JPH01502080A - Enhancement of gamma carboxylation of recombinant vitamin K-dependent proteins - 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] recombinant vitamin-dependent proteins Enhancement of gamma carboxylation λmitate 11 The present invention relates to vitamin-dependent proteins.

Vitamin-dependent proteins require vitamin K for their complete synthesis.

These proteins include prothrombin, factor Lotin S1 factor X and protein 2 (some of which are involved in blood clotting) ) and osteocalcin and bone matrix Gla found in bone. Contains protein. As a group, blood clotting proteins have amino acid sequences Has significant homology and is activated by enzymatic proteolysis of the zymogen to the active enzyme form (only protein S differs), all vitamin-dependent proteins are new It has a metal binding amino acid T-carboxyglutamic acid.

Glutamic acid residues in vitamin-dependent proteins are Not carboxylated.

Carboxylation occurs sometime before the protein is secreted from the cell.

Vitamin-dependent proteins are associated with liver disease, vitamin deficiencies, and warfarin Acquired deficiencies may occur, such as in the presence of antagonistic drugs to vitamins such as thorium (coumarino). be scarce Hemophilia B is a disease characterized by a genetic deficiency of the mlX factor. hemophilia Of the 25,000 Americans who suffer from hemophilia B, approximately 10 to 12% have hemophilia B.

Treatment of patients with diseases involving congenital or acquired deficiencies in blood clotting proteins is risky. Many are also expensive. For example, hemophilia B is currently treated in two ways: By using commercially available factor rear Factor X obtained by this method is of intermediate purity.

The human No. 1 gene is cloned into a plasmid and can be transformed into mammalian cells. has been done. However, the expressed factor In contrast, this partially carboxylated metal is only partially carboxylated. Boxylated factor It does not have any activity.

When vitamin-dependent proteins are first synthesized, they are converted into mature proteins (e.g. (propeptide), which is composed of adjacent amino acid sequences (propeptide) upstream of the mature protein. has been completed. When proteins are synthesized in the rough endoplasmic reticulum, prepeptides are Thiins (propeptides bound to mature proteins, e.g. proprothrombin) are It is cleaved during secretion into the endoplasmic reticulum of the vesicle. Following this secretion, gamma carbo Sylation occurs and, ultimately, the propeptide is cleaved and the mature protein is released from the cell. (In the present invention ai*, the prepeptide and propeptide are released together. (called prepropeptide).

Summary of the invention In general, the present invention relates to a first DNA sequence encoding a human vitamin-dependent protein. a sequence of DNA encoding the protein, fused to its 5' end; @2DNA2DNA not identical to the naturally associated propeptide coding sequence The second DNA sequence is characterized by a second DNA sequence in which the protein is derived from recombinant eukaryotic cells. Expressed within! Second, it has the ability to promote gamma-carboxylation of the protein. A non-naturally occurring propeptide sequence that can encode a propeptide sequence It is a co-ρ array.

(Recombinant cells are cells into which the gene for the protein has been introduced using an expression vector. It is. ) In a preferred embodiment, the second DNA sequence has a protein sequence in its amino acid sequence. propeptide of prothrombin rather than the propeptide that is naturally bound to protein. It encodes a similar propeptide. Most preferably, prothrombin A sequence encoding the propeptide itself is used.

The present invention is characterized by the fact that it is not sufficiently gamma-carboxylated and therefore has a low biological In contrast to other recombinant vitamin-dependent proteins that only express Our finding is that recombinant prothrombin is fully gamma-carboxylated. Based in part on observations. This difference is due to the dependence of other vitamins in recombinant cells. gamma-carboxylic acid that is superior to the propeptide of naturally occurring proteins due to certain effects. We believe that this is a function of the propeptide of prothrombin, which acts as a recognition site for ing.

The prepeptide is not gamma-carboxylated, therefore according to the invention , the prepeptide is naturally bound to either the protein or the propeptide or any other suitable prepeptide.

In another preferred embodiment, the vitamin dependent protein is factor Protein C Nibrotiin S: Factor X Nibrotiin 2: Osteocalcin : or bone matrix Gla protein.

The term "vector" as used here refers to a heterologous DNA (naturally known as a vector). a plasmid, virus, cosmid, or Contains 7 arge. The vector may be capable of self-replication and may contain heterologous DNA. A may be able to be incorporated into chromosome DNA. Vectors are normally starting point and at least one selectable gene, i.e., easily detectable or present. It contains genes that encode products that are essential for cell growth. Baek Other properties of tars are well known to those skilled in the art of molecular biology.

The present invention also provides the above DNA sequence; insertion into an expression vector of a eukaryote, e.g. a mammal; transfecting mammals, cells; and improving gamma-carboxylation. By culturing the cells to produce a protein that depends on the vitamin produces human vitamin-dependent proteins with improved gamma-carboxylation. It features a method of

In another aspect, the invention provides purified t-D encoding human prothrombin. N A sequence (especially cDNA sequence): A region encoding a prepeptide necessary for expression. It is characterized by a DNA sequence containing a region. The term "purified" here refers to This means that the DNA is separated from the DNA that is naturally associated with the gene within the cell. ing. The DNA sequence encoding prothrombin encodes the mature structural protein. It encodes the prepropeptide of prothrombin along with the DNA sequence that contains it. Contains DNA.

When the sequence encoding prothrombin is inserted into cultured eukaryotic cells, they Production of fully gamma-carboxylated human prothrombin by cells of can be incorporated into expression vectors that affect

FIG. 1 is the amino acid sequence of the prepropeptide of human prothrombin.

FIG. 2 is a DNA sequence encoding a portion of the amino acid sequence of FIG.

Figure 3 shows the DNA of the untranslated 5' end of the prepropeptide of human proI thrombin. It is an array.

Figure 4 shows four different anti-prothrombin antibodies and recombinant prothrombin. This is a graph showing the reaction.

structure FIG. 1 shows the amino acid sequence of the prepropeptide of human prothrombin.

The amino acid at position -1 (Arg) is the amino terminal (N-terminal) of mature human prothrombin. edge). The amino acids of the prepropeptide from position -36 to position -1 The acid sequence was previously published by Degcn et al. (Biochem, 20B?(19R 3)). cDNA encoding human prothrombin The 21 nucleic acids at the 5' end of, but not all, were also shown by Degen et al. P A complete cDNA containing the region encoding the repeptide is required for expression. Ru.

The prepropeptide shown in FIG. The pre-peptide consists of the ``gnar'' peptide. minute; the propeptide is the remaining portion (position -19 to position -43). protro The human prothrombin structural gene encodes the mature human prothrombin protein starting at position -1. is being coded. ``Proprothrombin'' has a propeptide attached to its N-terminus. It refers to prothrombin that is means prothrombin with a prepropeptide attached to its N-terminus. Ru.

D encoding residues -35 to -43 of the signal peptide of human prothrombin The NA sequence is shown in FIG. As mentioned above, the prothrombin structural gene and The DNA sequence of the DNA encoding residues -1 to -36 of the prepropeptide and As shown by Degen et al.

cDNA encoding preproprothrombin into a mammalian expression vector Cloning Chinese hamster ovary (CHO)! Transfection into cells I did it. Recombinant prothrombin is fully active and fully (naturally occurring human (compared to prothrombin) was expressed in a gamma-carboxylated form. So The observed complete gamma-carboxylation of the human factor (containing portions) were isolated from rat hepatoma cells (A++som et al., 31 5 Nature 683 (19ε5)): Human liver cancer cells (DeUsuL) (d et al., 316 Na1rc 26g (1985)): baby hamster fibroblasts (Anson, supra; de U Sale, supra); Kidney (B++5by et al., ηyNature 271 (1985)) ; and Chinese hamster ovary cells (Kaifmsn et al., S±J, Biol, Cbeya, 9622 (9116)) In the expression of human factor (compared to naturally occurring human factor), This is in sharp contrast to what is expected.

The propeptide of vitamin-dependent proteins binds the protein to its glutamic acid residues. This is the cause of the tendency toward gamma-carboxylation. recombinant expressed Prothrombin is believed to be fully carboxylated. The reason is that Does the propeptide of rotombin efficiently direct proteins toward carboxylation? It is et al.

In contrast, the expressed recombinant factor is not efficient in directing carboxylation, so it is not completely carboxylized. not be converted into

An improved targeting propeptide at its N-terminus, e.g. For example, the propeptide of prothrombin (instead of the propeptide of factor ■) Cloning the DNA encoding bound factor Ⅰ into a mammalian expression vector and when transfected into appropriate eukaryotic cells, the protein is converted into a gamma-carboxylic Substituted or modified in favor of xylation; factor II, which achieves improved gamma-carboxylation due to its reduced efficiency. can be expressed.

Fully gamma-carboxylated recombinant human proteins in recombinant cells. Expression of thrombin is described below, and in addition, prothrombin The preparation of Tido-Factor II eDNA is also described.

Affinity-purified anti-protron human liver cancer cDNA expression library Screened using Bin antibody I; Some selected clones are Tubing and nucleic acid sequence analysis revealed that the 3' end of the prothrombin cDNA It was shown to contain a 650 base pair insert. Complete prothrombin This fragment was screened from a fetal liver cDNA library to obtain the complete coding sequence. used for leaning. Restriction enzyme content of many isolated positive clones Analysis showed that these inserts were also incomplete at their 5' ends. restriction enzyme a fragment cut from the 5' end of the insert containing the most complete 5' sequence; The fragments are then used to rescreen the library. several Clones were recovered and they encoded the complete 5' end, including the initiator codon. I was doing it. Full length prothrombin encoding sequence (propeptide and (containing the prepeptide) is reassembled from two overlapping cDNA inserts. It was.

The resulting cDNA was 2.0 kilobases long, with approximately 100 −1 in the 3′ untranslated region. It contains 50 nucleic acids and 12 nucleic acids in the 5' untranslated region. 5' end nucleic acid sequence and the corresponding predicted amino acid sequence are shown in FIG. human prothrombin The leader sequence starts at residue -43.

The prothrombin expression vector pMT2-FT is an origin of replication of SV40, The major late promoter of noviruses, the prothrombin-encoding region, The region encoding dolo7oleate reductase, the early polyadenylation of 5v40. nylation sites, adenovirus virus binding genes, and E. coli (Eseber Contains the pBR322 sequence necessary for growth in iehii coli). child Nobe ct BIG (1985)). Plasmid pMT2- PT dihydro-7-oleate reductase-deficient Chinese hamster ovary cells dihydro7-oleate reductase by subculturing in selective medium. Cells showing a positive phenotype were selected. Culture solution harvested when cells have spread all over was measured by competitive radioimmunoassay using anti-total prothrombin antibody. Ta. Total prothrombin antigen is available at various concentrations up to 0.55 μg/ml. were found to be expressed by these primary transfectants. Fuse the same sample. conformational-specific antibodies, i.e. prothrombin in the presence of metal ions. Using antiprothrombin:Ca(I[) that binds to specific determinants appearing on It was measured. These determinants undergo metal-induced conformational changes. Appears when prothrombin is sufficiently gamma-carboxylated to , their presence is closely related to coagulation activity. measured using this assay The concentration of prothrombin obtained is similar to that measured for total prothrombin antibody. Equivalently, this means that all of the expressed prothrombin is carboxylated. It has been shown to be biologically active. In fact, anti-abnormal glothrombin antibodies Even if used, des-γ-carboxy (abnormal) prothrombin is 0.03 Hy No amount exceeding the measurement limit of mn was detected.

Lieb? Liebmaa et al. ($21ist, Ac1d, Sci, USA 317! (+985)) and Borowski CBarevski) et al. J-Biol.

Chew, 92N (19B5)) Prothrombin is isolated from conditioned medium using conformation-specific antibodies. Isolated by affinity chromatography. Tissue culture supernatant with Ca Antiprothrombin in the presence of (II): Ca(II)-Sepharose column Attached. All prothrombin antigens are removed from the culture medium by this method; The prothrombin antigen in the substance is anti-total glothrombin; It was not detected using antigen. Bound prothrombin is removed by EDTA. Prothrombin was eluted and quantitatively recovered from the solution.

Purified recombinant prothrombin was dehydrated in the presence of 2-mercaptoethanol. It migrated as a single band in the decyl sulfate gel. Its electrophoretic movement is It was identical to prothrombin obtained from human plasma.

Coagulation activity of recombinant prothrombin is measured directly using prothrombin-deficient plasma It was done. Purified plasma prothrombin of known concentration was used to generate the standard curve. I;.

Recombinant prothrombin increases the clotting activity of plasma prothrombin as shown in Table 1. It was found that 99±4% of the sexes were present.

Table 1 Coagulation activity and gamma-carboxylated gluta of recombinant prothrombin Minic acid Coagulation activity T-carboxylation (%) Glutamine a (mol) Plasma-derived prothrombin 100 10.0 Recombinant prothrombin 99±4 9.9±0.4 recombinant prothrombin and four different anti-prothrombin antibodies The interaction was investigated by competitive radioimmunoassay. Anti-total prothrombin anti The body is related to the degree of carboxylation and conformation of prothrombin. (combined with prothrombin. As expected, recombinant prothrombin, Plasma-derived prothrombin and abnormal (desu-T-carboxylated) prothrombin As shown in FIG. were replaced equally. Anti-aberrant prothrombin antibodies are desu-T-carboxylated It bound to antigenic determinants present only on types of prothrombin. As shown in Figure 4B Recombinant prothrombin and plasma prothrombin are derived from these antibodies. IISl-labeled aberrant prothrombin is not replaced, which means that in the recombinant product showed that there were also no traces of des-T-carboxylated prothrombin in . Different metal dependent structures of prothrombin conf.

Two groups of antibodies specific for r m e r ) have been described by Borowski et al., 261. J. Biol, Chem, (1986). anti-prothrombin :Mg(II) antibody has prothrombin that is sufficiently carboxylated. , which undergoes initial structural changes induced by many divalent and trivalent metal ions. It binds to antigenic determinants expressed on prothrombin only when it is decoding. anti pro Too7bin: Ca (11) 4>X antibody has prothrombin at the lipid binding site. It corresponds to the antigenic determinant that is exposed when it undergoes a second structural change accompanying the expression of . this The change is carried out only by Ca(II) and not by most other metal ions. As shown in Figure 4C and '\BAD, the recombinant prothrombi and plasma prothrombin from these antibodies. J-labeled prothrombin and from this criterion, recombinant prothrombin and plasma prothrombin Thrombin was shown to be substantially equivalent.

The amino-terminal sequence of recombinant prothrombin was determined by automated Edman degradation. As shown in Table ■, recombinant prothrombin and plasma-derived prothrombin The amino acid sequences of the first 16 amino acid residues of were identical.

Of particular interest are plays that have undergone incomplete processing, or No second sequence was detected at all. Additionally, during a standard sequencing cycle, Gamma-carboxylated glutamic acid derivatives are not released from the filter, leaving residues This undetectable degree of glutamic acid found in 6.7.14 and 16 Consistent with complete gamma carboxylation of these residues.

Table ■ Amino-terminal sequence residues of recombinant prothrombin Recombinant plasma Prothrombin Prothrombin I A l a 64 A 1 a 2 A s n 32 A s n 3 T h r 24 T h r 4 Phe 36 Phe 5 Leu 31 Leu 8 Vat 23 Va1 9　Arg　9　Arg lo Lys 19 Lys ll Gly 16 Gly 12 Asn 15 Asn 13 Leu 18 Leu 14 - - (C; Ia) 15 Arg? Arg Recombinant prothrombin T-carboxylated glutamic acid 9 red was treated with alkali. Determined by amino acid analysis of the hydrolyzate. Special recombinant prothrombin is shown in Table I. As mentioned above, the value per 1 mole of plasma-derived prothrombin was set as a standard value of 10 moles. 99 ± 0.4 mol of γ-carboxylated glucose per mol of protein. Contains tamic acid.

l Goodness] Groove c DNA library screening: Young and Davis, a o, Proc, Nat, Acad, 5ci-USA, 1194 (1983) The human hebatoma cDNA expression library was inserted into the λgtll vector. H was manufactured by Wet et al., 3. Chromosomes described in DNA, 437 (1984) using a genetic immunodetection system) , screened for clones expressing prothrombin antigen. Approximately 100 ．． 000 plaques were transferred to a nitrocellulose filter and soaked in 3% bovine serum alcohol. Bumin, 0.02% sodium azide, and 0.5 μg/ml immunoaffinity Tris-buffered saline containing rabbit anti-total prothrombin antibody purified by Tea method Incubated overnight. After several washes with Tris-buffered saline, this filament Luther was combined with 3% bovine serum albumin and horseradish peroxidase. Contains goat anti-rabbit immunoglobulin (manufactured by Bio-Rad, diluted 1:1000). The cells were incubated for 4 hours in Tris-buffered saline. After further washing, the substrate Add 1-chloro-2-naphthol (Bio-Land Co., Ltd.) to remove positive plaques. Detected. Seven positive clones were isolated by three rescreenings, and these Phage DNA was extracted from plate lysates as described by Helms et al., 4. DNA, 39 (1985). cDNA insert with EcoRl It was cut and subcloned for analysis by restriction enzyme mufting. one 6 The 50 base pair insert was inserted into the prothrombin cDNA based on its restriction pattern. I tentatively assumed that it was at the 3゛ end. This estimation is based on Maxam and Gi1ber t, 65+ Methods Enzymol, 499 (1980). Confirmed by nucleotide sequence analysis using chemical cleavage.

To obtain the full-length coding sequence of prothrombin, Charon 21 (Described in Toole et al., 313. Nature, 342 (1984) A cDNA library of human fetal liver in Bent.

n and Davis, 196. Screen using the method of Science 180 (1977). Leaned. cDNA obtained from human hebatoma expression library iii of prothrombin cDNA-A derived from the insert! lli! fragment ,” P-dCTP and Feinberg et al., 132. Anal, BLoche m-, 6 (1983). Random hexanucleotide priming using approximately IO”cpm/p radiolabeled to a specific activity of -90 mM sodium citrate, pH 7.0,900 5M Sodium Chloride, 5x Denhardt's Solution (Denhardt, 23.Bi.

chem, Biophys-Re5-Comm+ 541 (1966) ), with 10 mM EDTA and 0.5% sodium dodecyl sulfate. Hybridization for 16 hours at 68°C was followed by 3011M sodium citrate. 300mM sodium chloride and 0.5% sodium dodecyl sulfate Thoroughly washed with thorium.

The two positive substances were purified by the plaque method, and 71-di DNA was purified by the method of 1e1ms described above. was isolated from plate lysates.

Subclon the desired vi-restricted fragment of the cDNA insert into the appropriate M13 vector. As described in detail in Norrandes, 26, Gene, 101 (1983). 74.), restriction enzyme muffing and Sanger et al., 74. P roc, Nat-Acad, Sct, USA, 5463 (1977). Sequencing was performed using the deoxynucleotide chain termination method.

Prothrombin plasmid γ 1” raw Koni Mata 2-eni [”-full-length prothrombin coding sequence for each insert. Digest at one Hindl site and clone the appropriate fragment into mp18. It was reconstructed from two duplicate cDNA inserts by scanning.

Then, the EcoR1 fragment of lOkb, which encodes the complete prothrombin sequence, was (including DNA encoding the prepropeptide), Toole et al. (1986) P, N, A-i, U, S, A, , 83.5939. The vector was inserted into the EcoR1 site of pMT2, a mammalian expression vector. obtained pro The thrombin expression plasmid, pMT2-PT, was purified by restriction enzyme mufting. Correct orientation of the thrombin coding region relative to the adenovirus main late promoter It was found that it was included in

This prothrombin cDNA has the prepeptide coding sequence shown in FIG. The remainder of prothrombin c DN A given in Figure 2 of Egen et al. It was fused and included.

cell line T, nNAp” translocation, and selected cell lines with dihydrofolate reductase deficiency The Chinese hamster ovary cell line, CHODUKX-Bll, was hais et al., 77, Proc. Nat. Acad, Sci.

USA, 4216 (1980) and Kaufrnan et al.

159, Mo1. Bio, 601 (1982). cultivated and maintained. Transfer cells to 20 μl of prothrombin expression plasmid, pMT2. - In PT, Kaufman et al.* 195. Mo1. Bio, 601 (1982) 0, form transformed by calcium phosphate co-precipitation method as described in After transformation, cells were treated with 10% heat-inactivated baby bovine serum, 5 μg/l vitamin to * (Aquamephyton, Merck 5harp and Doh me), and thymidine, adenosine, deoxyadenosine, penicillin and and streptomycin (10 tzg/-1 each) without nucleosides. The cells were grown in α-modified Eagle's medium (Gibco). Nucleate cells after 2 days. Passaging in the same medium (selective medium) except that osside was removed and dialyzed* supernatant was used. did. Transformed cells should be grown until colonies become visible 10 to 12 days after passage. The selection medium was replaced every 3 to 4 days. These initial transformants were pooled and The cells were grown in selective medium until confluent. Confluent state Therefore, a 10c culture dish contains approximately 1-7X10 cells in medium 1 (+++1). Ta. Add medium to confluent JIrl in flasks every 3-4 days for several weeks. A large amount of conditioned medium was recovered by replacing the medium. This condition The digital medium was stored at -20°C until needed.

white” and Human prothrombin is extracted from plasma by adsorption of barium citrate and DEAE cellulose. chromatography, and Rosenberg et al., J. Biol, Chem-1 607 (1975) and Miletich et al., 253. J.

Biol, Chem, 6908 (197B). Purified by affinity chromatography using chistran sepharose . Abnormal (desu-T-carboxylated) human prothrombin is extracted from plasma by DEAE. Sephacel chromatography and Blanchard et al., 101. J-La b, CI in.

Anti-prothrombin Sepharose described in Med-242 (1983) Purified by affinity chromatography. The protein content of purified prothrombin is , the AI% at 280 nm was measured as 14.4. Prothrombin and abnormal proteins Rothrombin is from Morrison.

To Meth, by the method of Enzymol-214 (1980), Na"' Iodine-labeled with I.

Rabbit anti-prothrombin:Ca(II) antibodies are described in Blanchard, supra. on prothrombin-Sepharose in the presence of Ca(II) as described above. Purified by immunoaffinity chromatography and elution with EDTA.

Anti-prothrombin conjugated to prothrombin-Sepharose in the presence of EDTA. The antibody was eluted with 4M guanidine hydrochloride and named anti-total prothrombin antibody. Ta. Anti-aberrant protron antibodies are described by Blanchard et al., 101. J.La. b-CI in Med-242 (1983). Sequential immunoaffinity with γ-carboxylated prothrombin-Sepharose -Prepared by chromatography. Anti-brothrombin: Mg(II) and anti-brothrombin - Brothrombin: Ca(II)-specific antibody is According to the method described in i, either Mg(II) or Ca(II) is present. Continuous immunoaffinity chromatography with prothrombin-Sepharose in the presence of Prepared by elution with Claffy and EDTA.

i! Oymno Afuse Zui Displacement of IzS am prothrombin from anti-prothrombin antibody It was investigated using immunoassay.

Anti-total prothrombin antibody (1, IXI/10'M) was added to ItSl-labeled prothrombin antibody. Reaction mixture containing rhombin (1,7X1/X O”M) and various concentrations of competitor added to things.

All ingredients are 1mM benzamidine, 0.1% bovine serum albumin, and 3x MED. Diluted in Tris-buffered saline containing T, A and rabbit gamma globulin as carrier. did. Anti-brothrombin: Ca(II) antibody (3,4X1/10”M), anti- Prothrombin: Mg (II) antibody (1, OXI/IO'M> or anti- Rothrombin: Ca(II) specific antibody (1,3X1/10”M) was prepared using EDTA was added to the same reaction mixture except that 3mM calcium chloride was used instead. anti -X? Prothrombin antibody (4°Oxl/10'M) is '! SI-label abnormality Prothrombin (2,8x 1/10”M) and 3IIIM EDTA Added to a similar mixture containing After overnight incubation at 4°C, the bound +! 51-labeled prothrombin or abnormal prothrombin is a goat anti-rabbit immunoglobulin. Precipitated by addition of Robulin. Remove the formed precipitate by centrifugation and 8 quasi-curves using a known concentration of 11!5H on a mangamma 5ooo spectrometer. Created a line.

Recombinant prothrombin 1 Recombinant prothrombin was obtained from prepared media as described in B above.

by Rowski and Lieb m a n et al., 82° Proc- As described in Nat., Acad, Sci., USA, 3879 (1985). Immune affinity chromatography using sea urchin conformation-specific antibodies Refined with fee. The antibody used, anti-prothrombin: Ca (II), was Antigen determination expressed on T-carboxylated prothrombin in the presence of allotropic ions Binds to a fixed group. 10mM calcium chloride and 0.02% sodium azide The prepared medium containing anti-prothrombin:Ca(II) sepharose The sample was introduced into the ram at 4°C. 10mM calcium chloride and 0.5M sodium chloride After washing thoroughly with Tris-buffered saline containing It was eluted with 10+mM EDTA in the solution. Semen M81 recombinant prothrombin protein Concentration was measured with A+% at 280 nm as 14.4. Electrophoresis is Performed on gel. Samples were soaked in 2-mercaptoethanol before loading onto the gel. The samples were incubated at 100° C. for 5 minutes in a sample buffer containing Gel after electrophoresis was stained with Comassie Blue R-250.

Prothrombin clotting assay The activity of prothrombin is determined by the prothrombin activity described by BIanehard. A two-step assay using deficient plasma was performed. To create a standard curve, A known concentration of human prothrombin was used.

Amino terminal chain Automated Edman Digestion Applied Biosystems Model 470A Gas Attaching the model 120 PTH analyzer to the phase protein sequencing device went.

Recombinant prothrombin was purified using an RP-300 Brownlee Guard Car prior to analysis. High pressure using a Brownleeguard cartridge It was desalted by liquid chromatography.

Monocarboxylated glutamine Amino acid analysis was performed using a Beckman model 119CL amino acid analyzer. The test was carried out using a Model 126 data system. The protein is Hausc hka, 80. Ana 1. Described in Biochem, 212 (1977) Hydrolyzed in 2M potassium hydroxide at 110° C. for 22 hours as described above.

T-carboxylated glutamic acid composition is converted to ninhydrin after alkaline hydrolysis. A detection system was used to detect proteins bound to mature human factor II using an automated amino acid analysis method. The coding sequence of a hybrid molecule consisting of the prepropeptide of rotthrombin is Mietagenesis was performed in an age vector and constructed as follows.

The sequence is described in K.a.u.r.ma.n et al., supra. This plasmid pM T2-IX was cut with the restriction enzyme Pstl, and the resulting 2.5 kb fragment was Gel purified using standard methods. This fragment is a widely available phage vector. -Inserted into the Pst1 site of mp8. Place the factor ■ coding sequence in the desired vector. When clones containing the same orientation were digested with the enzymes EcoRV and Bam HI 2. It was identified by the presence of the Okb fragment. This vector was named mp8-■ did.

The CDNA sequence containing the coding sequence of the prepro region of prothrombin is mp18- PT was cut with restriction enzyme 1 Hindll and a 350 bp fragment was obtained using standard methods. Obtained by isolating . Mp1B-PT is described in this specification. However, in Jorgcnsen et al. is also listed. This 3501)P fragment was converted into H3ndl[ [Inserted into the site. A clone containing this insert in the correct orientation relative to factor , a 1.0 kb fragment when digested with the enzymes Xhol and Bg1 It was identified by the presence of The Hindnl flag of the coding sequence of this factor The construct containing the component was named mp8-PT/IX.

The sequence encoding the leader sequence of prothrombin encodes mature factor (1983) Nature, 304. ．． The intermediate nucleotide sequence was converted into a synthetic oligonucleotide using the method described in 456. removed with a "loop-out" mieta genesis using a That is, EcoR] A heteroduplex is formed between single-stranded mp8-PT/IX and single-stranded mp18. was formed. This heteroduplex anneals the primer for mutation. Contains a single-stranded portion for binding. Form one tftrnp8 PT/IX Therefore, we transformed E. coli (TG 1 strain) with this vector and picked up one plaque. Single-stranded templates were then prepared using standard methods. The second of this heteroduplex For chains, mp18 was linearized with the restriction enzyme EcoR1. This heteroduplex To form 2 pg linearized m p 18 and i-s μg m p 8- Mix PT/IX to a final volume of 1.5 μm and add 4 μl of IN NaOH. and denatured it.

and neutralized it. The mixture was incubated overnight to reanneal the denatured DNA (40 μl). It was incubated at 68°C and slowly cooled to room temperature over several hours. Obtained Ani A portion of the converted DNA contained the desired heteroduplex molecule.

Annealing of the appropriate mutant oligonucleotide to the heteroduplex A loop containing a single strand was formed. The remaining single-stranded gap is filled with primer - extension and end-joining (ligageron). Mutant ply used The mar is 31-mar, 5゛-GCGGGTCCGGCGATATAATTC AGGTAAATTG, to give the desired hybrid coding sequence: - From 13 base blocks and 18 base blocks complementary to the region to be combined It was something like that.

This oligonucleotide was synthesized on an Abrido Biosystems 380B synthesizer. synthesized and gel purified before use.

This primer was applied to the T4 polynucleotide kit in the standard reaction mixture of Maniatis et al. 5' phosphorylation using Nase, and 2. ! 11 (10 pmol) to 4 It was mixed with 0 μl (0.1 pml) of the heteroduplex mixture. This a The kneeling reaction was performed by heating at 68°C for 2.5 hours and gradually cooling to 15°C. I went further. To this primer, 20sM MgC1z: 40mM dithio Thretol; 211MATP, 1mM each (F)dATP, dTTP, dCTP , and dGTP: DNA polymerase I (Pharmacia) - fragment 5 units and 0.5 units T4 ligase (Bethesda, Re After adding 8 μl of 5earchLabs), incubate at 15°C for 4 hours. During the initialization, this primer was extended and transfected with pF (minus).

Plaques were generated from E. coli transformed with DNA containing the mutated mp1g chain. This is because mp8 has an amber mutation. F.A. DNA from the sexual plaques was used to transform E. coli. Picked up one positive plaque Then, a single-stranded template was prepared and the DNA sequence was determined.

Furthermore, this isolated clone contains only the desired mutation and no other mutations. In order to confirm this, sufficient restriction enzyme muffing was performed.

By using the above technique, the protonucleotide immediately adjacent to the amino terminus of the mature factor II coding sequence is Sequence encoding the rhombin prepro region (PR/IX , ) as the desired construct rI! I5I. Transpose this sequence into a mammalian expression vector p In order to insert into MT2, preproPR/IX is used with the restriction enzyme Eco Excise from mp18 vector using RI and insert into EcoR1 site of pMT2 can do. Clones were restriction digested with the enzymes Bgln and EcoRV. is identified by the presence of a 600 bp fragment. This plasmid pMT2-PT/IX is injected into mammalian cells, such as CHO cells, as described above. 0-expressed protein that can be transformed and used to produce mature processed factor Factor II is T-carboxylated and is biologically active.

blood rising 1 Other embodiments are within the scope of the claims, such as factor Ⅰ, protein C, protein protein S5 factor X, protein z1 osteocalcin, and bone Gla matrix Other vitamin-dependent proteins, including protein proteins, also proprotease their N-termini. It can bind to the propeptide sequence of thrombin. vector containing bound DNA - Expression in mammalian cells can be predicted.

Factor Ⅰ binds to the complete leader sequence of prothrombin, but the effect of the present invention To obtain complete carboxylation of the expressed protein, the proprotron We show that only the region encoding the /ropeptide portion of the bin peptide is essential. You should understand. Therefore, the DNA encoding the complete leader sequence of factor Instead of using DNA I’ia, which encodes the leader sequence of prothrombin. What is required is to convert the DNA encoding the profactor peptide into the proprotron. Simply replace the bottle with DNA encoding the propeptide.

MefAla H7s Val Arg Gly Leu Gin Leu P r.

Gly Cys Leu Ala Leu Ala Ala Leu's ts S erPm Gin Gin Ala Arq! ;er　Leu　Leu　Gl n　ArgMefAla　H7s　Vat　Arg　Gly　Leu　Gln　 Leu Pro, -.

5′ AGCTGA CACACT Purification (no change in content) 0001 OOI OJ ID 0OOI OOl aI LOooot oo t at to aoo’t aot cu t,.

[Bina” E, uq/ml [Slap J] Aoi IFIG, 4 Procedural amendment 1.Display of the incident PCT/US87103015 2. Name of the invention recombinant vitamin-dependent proteins Enhancement of gamma carboxylation 3. Person who makes corrections Relationship to the incident: Patent applicant address Name: Niney England Medical Society Center 4, Agent Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo 5. Subject of correction international search report INl, N, l1llA・l A Marshal b11b? ? ~・:'CT,'jS8710 30:5my+a++a*s°aeewa+,ae　Ne,,,,,,,2Sε , , O, O25

Claims (16)

[Claims] 1. The first DNA sequence encoding human vitamin K-dependent protein and its 5' end a protein naturally associated with said first DNA sequence which encodes said protein; A DNA sequence that includes a second DNA sequence that is not identical to the coding sequence of the peptide. hand; Said non-naturally occurring propeptide coding sequence can be used to encode said protein in recombinant eukaryotic cells. is expressed, a protein capable of promoting r-carboxylation of the protein. A DNA sequence that is capable of encoding a peptide. 2. The second DNA sequence is similar to the propeptide naturally associated with vitamin K-dependent proteins. Coating a propeptide with an amino acid sequence similar to that of prothrombin The DNA sequence according to claim 1, which encodes the following. 3. Claim 2, wherein the second DNA sequence encodes a propeptide of human prothrombin. DNA sequence described in section. 4. A DNA sequence according to claim 1, wherein the vitamin K-dependent protein is Factor IX. 5. DNA sequence according to claim 1, wherein the vitamin K-dependent protein is Factor VII. . 6. DNA sequence according to claim 1, wherein the vitamin K-dependent protein is protein C. . 7. DNA sequence according to claim 1, wherein the vitamin K-dependent protein is protein S. . 8. A DNA sequence according to claim 1, wherein the vitamin K-dependent protein is factor X. 9. A DNA sequence according to claim 1, wherein the vitamin K-dependent protein is protein Z. . 10. DN according to claim 1, wherein the vitamin K-dependent protein is osteocalcin. A array. 11. Claim 1 wherein the vitamin K-dependent protein is bone Gla matrix protein. DNA sequence as described. 12. A claim in which the first DNA sequence encodes a prepropeptide of prothrombin. The DNA sequence described in 1. 13. A vector comprising the DNA sequence according to claim 1. 14. Human prothromone, containing the sequence encoding the prothrombin prepeptide Purified DNA sequence encoding Bin. 15. A vector comprising the DNA sequence according to claim 14. 16. The DNA sequence according to claim 1 is prepared, and the DNA sequence is transformed into a mammalian expression vector. transform the vector into mammalian cells, and culture the cells. to produce a vitamin K-dependent protein with improved r-carboxylation, A human vitamin K-dependent protein with improved r-carboxylation. manufacturing method.