenue, New Raven, Con necticut 06535+ Available from USA.

pDHFR-I was obtained from Dr. Niru Berkner, ZymoGenetics Inc, +4225 Roosevelt Way NE, 5eattle , Washington 98105. (The structure of this plasmid Built by Berkner, L., and Harp, P., H. (1984) N. detailed in ucleic Ac1ds Res, 12: 1925-1941. ing. ) Molecular cloning of the DHFR cDNA present in this plasmid, Its subcloning in mammalian expression vectors under the control of a denovirus-derived promoter -ing is detailed. (Chang, A, C, Y, etc., Nature 275: 617-624 and Kaufman + R, J and 5harp, P , A. (1982) Mo1. Ce11. Biol, 2:1304-13 19) The backbone plasmid of pDHF R-I is pBR322 ( Sutcliffe, J.G. (1979) Co1d Spring Ha rborSymp, Quant, Biol, 43: 77-90; 5ut Cliffe, J.G. (197B) Nuc-1eic Ac1ds Re s, 5:2721-2728) pUc13 Vieira, J. and Mes. sing+1. : 1982. Gene 19:259-268 , Pharmacia LKB Biotechnology (Catalog #2 7-4954-01).

pUc19 Yanisch-Perron, C., and Messing, J. , 1985+Gene 33: 103-119, Pharmac. ia LKB 8iotechno-1ogy (Catalog #27-4951- 01).

growth medium LB-broth Mix the following ingredients in a sealed plastic container to make a mix.

227g of Batatotributon (Dirco O123-01) 113.5g Yeast extract (Dirco O127-01) 227g NaCl Add 12.5g of the mix to 500ml of water in a 100 On+1 container (7), Shake well and sterilize in an autoclave.

For example, Dargenokos Modified Eagle Medium is available from Gibco Ltd. P, 0. Box 35. Trident House, Renfrew Road.

Pa1sley PA34HF, Renfrewshire, 5cotla nd, Cat, #042-250M (10x concentrated).

antibody Anti-α, MAO33 and peroxidase-conjugated anti-α2MPE326 Obtained from Mark's Cohenhage 7 DAKOPATTS A/S.

Fallen knee [ Cloning and distribution of human αM Human “’, HeG2 or” mRNA =. I+ human liver tumor cell line HepG 2 (ATCC No. HB 8065, freely available) was used as the source for mRNA preparation. 1 Dulbecco's Modified Eagle Medium with 0% Fetal Bovine Serum and Antibiotics HepG2 cells were cultured in soil until the total cell number was 15×10′.

Total RNA was extracted using the guanidinium thiocyanate method (Chirgivin et al., (197 9) Isolated by Biochemistry 18: 5293 5299), Purified on CsC1 gradient. A total of 3000 μg of RNA was obtained. Oligo (dT) -Cellulose column (Aviv & Leder (1972) Proc.

Natl, Acad, Sci, USA 69: 1408 1412) mRNA was isolated. After one cycle of affinity chromatography, 60 μg m RNA was obtained. After ethanol precipitation, the prepared mRNA was diluted with 10mM Tris. -HCI (pH 7, 5, 0, 1mM EDTA - Final concentration 1dg/μI N a) Resuspend at 80 °C until next use to construct cDNA library. Stored.

-cDNA library of cDNA live-1 from He G2 mRNA, The method described by Okayama & Berg (Mol.

Ce! 1. Biol, 2: 161-170 (1982); Mo1. Ce Biol, 3: 280-289 (1983) pCDVI-PL/ psP62-2 vector (Noma et al. (1986) Nature, 3 19: 640 646. 606 Dr. Tas of Kyoto University School of Medicine in Kyoto, Japan Can be obtained from Ku Hongjo. ).

E, co 1 i Kl 2 (MC1061) (cAdaban & Coh en (1980) J, Mo1. Biol, 138: 179-207) Used for transformation.

MC1061 0Dbb in L-broth at 37°C. Proliferated until -0.5 . Centrifuge 20ml and add the pellet to 7ml of water-cooled sterile 0.1M CaCl□. Resuspend, leave on ice for 30 minutes, centrifuge briefly, and finally keep in the cold room overnight. did.

Prepare a 95 μm suspension of transformation-reactive E. coli MC1061 cDNA 1. Added per O1 and II. Incubate on ice for 30 minutes and incubate at 43.5°C for 45 minutes. A heat shock was applied for 2 seconds, and finally, the cells were incubated at 37°C for 30 minutes after adding L-broth. .

After resuspension, plate broth containing ampicillin (50 μg/ml) for 8 hours. The cells were grown for 8 hours at 37°C. A total of 2.9X105 colors from this library Got a knee.

- Anal U Hisakage i Sakapu Dan Yu Sat jg hit α2M ecoff engineering and 2NA cloning Sukug 2 = 71” Standard colony high U using nitrocellulose filter Dization technology (Maniatis et al. (1982) Molecular Cl, on-ing -A Laboratory Manual, Co1 d Spring Harbor, Ne14York) with 5 x 10' colonies. was screened.

A 20-mer oligonucleotide mixture 5' CC (T/C) TTCAT (G/A)TC(T/C)TC(T/C)TG(T/C)TT3' [In the formula, ( X/Y) is the amino acid residue Lys-Gin-Glu-Asp-Met- Lys-Gly (Sottrup-Jensen et al., J, Biol, Che m.

259: Residues 493-499 as described in 8318-8327 (1984) Nucleic acid X or Y complementary to human α2M mRNA can be used in the region encoding means. ] (Applied Biosystems, USA DNA synthesizer) ) was synthesized (using T4 polynucleotide kinase and γ-32P-ATP) 32 P-labeled with 3×10”cpm/pmol oligonucleotide specific activity. did. This labeled oligonucleotide was purified by gel chromatography and cDNA It was used in the screening of A library.

Hybridization solutions were 6X SSC, 5X Denhardt's solution 's 5 solution), 0.05% SDS (Maniatis et al., ( 1982) Molecular Cloning-A Laborator y Manual, Cold Spring Harbor, New Yor k), contained a labeled oligonucleotide mix at 10'cpm/ml.

Hybridization was carried out at 45°C for 3 hours. Filter the filtrate in 6xSSC, 0,0 Washed with 5% SDS for 3 x 10 minutes at 45°C.

After autoradiography, the filtrate was washed under the same conditions (but at 52°C). This temperature Colonies that still showed hybridization were isolated and the corresponding plasmid The sequence of the cDNA insert (pα2M) was determined. cDNA sequence and its origins In the attached sequence list, enter the amino acid sequence of SEQ TDNo: 1. SEQ ID Shown as No. 2.

”-Color LyJ111 ratio Pa2M had a cDNA insert of approximately 4.6 kb.

The sequences are shown in Table I.

The sequences in Table I show that the entire coding region of a single peptide, including α2M, is in the inserted sequence. It is shown that.

In addition to the coding region, the insertion sequence includes 7. M 5'-, 3'-untranslated mRNA molecule Also includes translation area.

The amino acid sequence of human α2M derived from the cDNA in pα2M is the published sequence ( Sottrup-Jensen et al. (1984) J. Biol.

Che11+, 259:831B-8327). codon Number 1000 (numbered from the first methionine codon of the signal peptide) is ATC encodes isoleucine, and human liver mDNA (Kan et al. , (1985) Proc, Natl, Acad, Sci, tlSA, 82: GTC (2282-2286) found in α2M cDNA synthesized from It turned out that it was not a valine (coded for valine).

For the α2M cDNA sequence from the HepG2 library, the α2M cDNA sequence from the liver library Ten silent changes were identified compared to the sequence. Please refer to the following table ■.

Table I Codon Liver HepG2 413 (Asn) AACAAT 495 (Phe) TTT TTC 750 (Giy) GGG GGT 796 (Leu) CTT CTC 835 (Leu) CTT CTA 1266 (Ala) GCCGCA 1296 (Asn) AAT AAC1326 (Thr) ACCACA.

1442 (Leu) CTCCTG 1460 (IIe) Hybridization in ATCATT colony screening The position of the oligonucleotide mixture used as an ization probe is 157. 4 to 1594, and the position of the reactive thiol ester is 5EQID No: 1 It was 2939-2953.

pα2M was cleaved with Xbal and EcoRI (Figure 1a), and 2 markers were added to pSP62-. A 1.2 kb fragment containing the 5' portion of α2McDNA along with the cloning site was added. Garose gel isolated and Xbal/EcoRI cut Ml3mp19 vector was cloned to generate Ml3mp19A. α2McDNA sub The unique EcoRV site was added to the 1.2kb fragment to facilitate cloning. 10 nucleotides 5' to the translation initiation ATC (methionine) codon. Towards natural mutagenesis. (Kunkel et al., (1987) Methods En zymol, 154:367-382) In similar mutagenesis experiments, suddenly Mutagenic oligonucleotide N OR593: 5' (TTCTTCCCCC ATGGTGGATATCGAAGGAGCTG) The leotide 5'-methionine codon was changed to CCACCATG. As a result, A A new Nco1 site is created that targets the TG codon. Digestion with restriction enzymes confirmed the correct mutant M13mp19B through DNA sequencing. .

The mutated 5' end of azMcDNA was transformed with HindlII and Eco Ml 3mp 19A replication type was isolated through RI digestion and agarose gel electrophoresis. isolated from a child. This isolated DNA fragment was ligated to Hindll[/Eco It bound to RI-cleaved ptx2M to generate a pH of 36. In this plasmid, α 2M cDNA is reassembled over its entire length, but there is a unique EcDNA at the 5' end. It has an oRV site. Digest pH36 with EcoRV/DraI to obtain the α2M fragment. Isolated on agarose gel, the adenovirus 2-miyarate promoter (Ad 2MLP) was cloned in a mammalian expression vector.

An adenovirus-promoter based vector.

Berkner's teachings (ZymoGenetics Inc, +5eatl Constructed by e, WA,). This is a detailed description of the functional elements in mammalian expression vectors. A detailed description can be found in Po1vell, J.S., et al. (1986) Proc, Natl. , Acad, Sci, USA83: 6465 6469. Boel et al. ( 1987) FEBS Lett, 219: 181-188.

The expression vector used to express human α2M was a mammalian expression vector (Boel, E. , et al. (1987) FEBS Lett, 219: 181 188) caused it to occur. Ad 2 Human pancreatic polypeptide cDNA was cloned under the control of MLP. I took a loan.

ppp was cut with BamHI (Figure 1b), and the resulting complementary ends were digested with DNA polymerase. Repaired with (Flenow fragment and 4 deoxynucleotide triphosphates) Ta. By ligation, a 4.5 kb EcoRV/Dra I αz M cDNA fragment into this vector and the correct recombinant was isolated from a small amount (minipr ep,) plasmids were characterized by restriction enzyme analysis.

αzM transcribed from the resulting 8,76kb plasmid (referred to as pH67 in Figure 2) m D N A is adenovirus 2-rate tripartite reader (LL-3) has an mRNA splicing signal (SS) at the 5' end. At the 3' end, the transfer body is terminated by the SV40 rate termination and polyadenylation signals.

SV40 enhancer (ENH) and adenovirus from 5′ to Ad2MLP 5.

α2M in cells Cultured BHK cells [Syrian Hamster kidney] , thymidine kinase mutant system tK”s 13 (Waechter and Ba serga (1982) Proc, Natl, 8cad, sci, UsA 79: 1106-1110); American model bacterial culture collection (Aa+erica n Type Culture Co11ection) CRL 1632) For expression of human α2M in pDHF R-1, the expression vector pH67 was (Berkner, L. and Harp, P.A.

(1984) Nucleic Ac1ds Res, 12: 1925 1941. K. L. Berkner.

Zya+oGenetics Inc., obtained from 5eattle), along with Calcium phosphate-mediated transfection step (Grahata and Van de r　Eb(1973)　Virology　52:　456　467) by , and co-infected semiconfluent cells. In this transfection experiment, pH The molar ratio of 67 and pDHFR-1 was 1oll. Cells were mixed with 10% fetal bovine serum. The cells were grown in modified Eagle's medium supplemented with (Fe2). .

48 hours after transfection, the cells were trypsinized and It was diluted into medium containing 400 nM methotrexate (MTX). 10-1 After 2 days, individual colonies were cloned and propagated separately. this The propagation culture was grown for 24 hours as described above, and the producing clones were grown in this growth medium. Enzyme-linked immunosorption assay for secreted human α2M (ELIS) A) (Munk Petersen C, et al. (1985) 5cand , J. Cl1n, Lab. Invest. 45: 735-740). Identified it.

α2MELISA assay: The materials utilized for EL (SA) were as follows: capture antibody A033 anti-α2M; Peroxidase-conjugated anti-α2M antibody PE326.1.2-hue Nylene diamine dihydrochloride (OPD) (all of these are DAKOPATTS A/ S, obtained from Copenhagen, Denmark), Urea peroxide 125n+g; Obtained from Organon Teknika.

96-well ELISA plate; obtained from NUNC, Copenhagen.

Coating buffer: A 10011M carbonate buffer pH 9.6 was prepared as follows: water 1000+w Naz COz 3. 18 g and 5.96 g of N a HCOs were added. .

Buffers for standards and samples: 50 μm Tween 20. 2 g of bovine serum albumin (Sig+AA 7030) was added.

Wash buffer: 10mM sodium phosphate pH 7,4, 145mM sodium chloride, 0.1% Tween 20° Citric acid-phosphate buffer, pH 4,9: The following reagents were added to 10,100 O of water; 7.3g of citric acid.

Naz HPO4・12Hz O23, 88g 1 Tween 200.5ml.

This buffer solution was stored at 4°C and used for up to 14 days.

Urea peroxide solution: 125 mg of urea peroxide was dissolved in 8.93 ml of water.

This solution was stored in the dark at 4°C.

Coating the plate for assay: Coating this 96-well plate 175μ of DAKOAO33 antibody diluted to I:10000 in buffer It was coated with l. The plate was incubated overnight at 4°C. . The plate was washed four times in wash buffer before use.

Usage of standard products and samples: A 100 μm standard or sample was added to each well. As this standard product, purified h a:z M 2 mg/ml (Sottrup-Jensen et al., (198 3) Ann, N, Y, Acad, Sci, 421: 41-60. described in (prepared as described above) was utilized. This standard curve contains two 500 μg serial dilutions of /L and below corresponds to the maximum concentration of 1.95μg/L, I:4000, 1:8 000. From l:16000 and others to 1:1024000. For standard products and samples All dilutions were made with buffer. Incubate the plate overnight at 4°C. and then washed four times with wash buffer before the next step.

Addition of conjugated antibody: PE326 diluted 1:6000 in standard and sample buffer 100J! /] was added to each well. Incubate the plate for 2 hours at 20°C. The cells were dried and then washed four times with washing buffer.

Enzyme activation: 8 mg of OPD was dissolved in 12 ml of citric acid-phosphate buffer. in this solution 500 μl of urea peroxide solution was added and the mixture was used immediately. 100 Add μl of this final solution to each well and place the plate in the dark for 6 minutes. Incubated for a while. Then 100 μl of 2M H2SO4 was added to each well, Then, A4az was measured in an automated ELISA plate reader. .

The above ELISA is based on medium supplemented with 10% Fe2. showed no background in BHK cell conditioning medium. single Of the 24 isolated MTX-resistant colonies, 16 had detectable amounts of recombinant α2M. was produced.

Selected cell lines containing 12.3 mg/L in supernatant over 48 hours. (Kl 6-6) and 19. 1mg/L (K17-6) secreted (grown in 6-well NUNC-plates) in large quantities of recombinant human α2M (rαzM). bred for production.

Human α2M I+ Cell lines 16-6 and 17-6 each have a growth surface of 6000 cm". 1 ten double tray (NUNC+D enmark). Based on this cell at 80% confluency Change the medium from one containing 10% fetal serum (Fe2) to one containing 2%. Ta. After 48 hours of growth in medium containing only 2% (Fe2), this medium was removed. The cells were then washed twice with serum-free medium.

The cells were then grown in a blood-free environment for 4-5 days, changing the serum-free medium every 2 days. It was grown in purified water. Pool conditioned media and test by ELISA. We analyzed rα2M.

The pooled conditioned media from K16-6 and K17-6 were respectively It contained 7.15 mg/L and 21.5 lmg/L of 172M.

172M was purified according to published methods (Sottrup-Jense n et al. (1983) Ann, N. Y., Acad, Sci., 421: 4l. -60), briefly stated, this conditioned medium was 10 ml of Zn-chelate color equilibrated with Li-HCl pH 8.0 Mu (Zn”-Iminoniacetic acid Sepharose 4B (Porath, J, et al., 1975) Nature 258: 598-599), Ats Phosphate buffered saline (PBS) pH 7,2100m until o is less than 0.036 1. Add 20mM sodium phosphate until Azeo<0.033. A second wash was performed with thorium, 500mM NaCl pH 6.2. The flow rate is 1 00 ml/hr and both 3 ml aliquots were collected. rcx, M 100mM Elution was performed with EDTA pH 7.0 at a flow rate of 40 ml/hr. During elution Both 1 ml aliquots were collected.

The recovery rate of 172M was 44%. The rα2M-containing fraction was transferred to a PMIO membrane. Concentrate to 1 ml in an Amicon apparatus and then ose) 12 gel filtration columns (25mM Tris-HCl, 150mM Na C1 pH 8,0). The rα2M-containing fractions were pooled and analyzed. Stored at 20°C.

劃-”Yu , human rα M's A, heart with thiol ester: locus/fragment and methylamine Human r compared to a preparation of human plasma-derived α2M (labeled preparation LSJ39) A number of different analyzes were performed to evaluate the structural and biological properties of α2M.

An important structural feature of α2M is the presence of a thiol ester. 15 at 95°C When heated for a minute, this thiol ester forms a thiol in the backbone of α2M. It will induce cleavage of the peptide bond at the esterified Glx-residue. this thing is a 180 kD α2M monomer with two polypeptides of 120 kD and 60 kD. resulting in fragmentation into chidos. Figure 3 shows 10-20% SDS polyacrylic Purified rα2M (derived from two transformed BHK cell lines) and The results of both analyzes of purified human plasma-derived preparation LSJ39 are shown. put it on this gel different preparations, i.e. natural All recombinant α2M derived from BHK or BHK cells were heat treated. Molecular weight marker ( From the top, boarding house, 180, 120, 92, 60, 43, 26.14 and 6kD) This was applied to zones 1 and 8. Samples in lanes 2, 3 and 4 were subjected to electrophoresis. Not previously reduced and samples in lanes 5.6 and 7 were reduced. tone Product LSJ39 was applied to lanes 2 and 5. rα, M K16-6 is lane 3 and 6, and rα, M K17-6 was applied to lanes 4 and 7.

From the pattern of protein fragments on the gel, α2M and two types of rα2M were identified. Both products were found to exhibit a significant degree of thermal fragmentation. Forecast As expected, only the reduced sample showed this fragmentation. For non-reduced samples In this case, this molecule migrated as a 360 kD dimer.

In the human plasma-derived preparation LSJ39 (lane 5), a 60kD fragment was detected. A fragment was observed that migrated slightly faster than the sample. Lanes 6 and 7 are similar The results showed that the recombinant material had a fragment with a fast electrophoretic mobility. This fragmen The figure shows a slightly glycosylated 60 kD fragment variant. considered to be a thing.

Methylamine (MA) and other low molecular weight nitrogen-containing nucleophiles are thiol esters. would cause the ester to be cleaved and thus inactivate this ester (Sott up-Jensen, L. et al. (1980) Thermal flag of αzM after activation Mentation was not observed.

Figure 4 shows a 5DS-PAGE (spiked sample) performed similar to that shown in Figure 3. α2M and 172M applied here are predicted by MA. Preparations are being made. What can be considered from this gel is that 172M Teru is photosensitive to cleavage by MA, just like the natural αzM thiol ester. It is a certain thing. Based on the reduction, MA-treated α2M and 172M form a single 18 It was run as a 0 kD heptamer substance.

Lane 5 of both Figures 3 and 4 showed an additional band of approximately 85 kD. α2M is cleaved in the bait region by proteinases present in the blood. When combined, this results in the generation of two fragments each with a molecular weight of 85 kD. .

This human 72M preparation LSJ39 (purified from serum) does not contain such cleavage products. but they were not detected in the gels of these two rα2M preparations. It was. This indicates that the substances secreted from the transformed BHK cell line are primarily natural. This suggests that it is uncomplexed α2M. All α2M reacted with brotinase The molecule is inactivated and forms further complexes with other proteinases. I can't do it.

BHK cells are free from any proteinase that forms a complex with the rα2M product. This cell is well suited for the production of recombinant human α2M.

B, multiplication with l-lipsin Reaction with trypsin is a standard assay for the ability of α2M to form a blockinase complex. method (Sottrup-Jensen, L. (1987), 'Th e Plasma Protein” (Putnan, F. W. [Collection) 2nd edition, i:191-29L AcadellIic Press, 0rlando, FL; Harpel, P.C.

J, B. (1979) J. Biol, Chen+, 254: 4452 -4456), in this reaction, trypsin is activated in the decoy region of α2M. The target site(s) are cleaved and the resulting reduced cleavage product (85 kD) will migrate as a double band. Under non-reducing conditions, this Pushin-α, M? Ji coalescence will migrate as a high molecular weight product.

Figure 5 shows the natural human α2M preparation LSJ39 (lanes 2 and 5) and the cell line LSJ39. 6-6 (lanes 3 and 6) and 172M from 17-6 (lanes 4 and 7). We present the results of such an analysis ((Sottrup-Jensen, L., (1 987), 'The PlasmaProteins' (Putnam, F, W,,! ) 2nd edition, 5: 191 291. Acade-mi c Press, 0rlando, FL; Harpel, P, C, (197 3) J, Exp, Med.

138: 508-521; Harpel, P. C. et al. (1979) J. Biol, Chem, 254:8869-8878; Swenson, R , P., and Holvard, J. B. (1979) J. Biol.

Chem, 254:4452-4456). lane 2 Samples in °3 and 4 were not reduced before electrophoresis and were Samples in sections 5.6 and 7 were reduced. Lane 5 shows Haton All human natural α2M was cleaved by trypsin, but the two 172M The preparations are cut with an efficiency of approximately 80% or more. This complex Without reduction, trypsin and natural α2M or BHK cell-derived 172M No low molecular weight products from the reaction were observed on the gel. This recombinant substance The derived 85 kD fragment migrated somewhat faster than the human standard; As expected, this recombinant material may have undergone slight glycosylation.

When α2M is reacted with methylamine, this thiol ester is inactivated. , and α2M will change its form from the “slow” to the “fast” form ( Sottrup-Jensen+L, (1987), The Plasma Proteins (Putnaa+, F, W+m,) 2nd edition, 5: 19 1-291. Academic Press+ 0rlando, FL; V anLeuven, F., + Cassiman, J.-J., and Van Den. Berghe, H. (1981) J.

Biol, Chem, 256: 9016-9022), in this form In some cases, they react rapidly or in complexes with proteinases, e.g. trypsin. may become unable to form.

Figure 6 shows the results of a set of experiments conducted in parallel with the experiment shown in Figure 5 above. vinegar. However, the natural human α2M and 172M used in this experiment are It was treated with methylamine before the reaction with syn (Sottrup-J ense, t,.

(1980) FEBS Lett, 121: 275 280), Under these conditions, both native α2M and 172M are targets for trypsin. showed a favorable reactivity reduction (80% or more of α2M and rα2M monomers). - was run as a 180kcl polypeptide. From this, trypsin Adults in ra, M derived from methylamine-treated human α2M or BHK5 cells This suggests not to perform rapid cutting in the area.

In these types of experiments, BHK cell-derived 172M is similar to natural human α2M. showed similar characteristics.

C,) Conformation in α2M excited by lipsin and methylamine N・1 As mentioned above, α2M molecules are involved in complex formation with proteinases and thiol esters. Methylamine-induced splitting of the tail results in a conformational shift. saw in the structure displacement results in a change in the mobility of the rate gel (Sottrup-Jense n, L., 1987, 'The Plasma Proteins (Put nam, F.W., ed.)'2nd edition, 5゜pages 191-291, Acade. mic Press, 0rlando, FL; Van Leu-ven, F., Cassiman, J.-J., and Van Den Bergh e, tl, 1981, J, Biol, (:hem,, 256:9016- 9022 pages; unreacted αz M moves in a “slow” manner, and reacted α2 M moves in a "fast" manner.

Figures 7 and 8 show these conformational displacements, and these displacements are (5-10% level) after reaction with trypsin and methylamine respectively. (by gel analysis).

Lane 1 of both gels is purified human pregnancy zone protein (PZP) C3. and, 0 et al., (1985) J. Biol. Chem. 260: 157 23-1.5735), which has dimeric (D) and tetrameric (T) structures. It is known that it appears to have both.

Lane 2 for both gels contains unreacted human αzMiFI agent LSJ39. both Lane 3, which involves the gel, shows the fast migrating form and is associated with trypsin and methylamine, respectively. Resulting from reaction with min. Lane 4 for both gels is for unreacted 172M preparation. 16-6 and lane 5 shows the corresponding early form. Lane 6 for both gels shows the unreacted 1・α2M preparation K 17-6, and lane 7 shows the corresponding early form. show.

Complex formation of 172M and trypsin and reaction of 172M with methylamine It can be concluded that the emergence of faster moving structures occurs in both . . These structures allow human α2M to react with trypsin and methylamine. The structure is very similar to that obtained when I can do it. These figures also show that the rα2M protein is present in the gel dimeric and tetrameric PZP. When compared to the movement of these molecules in the active tetrameric conformation, We demonstrated that BHK cells exhibit a migration pattern that is consistent with the fact that they are produced and secreted from BHK cells. it is obvious.

D, α2M chromatography using Superrose 6 column = Z/Nini The Superrose 6 column converts molecules in a tetrameric structure to α in a dimeric structure. 2M molecules can be partially decomposed (Sottrup-Jensen, L. ,unpublished). Human standard α2M and 172M are 241n1 Superrose 6 colors (Buffer: 25mM Tory HCl, 125mM NaCl, p H8,0; Flow rate: 1 precept/min; Fractional size: 1d). Figure 9 shows KIT-6 and NI-1. 6-6 Human standard α2M and 172M chromatograph purified from BHK cell line A diagram obtained by Rafi is shown. Tetramer cxz M (Sottrup-Jens en, unpublished record) elutes in fraction 12 of this type of column. This horn The romatogram shows that the 172M preparation was It is clear that the fraction 12 was eluted. With this type of column, dimer α2M molecules elute in fractions 14 and 15 (Sottrup-Jens en, unpublished record).

The results of this analysis indicate that 172M was secreted from BHK cells in a tetrameric structure. This supports the results obtained from the rate gels (Figures 7 and 8).

E, l-ribusin＼ When trypsin is entrapped in an α2M molecule, its catalytic ability is enhanced by a low molecular weight substrate, e.g. S-2222 (N-benzoyl-L-11e-L-Glu-Gly- L-ArgI)-nitroanilide). Trypsin is α2 When effectively complexed with M, high molecular weight inhibitors such as soybean trypsin inhibitor ( STI) (Sottrup-Jensen, L., 1987, 'The Plasma Prote-ins”, Putnam, F.W., ed. 2nd edition, 5.191-291 pages, Academic Press+ 0rla ndo, FL: Ganrot+P, 0.1966, Cl1n.

Chim, octa, 14.493 501 pages; 5ottrup-Je nsen, L. et al., 1981, FEBS Lett, 128.127-1 page 32).

rcx2M derived from K16-6 and 17-6 was detected in humans by protection assays. It was compared with Toplasma α2M. 100ul of (rzM (25mM tree) HCl, 125mM NaC1° in pH 8,0) and 30μM trypsin ( 0.0 in 20mM acetate acetate, pH 5.0. 5mg/rd! ,) mixed with did. After 2 minutes of incubation, 30 μl of 1 mg/m 5TI (in PBS) was added. . 10 μm aliquots were removed after 2 and 4 minutes, each injected with 750 μl, 0 ．． 12mM S-222 (0.1M sodium phosphate pH 8, 0.5% dimethyl (dissolved in Rusulfoxide).

Absorption changes at 405 nm were recorded for 2 minutes. Table 1 shows the results of this assay. Shown below.

Table 1 Human LS J 39 0.140 5.00 0.02BK 16-6 0. 111. 4.62 0.024K 17-6 0.119 4.87 0.0 24 From this result, 172M has a higher protective ability in ST than human plasma α2M. It is concluded that the protective potency against I is substantially the same.

Treatment of α2M with methylamine before the protection assay dramatically reduced the protective capacity. Ru. In an assay similar to that described above, human plasma treated with methylamine Only α2M retained 17% of its protective capacity, while 16-6 and 17-6 72M were 16% and 14%, respectively. Thus protected by 172M , Trypsin for STI has almost the same function as human plasma α2M. Theoretically, α2M characterized by this study is simply a hum Calf α2M (serum impurity) from star (endogenous material from BHK cells), Alternatively, it may be derived from the transformation plasmid pH67. Adopted ELI According to the SA assay, BHK α2M cannot be observed in the medium of the cellular state. The α2M studied is human α 2M and to characterize the amino-terminal processing of the recombinant. , amino-terminal amino acid sequencing was performed as described (Sottrup -Jensen, L et al., 1984. J, Biol, Chem, 259 ; 8293-8303), Kl 6-6 and 17-6 ra, M Ta.

The Edman decomposition is repeated for 16 cycles, and the detected atoms in each cycle are The results of the identification of amino acid derivatives are the amino terminal sequence of human α2M: Ser-Val -5er -Gly -Lys -Pro -Gin -Tyr -Met -Val　-Leu　-Val-1, while calf α2M is Amino terminal sequence: Ala-Val-8sp-Gly-Lys-Pro -Gin -Tyr -Met -Val -Leu-Val ( Unpublished, Dr. Torsten Christensen, Department of Molecular Biology, Arhas University, Denmark. ).

■In this experiment, human α2M bait region The area is the bait region of human pregnancy protein (PZP) (Sottrup Jens en, L, , Folker-sen+J, , Kr1stensen, T, O and Tuck, B.F., partial-substructure of human pregnancy protein: human alpha. Extensive sequence homology with macroglobulin (Partial primary y 5 structure of human pregnancy zone p rotein: Extensive 5equence homology inith human alpha 2-macroglobulin), Proc, Natl, Acad, Sci.

U, S, A, 81 7353 7357+ 1984; 5 and 0. , F olkersen, J.

Wes tergaard lJ, G, and 5ottrup Jens en, L, characteristics of human pregnancy protein, human alpha 2-macroglobulin and Comparison (Charactertzation of hu+wan pregna ncy zone protein.

Comparison with human alpha 2-+++ acr oglobulin), J. Biol.

Starvation, casting 0.15723-15735.1985), obtained α2M bait area The mutant exhibits similar behavior to that of human pregnancy protein protease inhibitory activity. show movement.

Promotes replacement of the DNA fragment encoding the bait region of human αtM cDNA For further development, target sites for the restriction enzymes PstT and 5acU were added to the bait region. was inserted into the 5' and 3' ends of the cDNAjii region encoding.

Human α2M expression plasmid pH67 was digested with BamHT and C1al, and human α, A 2660bp plasmid carrying the central part of M cDNA was added to BamHr and CIa It was subcloned into the T-digested vector psXL91.

This pre-constructed vector is a derivative of pUcl9. This thing is not listed Constructed as shown. In other words, erase PUC19 with EcoRI and HtndI[I. and cloned a synthetic linker with the following sequence into the digested pUc19 vector. I clicked. Annealin derived from synthetic oligonucleotides N0R781 and N0R782 This linker, which is a product of pUcl, EcoRI and HindI [[parts of pUC in such a way that the ligation sites at the position are reproduced in the psX191 vector. It has a cohesive end that can be linked to each of the 19 sites. In this way, psX19 1 is x engineering'', P s t I, E c o RI.

HindIII, C1a I, 5. Parts for p2-EI and 13amHI have the rank of

The resulting plasmid pSX191α2M was digested with BamHI and HindI[I. The purified BamHI/HindnIα 2M fragment 2,6 kb was then purified in Ml 3mp1.M for mutagenesis by the method described. 13mp18α2M was produced. The following array: 5’ (TTCATACTGCTGCAGCTGTGGACAC) 3’ 2 of the above cDNA sequence using a synthetic oligonucleotide N0R973 with Insert into the PstI site at position 102 (SEQ ID No: 1), then insert the Sequence: 5’ (AGCCACCCCCGCGGAGTTTACCAC) 3 of the above cDNA sequence using an oligonucleotide (NOR974) with No mutation in the amino acid sequence at position 2271 (SEQ ID NO:l) and they are convenient for the bait 6N region of the human α,M cDNA. within good distance. Both primers were used in the mutagenesis experiments described above (Kun kel, T., 8, + Roberts, J.D., and Zakciur, R. , A, rapid and effective site-directed mutagenesis without the need for phenotypic selection (R apid and Efficient 5ite-5specific Mut genesis with Phenotypic 5 selection ), Methods in Enz mol, 15C367-382, 198 7), ds DNA was mutated from Ml 3mp 18a2M plaque. This DNA was then digested with restriction enzymes PsjI and 5acll. 160bp i Correctly mutated recombinants containing the insert were further analyzed by DNA sequencing. (Tabor, S., and Chardso, C.C.) DNA sequence analysis using Liophage T7 DNA polymerase (DNAsequ ence analysis with a modified bacter iophage T7 DNA polymerase), Proc, Natl, Acad, Sci, U, S, A, 84+ 4767-4771+1987) . Correct (XzM cDNA variant (M13mp18αz M# 212, 1) BamHT/HindI[I fragment 2.6kb derived from BamHI /HindlII digested pUc13 vector and then the appropriate Subclone p1308 was isolated and BamHI/HindII [and S　a　c　Specialized by IT double digestion and DNA electrophoresis The sex has been determined.

Excise the PstI/5acII7 fragment of p130B and then insert the bait region. It can be replaced with another DNA fragment encoding the variant. α obtained in this way A novel variant (bait region variant or analogue) of 2M cDNA is BamHI / for Hi-1-PZP (5ottrup-Jensen et al., mentioned above) in this example. The DNA encoding the amino acids of the bait region is composed of eight synthetic oligonucleotides. obtained from ligage gin, annealing and cloning.

This synthetic fragment and the encoded amino acid as inserted into the α2M clone The DNA sequence of the amino acids is given as SEQ TDNO:3, and is 2107-2. Contains the amino acid corresponding to position 305. Ps at the 5' end of the synthetic fragment A tI site was inserted, and a 5acII and BamH1 site was inserted at the 3' end.

This synthetic NA fragment of 0.2 kb was used for DNA sequencing as Pstl/ Cloned into BamHI digested Ml 3mp I F3 hector. Appropriate distribution DNA from clones containing the sequence was digested with PstI and 5acII, then The purified 0.2 kb fragment was digested with PstI/5acff and run on a gel. Cloned into purified p1308 vector. Proper recombinant p267P ZP was characterized from this plasmid with restriction enzymes, and the bait 6N region was mutated (α 2M-+PZP) cDNA is a 2.7 kb BamHI/C1aI fragment. and then BamHI/C1a digested α2M expression hector pH 67. subcloned. Digested p1365 of the resulting plasmid was analyzed on a large scale. plasmid preparation method, purified by CsC1 centrifugation, and then pDH BHK cells were co-transfected with FR-1.

Through this operation, nucleotides 2102-2275 in SEQ ID NO:3 and replaced with nucleotides 2102-2305 in SEQ ID NO:3 did.

Transfection, bait region mutation α2M (digested rtx2M-PZP) Selection of recombinants (by crz M-specific ELISA), large-scale production and Purification of mutant α2M is described elsewhere in this application (Example 2).

Protein of human α2M bait 2, 91 pi L round size Purified recombinant α2M mutant rα2M-PZP was purified using a previously described method (Sa nd et al., 1985) to achieve inhibitory specificity for various proteolytic enzymes. I characterized it. In a comparison of α2M and PZP derived from human plasma, they were treated with a series of proteolytic enzymes in parallel reactions. Proteolytic enzyme used is chymotrypsin, elastase, trypsin and Staphylococcus aureus. Glu specific protein of Staphylococcusaureus It was a degrading enzyme. Chymotrypsin and elastase are associated with PZP and αzM. also shows a rapid reaction, but two protease inhibitors and trypsin and starchase The reaction between the Glu-specific protease of Phyllococcus aureus is P ZP and α2M are completely different (both proteases rapidly interact with α2M). but the reaction with PZP is slow) has already been reported (Sand et al. 1985). The reason for this difference in reaction rate with different proteolytic enzymes is , the PayFeI region in PZP is potent against chymotrypsin and elastase. Contains specificity determinants, but trypsin and Staphylococcus aureus G This is believed to be due to the fact that this is not the case for lu-specific proteolytic enzymes. It is.

The results of the analysis are shown in Figures 10-13.

Figure 10 shows chymotrypsin-treated human α2M, human PZP and 172M-PZP. Discloses gel electrophoresis (10-20% reduction 5DS-PAGE) of reaction products from do. Molecular weight markers (from top to bottom: 180, 120, 92°60. 43, 26.14 and 6kD) were placed in lanes 1 and 8. all samples was returned. Lanes 2, 3 and 4 are for chymotrypsin and human plasma, respectively. Next PZP.

Cleavage product obtained by reaction with 172M-PZP and human plasma-derived α2M show something The ratio of proteolytic enzyme to inhibitor was 1:1. lane 5 , 6 and 7 are the same ratio of 2=1 between the proteolytic enzyme and the above three inhibitors. shows the cleavage products of various reactions. In all six lanes, the cleavage product (85 kD) could be identified. This word, 172M-PZP reacts with chymotrypsin. showed that it reacts in the same manner as α2M and PZP derived from human plasma.

Figure 11 shows elastase-treated human α2M1 human PZP and 172M-PZP derivatives. Gel electrophoresis of the previous reaction product (showing 10-20% denatured 5DS-PA (1,E)) vinegar. The molecular weight markers were the same as those applied on the gel in FIG. all sample was reduced. Lanes 2.3 and 4 are human plasma-derived P Reaction of ZP, rα2M-PZP and human plasma-derived α2M with elastase The decomposition products obtained from The ratio of protease to inhibitor is It was 1:1. Lanes 5, 6 and 7 show protease and three test inhibitors. This shows a decomposition product obtained by similarly reacting with a 2:1 ratio of 2 to 1. all 6 lanes A degradation product (85 kD) was identified in the sample. This means that 172M-PZ P has similar properties as human plasma-derived α2M and PZP reacted similarly. indicates that it has reacted with elastase.

Figure 12 shows the reaction of trypsinized human α2M1 human PZP and rα, M-PZP. Gel electrophoresis (10-20% denaturing 5DS-PAGE) of the reaction product is shown. molecular weight The markers were similar to those applied on the gel in Figure 2. Lane 2.3 and 4 are human plasmid-derived PZP, human plasmid-derived α2M and and shows the decomposition products obtained by the reaction of 172M-PZP and trypsin. . The ratio of protease to inhibitor was 1:1. lane 5゜6 and 7 for a similar 2:1 ratio of protease to three test inhibitors. It is a decomposition product obtained from the reaction.

In lanes 3 and 6, the degradation product (85 kD) was synthesized by trypsin and α2M. Identification was possible from the reaction. In lanes 2, 4.5 and 7, trypsin and PZP No decomposition products were observed from the reaction with PZP and 172M-PZP. As a result does not react as much with trypsin as human plasma-derived PZP did. demonstrated that α2M was degraded during the reaction with trypsin.

Gel electrophoresis of reaction products from h1-PZ, P and rα2MPZP (10~ 20% denaturing 5DS-PAGE). Molecular weight markers applied to the gel in Figure 2 It was the same as the one I did. All samples were reduced. Lanes 2, 3 and 4 is obtained by reaction with PZP, 172M-PZP and human plasma-derived α2M. The decomposition products are shown below. The ratio of protease to inhibitor is 1:1 Met. Lanes 5°6 and 7 are two of the protease and three tested inhibitors. Decomposition products reacted in a similar manner at a ratio of 1 to 1 are shown. In lanes 4 and 7, minutes The decomposition product (85 kD) From the reaction with Glu-specific protease and α2M I was able to identify it. In lanes 2°3.5 and 6, this protease, PZP and 1 Much fewer decomposition products could be identified from the reaction with 72M-PZP. As a result As human plasma-derived PZP reacted, 172M-pzp reacted with staphylococcus. Aureus (Stah once ococcus...) protease and remainder It has been demonstrated that α2M does not react with this protease, whereas α2M does not react with this protease. Disassembled.

172M-PZP is similar to human plasma-derived PZP and four types of proteases. It was concluded that the reaction pattern was as follows. This reaction pattern is similar to α2M. This was different from the correspondence pattern obtained from the reactions. Therefore, 172M-PZP is a natural Has protease inhibitor tendencies similar to PZP and not similar to α2M This has been proven. Therefore, the protease inhibitor tendency of α2M is It has been demonstrated that the region can be modified by substitution of the DNA encoding the region.

As explained in the present invention, the substitutions do not impair the activity of the protease inhibitor. There wasn't. Furthermore, the functional macroglobulin hybrid therefore contains in the beta region, It is demonstrated that it can be constructed by substitution (mutation). This finding provides a new and desired This will guide the design of α2M-derivatives with proteases. Without a doubt, These results can be extended to other macropurine-based hybrids, and this /’t The substrate region can be freely modified to create novel inhibitor properties. You can get the opposite sex.

The aggressive activity of proteases is often a problem with respect to various diseases (e.g. For example, the activity of elastase and cathepsin G in some inflammations (leading to organ destruction and deterioration). Inhibitors of such proteases may be useful in drug design. The target site for protease is In the known situation, no inhibitors can be identified and α2M has the desired specificity. can be manipulated to obtain specificity (can be mutated in the beta region). of the protease in question. In situations where target specificity is not known, saturation mutagenesis or randomization of the base region is recommended. dam synthesis is an unrelated protein that can be introduced and further expressed into hybrid ndomacroglobulin. yielding a specific number of target sequences). These hybrids are protea can be screened for enzyme inhibition. The target sequence can then be identified. obtained α2M congeners can be prepared and purified as described elsewhere in the invention. When injected into the ring, such a 72M congener binds a protein with a given specificity. It will prevent aase from the blood and make it clear.

When a protein homolog or variant is introduced into the human body, the potential for antigenicity is Always rising. Panel of 45 mouse monoclonal antibodies against human α2M The occurrence of 1988). None of these antibodies were directed against the bait region. child This indicates that the bait region is not highly antigenic and furthermore Variants have arisen in the region that can be used therapeutically without further risk to antibody development. It shows that it can be used.

Sequence list (1) General information (i) Applicant: Novo Nordisk A/5 (11, Title of invention: Plasma Guru Coprotein expression (iii) Number of sequences: 4 (iv) Communication destination (A) Address Ninovo Nordisk A/S, Patent Department (B) Street Ninobo Alley (C) City: Hazgpaert (D) Country: Denmark (F) Gimp: DK-2880 (vi) Priority filing date: (A) Application number: DK 4285/89. DK 4236/89゜DK 4 237/89 (B) Application date: 29-AUG-1989 (2) Information for sequence ID NO:1 :(i) Sequence characteristics (A) Length: 4569 base pairs (B) type: Nucleic acid (C) Number of chains (D) Topology: linear (ii) Number of molecules: cDNA (iii) Hibocetical 2N (vi) Origin (A) Organism name: Homo sapiens (F) Type of Mll weave: hepatic (G) Cell type: Germinoma (H) Cell type: HepG2 (ix) Characteristics of arrays (A) Name/Symbol: CD5 (B) Position: 29. ．． 4450 (D) Other information: (xi) Sequence: SEQ ID NO: TACACA GGG AGCCGCT CT GCCTCCAACATG GCG ATCGTT GAT GTG A AG 4180AGTGCTTTGCTGGAGTCCTG TTCTC Ding GA GCTCCACAGAAG ACACGTGTTT TTGTATCTTT@4 530 AAAGACTTGA TGAAdingAAACA CTTTTTCTGG TCA AAAAAA 4569 (2) Information for SEQ iD NO:2: (i) Distribution Column characteristics (A) Length: 1474 amino acids (B) type diamino acid (D) Topology: linear (E) Features: Bait area: 69o~73° (ii) Molecule type: Protein analogy (xi) Sequence: SEQ ID NO: 2: Met Gly Lys Asn L ys Leu LE! 11 His Pro Ser Leu Vat Leu Leu Leu L■■ l　5　10　15 Lys Phe Arg Val Va] Ser M Asp Glu Asn Phe His Pro Leu Asn GluArg Ala Ser Val Ser Val Leu Gly Asp Ile Leu Gly Ser ATa Met G1nPhe Ile Asp Glu Ala H is　Ile　Thr　Gln　A]a　Leu　Ile　Trp　Leu　S er　G1n0Ar　(+In　Lys　Asp　Asn　GTy　Cys　P he　Arg　Ser　Ser　Gly　Ser　Leu　Leu　As■ VaT Arg Asn Ala Leu Phe CyS Leu Glu Ser Ala Trp Lys Thr Ala Gin]]25]Co30 1135 (2) Information for SEQ ID NO:3 = (i) Characteristics of array (A) Length: 4599 base pairs (B) type: Nucleic acid (C) Number of strands Knee end strand (D) Topology bilinear (ii) Molecule type: cDNA-mRNA (ji) Hybocertical: Y (iv) Antisense IIN (vi) Origin: (A) Organism name: Homo sapiens (ix) Features: (A) Name/Symbol: CD5 (B) Position=29. ．． 448'0 (D) Other information: (ix) Features (A) Name/Symbol: Insert 5eq (B) Position 82102. ．． 2305 (D) Other information: (xi) Sequence: SEQ ID NO: 3: GAA GAT TCA CAG G CT TTCTGT GAG AAA TTCAGT GGA CAG CTA AACAGC916TCA AAT CAG ACA CTG AGCTTG TTCTTCACG GTT CTG CAA GAT GTCCCA 43 7Q seu GTy Asn Ala TCTCTGAGCTCCACAGAAG ACACCTGTT TTGT ATCTTT AAAGACT C, A TGAATAAAAbA -4580 CTTTTTCTGG TCAAAAAAAA 4599 (2) SEQ ID N Information for O:4: (i) Characteristics of the array (A) Length: 1484 amino acids (B) type diamino acid (D) Topology: linear (E) Features: Bait region: 690-740 (ii) Molecule type: Protein (xi) Sequence: SEQ ID NO: 4:11e Leu Asn Gly G ly　Thr　Leu　Leu　Gly　L[! 11 Lys 1-ys Le u　Ser　Phe　syr Tyr Leu rle Met Ala Lys Gly Gly Tle Va] Arg Thr Gly Thr His GlySer Val Cy s Ala Leu Arg ATa VaT Asp G1r+ Ser V at Leu Leu Met Ly■ Asp Cys Ile Asn Arg His Asn Val Tyr lie Asn Gly Ile Thr Tyr Thr Gly Thr T yr Asn Val ITe Pro Leu Asn Asn Glu G in Ser Ser GTy Pr.

Val Pro Glu Thr Val Arg Lys Tyr Phe Pro Glu Thr Trp ITe Trp AspPro Phe P he Val GTu Leu Thr Met Pro Tyr Ser V aT Ile Arg Gly Glu805 8] 0 815 Leu Gly Asp Ile Leu Gly Ser Ala Met Gln　Asn　Thr　Gln　Asn　Leu　LeuVal　Lys　L ys Asp Asn Ser Val His Trp Glu Arg P ro Gln Lys Pro LysArg Thr Gly Lys Al a Ala Gln Val Thr Ile Gln Ser Ser Gl y Thr PheSer Ser Lys Phe Gln Val Asp Asn Asn Asn Arg Leu Leu Leu Gln G1n 1300 1305 +310 Glu Lys Glu Glu Phe Pro Phe Ala Leu Gly Val G]n Thr Leu Pro G1n1345, 13 50 1355 1360Thr Cys Asp Glu Pro Lys Ala　His　Thr　Ser　Phe　Gln　I]e　Ser　Leu　 5erVal Leu Ile Tyr Leu Asp Lys Val S er　Asn　Gln　Thr　Leu　Ser　Leu　PheHindll l F Engineering G, IA FIG, IB FIG.2 11-111” 360 KD FIG.3 FIG.4 FIG.5 FIG.6 FIG.7 FIG.8 Fraction 12 ↓ FIG. 10 FIG. 11 1 23'45678 FIG. 12 FIG. 13 Procedural amendment (formality) March 13, 1992

Claims (1)

[Claims] 1. In a method for producing recombinant α-macroglobulin, its variants, fragments, and derivatives hand, alpha-macroglobulin, variants, fragments, derivatives thereof, or alleles of such genes; A functional expression vector containing a gene encoding the expression of the gene is Introduce it into a suitable host that can express The host is cultured in a suitable nutrient medium containing assimilable carbon, nitrogen sources and other essential nutrients. death, Collecting expressed α-macroglobulin or its fragments and derivatives The method comprising: 2. If the gene is human α2-macroglobulin, or a variant, fragment, or derivative thereof 3. The method of claim 2, wherein the method encodes the expression of a protein. 3. The human gene comprises the amino acid sequence identified as SEQ ID NO: 2. The claimed material encodes the expression of α2-macroglobulin, or a fragment or derivative thereof. The method described in Scope No. 2. 4. The gene has a DNA sequence specified as SEQ ID NO: 1, or 4. A method according to claim 2 or 3, comprising a fragment. 5. The gene has a changed amino acid sequence in the prey region of α-macroglobulin. 3. The method of claim 1 or 2, wherein the method encodes a variant. 6. The bait area has been altered by the introduction of additional attack target sites for proteinases. 6. The method according to claim 5. 7. The bait area has been altered by removal of the proteinase attack target site. The method described in item 5 of the scope of the request. 8. The bait region targets one or more specific attack target sites for the proteinase. The claimant has been altered by substituting one or more other specific attack target sites of the The method described in item 5 of the scope of the request. 9. The specific attack target sites of proteinases are bovine trypsin, streptococcal Mycis chryseus trypsin, papain, porcine elastase, bovine chymosin, Bovine chymotrypsin, Staphylococcus aureus strain V8 proteinase, human plasmin , bovine thrombin, thermolysin, subtilisin NOVO, and/or stress Claim no. 10. The bait region connects the bait region or a portion thereof to another α-macroglobulin bait region or a portion thereof. 6. The method of claim 5, wherein: is changed by substituting a part thereof. 11. The bait region contains human α2M, pregnancy zone protein (PZP), rat α1M, rat α2M, rat α1I3 mutant 1, or rat α1I3 mutant 2 (α1I3=α1-inhibitor 3), in particular PZP, claim 10 The method described in section. 12. The gene has the amino acid sequence supported by SEQ ID NO: 4. 2- Encoding the expression of a variant of macroglobulin, or a fragment or derivative thereof 12. The method according to any one of claims 5 to 11. 13. If the gene is a DNA sequence specified as SEQ ID NO: 3 or 13. A method according to any of claims 5 to 12, comprising a fragment of. 14. Any one of claims 1 to 13, wherein the gene is a synthetic gene. Method described. 15. The α-macroglobulin, its variant, fragment or derivative is a desired gene. A method according to any of claims 1 to 14, expressed with a product. . 16. The claimed gene is a human gene or is derived therefrom. The method according to any one of the ranges 1 to 15. 17. The host is a bacterial strain, a fungal strain, a mammalian cell line, or a mammal. 17. The method according to any one of claims 1 to 16. 18. 18. The method of claim 17, wherein the host is a fungus. 19. 19. The method according to claim 18, wherein the fungus belongs to the genus Aspergillus. 20. 19. The method of claim 18, wherein the host is yeast. 21. 21. The method according to claim 20, wherein the yeast belongs to the genus Saccharomysis. 22. 18. The method of claim 17, wherein said host is a mammalian cell line. 23. The mammalian cell line is the Syrian baby hamster kidney (BKH) cell line. 23. The method of claim 22, wherein the method is a system. 24. The cell line was awarded No. 1 by ATCC. Available as CRL1632, Claim The method according to item 23. 25. Encoding the expression of α-macroglobulin, its variants, fragments or derivatives. A DNA sequence containing a gene. 26. Claim 2, wherein the gene encodes human α2-macroglobulin. DNA sequence according to item 5. 27. The gene has an amino acid sequence identified as SEQ ID NO: 2, or 26. A DNA sequence according to claim 25 encoding a fragment or derivative thereof. 28. If the gene is a DNA sequence specified as SEQ ID NO: 1 or 28. A DNA sequence according to claim 26 or 27, comprising a fragment of. 29. The gene has a changed amino acid sequence in the bait region of α-macroglobulin. The DNA sequence according to claim 25 or 26, which encodes a variant of . 30. The bait area is altered by the introduction of additional attack target sites for proteinases. 30. The DNA sequence of claim 29. 31. The bait area has been altered by removal of the proteinase attack target site. , the method according to claim 5. 32. The bait region targets one or more specific attack target sites for proteinases. has been altered by replacing one or more other specific attack target sites of the A DNA sequence according to claim 29. 33. The specific attack target site of proteinase is bovine trypsin, strep tomysischryseus trypsin, papain, porcine elastase, bovine chymosin , bovine chymotrypsin, Staphylococcus aureus strain V8 proteinase, human plasmid bovine thrombin, thermolysin, subtilisin NOVO, and/or The claim is specific for Leptomyces chrysaeus proteinase B. DNA sequence according to paragraph 29. 34. the bait region or the bait region or a portion thereof to another α-macroglobulin bait region or DN according to claim 29, wherein the DN is changed by replacing a part thereof. A array. 35. The bait region contains human α2M, pregnancy zone protein (PZP), rat α1M, rat α2M, rat α1I3 mutant 1, or rat α1I3 mutant 35. A DNA sequence according to claim 34, derived from PZP. 36. human α2 macroglobulin, its variants, fragments, derivatives, or such derivatives; According to any one of claims 25 to 35 for the expression of alleles of a gene. Functional expression vectors containing the described genes. 37. It further contains regulatory elements necessary for stable maintenance of the vector in mammalian cells. , the vector according to claim 36. 38. The claimant further comprises sequences corresponding to processing and secretion of the expressed product. The vector according to claim 36 or 37. 39. Claims further comprising one or more genes encoding the desired gene product. The vector according to any one of Items 36 to 38. 40. alpha-macroglobulin, a variant thereof, essentially as described in the specification; Genes encoding fragments, derivatives, or allelic expressions of such genes. A functional expression vector. 41. human α2-macroglobulin, its fragments, derivatives, or such genes; Transformation containing a functional expression vector containing the gene encoding the expression of the allele replaced host. 42. The vector is the vector according to any one of claims 36 to 40. 42. The host according to claim 41, which is 43. The host is a bacterial strain, a fungal strain, a mammalian cell line, or a mammal. The host according to claim 41 or 42. 44. 44. The host according to claim 43, wherein the host is a fungus. 45. 45. The host according to claim 44, wherein the fungus belongs to the genus Aspergillus. 46. 45. The host according to claim 44, wherein the host is yeast. 47. If the host 47. The host according to claim 46, which belongs to the genus Calomysis. 48. 44. The host of claim 43, wherein said host is a mammalian cell line. 49. the host is a Syrian baby hamster kidney (BKH) cell line; The host according to claim 48. 50. The cell line was awarded No. 1 by ATCC. Available as CRL1632, Claim The host according to item 49. 51. of the active form, identified as SEQIDNO:2 or SEQIDNO:4 Recombinant α2-macroglobulin. 52. A recombinant product produced by the method according to any one of claims 1 to 24. α-macroglobulin, variants, fragments or derivatives thereof. 53. By using the vector according to any one of claims 36 to 40. Recombinant α-macroglobulin according to claim 52 produced, a variant thereof , fragment or derivative. 54. alpha-macroglobulin, a variant thereof, essentially as described in the specification, Fragment or derivative. 55. recombinant human α2-macroglobulin, as essentially described herein; Variants, fragments or derivatives thereof. 56. Growth medium containing one or more types of α-macroglobulin. 57. Recombinant α-macrogroglos according to any one of claims 51 to 55 A growth medium comprising Brin, a variant, fragment or derivative thereof. 58. Recombinant α-macrogroglobin according to any one of claims 51 to 55 Purin, its variants, fragments or derivatives as protein carriers in enzyme exchange therapy Use as. 59. Recombinant α-macrogroglobin according to any one of claims 51 to 55 Brin, its variants, fragments or derivatives as DNA carriers in gene therapy Use as.