WO1991001372A1

WO1991001372A1 - VITAMIN K-DEPENDENT η-CARBOXYLASE

Info

Publication number: WO1991001372A1
Application number: PCT/US1990/004015
Authority: WO
Inventors: Barbara Furie; Bruce Furie; Brian Hubbard; Margaret Jacobs; Magda Ulrich; Christopher Walsh
Original assignee: New England Medical Center Hospitals, Inc.; President And Fellows Of Harvard College
Priority date: 1989-07-17
Filing date: 1990-07-17
Publication date: 1991-02-07

Abstract

Substantially purified vitamin K-dependent η-carboxylase, methods for purifying it or other polypeptides, a vector containing the coding sequence for vitamin K-dependent η-carboxylase, and a cell containing such a vector are disclosed.

Description

VITAMIN K-DEPENDENT γ-CARBOXYLASE

BACKGROUND OF THE INVENTION

This invention relates to vitamin K-dependent γ-carboxylase.

The vitamin K-dependent proteins ("VKD proteins") are a class of calcium-binding proteins that contain a modified amino acid, γ-carboxyglutamic acid. These proteins, which include the blood coagulation and coagulation-regulating proteins prothrombin, Factor VII, Factor IX, Factor X, protein C, and protein S ("coagulation proteins") are

synthesized in the liver in a precursor form comprising, from the amino terminus, a signal peptide, a propeptide, and the "zymogen polypeptide": the amino acid sequence comprising the mature VKD protein that circulates in the blood (but without the latter protein's posttranslational modifications). After translocation from the site of mRNA translation to the rough endoplasmic recticulum of the liver cell, these proteins undergo post translational modifications that include the γ-carboxylation of a specific set of glutamic acid residues within the amino terminal region of the zymogen polypeptide. If these glutamic acid residues are not so converted into γ-carboxyglutamic acid, the descarboxy VKD protein will be unable to bind calcium and biologically inactive. This γ-carboxylation, which is catalyzed by a vitamin K-dependent carboxylase ("carboxylase") located in the rough endoplasmic reticulum, requires the reduced form of vitamin K, molecular oxygen and carbon dioxide, as well as a substrate polypeptide containing glutamic acid. Studies on various putative substrate peptides, including thermally-decarboxylated mature prothrombin and acarboxy peptide fragments of that protein, have indicated that polypeptides which lack the propeptide region are relatively poor substrates for the carboxylase enzyme. These results have led to further investigations of the nature of the propeptide region and its role in posttranslational processing of the VKD proteins. Based upon cDNA sequence analysis, considerable amino acid sequence homology has been found to be present among the propeptide regions of each of the VKD coagulation protein precursors. Certain deletions and point mutations within the propeptide region abolish in vivo γ-carboxylation of the precursor protein, suggesting that the propeptide provides a recognition site for the carboxylase enzyme (Jorgensen et al., Cell 48:185-191, 1987). This interpretation was further strengthened by experiments utilizing as substrate a 28-residue synthetic peptide (termed "proPT28") representing the complete 18-amino acid propeptide of prothrombin linked to the first 10 amino acids of acarboxy prothrombin (i.e., residues -18 to +10 of proprothrombin). It was found that the glutamic acid residues within the zymogen polypeptide portion of the

synthetic peptide were efficiently γ-carboxylated by a

partially purified carboxylase enzyme, while those on peptides representing residues +1 to +10 or -10 to +10 of proprothrombin were not (Ulrich et al., J. Biol. Chem. 263:9697-9702, 1988). In addition, a synthetic peptide corresponding to residues -18 to -1 (i.e., the propeptide region) of proprothrombin, termed "proPT18", competitively inhibited γ-carboxylation of the

28-residue peptide substrate, but "modestly stimulated" the γ-carboxylation of other peptide substrates containing only short segments of acarboxy zymogen polypeptide sequences.

Investigation of the details of the carboxylation reaction has been hampered by difficulties encountered in attempts to purify the carboxylase enzyme, which is an integral membrane protein (see, e.g., Suttie, Ann Rev. Biochem. 54:459-477, 1985; Ulrich et al., 1988; Canfield et al., Arch. Biochem. Biophys. 202:515-524, 1980; and Metz et al., FEBS Lett. 123:215-218, 1981).

SUMMARY OF THE INVENTION

In general, the invention features substantially purified vitamin K-dependent γ-carboxylase, which can be isolated from liver cells, such as bovine liver cells.

Also featured is an affinity resin for separating, from a mixture of polypeptides (a polypeptide being defined as two or more amino acid residues linked by peptide bonds), a first polypeptide which is not an antibody, and which is capable of binding to a ligand which is not an antibody, where the affinity resin is made of an insoluble matrix material linked to a second polypeptide which is a portion of the ligand, which portion is capable of binding to the first polypeptide. The polypeptide to be isolated is preferably a vitamin K-dependent γ-carboxylase and the ligand portion preferably contains the carboxylase-binding segment of the propeptide region of a substrate for the carboxylase, which segment may be, for example, a portion or all of the 18-amino acid sequence HVFLAPQQARSLLQRVRR; the affinity resin is preferably cyanogen bromide-activated agarose or,

alternatively, 2-thiopyridyl-activated thiol-agarose.

The invention also features an affinity column, comprising insoluble matrix material linked to a polypeptide containing the carboxylase-binding segment of the propeptide region of a substrate for the carboxylase, for separating a vitamin K-dependent γ-carboxylase from a mixture of

polypeptides, and a method for so using the column that

involves (1) applying the mixture to the column under

conditions permitting the binding of the carboxylase to the affinity resin, (2) washing unbound constituents of the mixture from the affinity resin, and (3) eluting the carboxylase from the affinity resin. Elution of the carboxylase from the column is preferably accomplished by applying to the column a solution containing a molecule capable of binding to the ligand-binding site of carboxylase, or, alternatively, by subjecting the column to conditions capable of cleaving the linkage between the carboxylase-binding polypeptide and the matrix material.

The invention also features substantially purified cDNA encoding vitamin K-dependent γ-carboxylase, and a vector containing DNA encoding vitamin K-dependent γ-carboxylase, such as bovine or human vitamin K-dependent γ-carboxlyase, and a cell transfected with such a vector. The cell may be a eukaryotic cell or a prokaryotic cell; preferably the cell is capable of expressing the carboxylase and, more preferably, also a VKD protein (e.g. a coagulation protein such as

prothrombin. Factor VII, Factor IX, Factor X, protein C or protein S), and is, most preferably, capable of

γ-carboxylating glutamic acid residues on the VKD protein.

Also featured are (1) an affinity resin for

separating, from a mixture of polypeptides, a first polypeptide which is capable of binding to a second polypeptide, which affinity resin comprises an insoluble matrix material linked via a disulfide bond to the second polypeptide; (2) a column incorporating the affinity resin; and (3) a method of using the column to isolate the first polypeptide from the mixture, which method includes applying the mixture of polypeptides to the column under conditions permitting binding of the first

polypeptide to the affinity resin, washing unbound constituents of the mixture from the affinity resin, subjecting the affinity resin to conditions uηder which the disulfide bond is cleaved, and eluting the desired polypeptide from the affinity resin.

The invention provides a method for purifying

10, 000-fold an enzyme, vitamin K-dependent γ-carboxylase, which previously had proven difficult to purify. The existence of a supply of purified carboxylase permits, for the first time, sequencing of the enzyme and construction of a family of DNA oligonucleotides representing codons for part of the protein. Use of this oligonucleotide family as a probe for cDNA corresponding to the carboxylase coding sequences will allow carboxylase cDNA to be selected from a cDNA library, cloned, and ultimately expressed in a prokaryotic or eukaryotic expression system. A plentiful source of pure, active

carboxylase, whether derived from affinity chromatography of crude extracts or from cloning and expressing the carboxylase coding sequence, will permit the large-scale in vitro

carboxylation of coagulation protein precursors produced by, for example, expression of a cloned coagulation protein coding sequence. Alternatively, carboxylase and coagulation protein coding sequences could be cloned into a single cell line, resulting in a cell line capable of producing large quantities of fully carboxylated, and therefor biologically active, coagulation proteins useful for treatment of hemophilia and other blood coagulation disorders. A non-blood source of these medically-important proteins is vitally needed to reduce the spread of blood-borne viral diseases such as hepatitis and Acquired Immune Deficiency Syndrome (AIDS).

The affinity chromatography methods of the invention have general applicability to the isolation of polypeptides other than carboxylase, and should prove to be useful where a ligand (or a portion of a ligand) which binds to the

polypeptide of interest is available in significant quantities.

The demonstration that the disulfide bond linkage between the ligand and the matrix material in one of the affinity resins of the invention is cleavable by reduction, without simultaneously destroying the activity of the protein of interest, provides a novel method of dissociating a bound polypeptide from an affinity resin, one that will be useful in some applications that have resisted alternative affinity chromatographic techniques. DESCRIPTION OF THE PREFERRED EMBODIMENTS The drawings are first described.

Drawings

Fig. 1 is a schematic illustration of each of the four types of affinity resins described in the preferred embodiments.

Fig. 2 is a photographic print of three separate Coomassie blue-stained SDS-polyacrylamide gels, each of which was run in parallel with a similar gel containing size markers (not shown): in lane A, protein eluted from a column of

affinity resin-A according to Example 1 was electrophoresed on a 10% gel; in lane B, protein eluted from a column of affinity resin-C according to Example 2 was electrophoresed on a 7.5% gel; and in lane C, partially purified carboxylase was

electrophoresed on a 7.5% gel.

Fig. 3 is a graph demonstrating the inhibitory effect of anticarboxylase antiserum (closed symbols) or preimmune serum (open symbols) on the γ-carboxylase activity of either a partially purified carboxylase preparation subjected to detergent solubilization and ammonium sulfate fractionation (circular symbols), or carboxylase purified on a column of affinity resin-C (square symbols).

Fig. 4 is a photographic print of a Western blot showing the presence of carboxylase antigen in co-migrating bands from each of three carboxylase fractions: in lane A, protein eluted from affinity resin-A according to Example 1; in lane B, protein eluted from affinity resin-C according to

Example 2; and in lane C, partially purified carboxylase.

Example 1. Affinity purification of the carboxylase using a propeptide elution system.

A. Preparation of affinity columns.

Two synthetic "affinity peptides" were prepared by the solid phase method using t-BOC chemistry in an Applied

Biosystems (Foster City, CA) Model 430A peptide synthesizer, as described by Ulrich et al., 1988. The use of a "linker segment" between the amino-terminal lysine and the propeptide sequence of the affinity peptide was found to be necessary to permit binding of the matrix-linked propeptide to carboxylase, perhaps due to steric considerations. One of the peptides, termed "affinity peptide-A", has a trilysine amino-terminus, a linker segment comprising eleven amino acids, and the

propeptide of proprothrombin from residue -18 to residue -1 (proPT18); this affinity peptide-A has the overall sequence KKKGGGIGGKAAAAHVFLAPQQARSLLQRVRR, and, whether free or linked by its amino-terminus to a matrix material, has been shown to bind to carboxylase.

The second peptide, termed "affinity peptide-B", contains a single amino-terminal lysine, a short dipeptide linker segment, and proPT18, with the overall sequence

KGGHVFLAPQQARSLLQRVRR. Like affinity peptide-A, it will bind to carboxylase in both its matrix-bound and unbound states.

After synthesis, the cleaved, deprotected peptides were purified by standard techniques using high performance liquid chromatography (Waters, Milford, MA) with a reverse phase column. The sequence of each peptide was verified by automated Edman degradation using an Applied Biosystems

Model 470 protein sequencer and Model 120A PTH analyzer.

Purified affinity peptide-A or -B (40mg; 2mg/ml in PBS [20mM sodium phosphate, pH 7.4/0.15M NaCl]) was coupled to cyanogen bromide-activated Sepharose 4B (5 ml, Pharmacia,

Piscataway, NJ) for 16 hr at 4ºC. The coupling efficiency was estimated from the OD₂₈₀ of the eluate according to the

method of Cuatrecasas et al., Proc. Natl. Acad. Sci., USA

61:636-643, 1968. The coupled affinity resins (illustrated in Fig. 1A and 1B) were washed with PBS, neutralized with 1M ethanolamine (pH 8), rewashed with PBS, and stored at 4ºC in PBS containing 0.02% NaN₃. A column (1 × 10 cm containing 5 ml of resin) of each type of affinity resin was equilibrated in buffer A [20mM sodium phosphate, pH 7.4/0.15M NaCl/0.1% (w/v) 3-[ (3-cholamidopropyl )-dimethylammonio]-1-propane sulfonate ( "CHAPS " ) (Sigma, St . Louis , MO) / 1mg/ml L-α-phosphatidyl-choline (Type V-E , Sigma) ] .

B. Purification of carboxylase.

Carboxylase was partially purified (approximately 100-fold--see Table I) from bovine liver microsomes as described by Soute et al., Thromb. Haemostas 57:77-81, 1987, and stored in 0.5M NaCl/20mM Tris-HCl, pH 7.4 at -80ºC until use. An aliquot of the active fraction (herein termed "partially-purified carboxylase") (4 ml, 41.8 mg protein/ml) was applied to the affinity column and the flow stopped for 3 hr, after which the column was washed (first with buffer A, then with buffer A having a NaCl concentration adjusted to 1M) to separate unbound proteins from the matrix-bound carboxylase. The carboxylase was then eluted from the column by equilibrating the resin for 3 hr with 4ml of buffer A containing 10mM proPT18. The eluted carboxylase, washed from the column with buffer A, was stored at -15ºC.

C. Assay for carboxylase activity.

The carboxylase assay mixture contained the

carboxylase preparation to be assayed, 0.8mM vitamin K

hydroquinone (Merck, Sharpe and Dohme, Rahway, NJ),, 1.5mM

NaH ¹⁴CO₃ (10μCi; Amersham, Arlington Heights, IL), 8mM

dithiothreitol (DTT), and 10mM FLEEL (a pentapeptide substrate, Phe-Leu-Glu-Glu-Leu, which is γ-carboxylated, albeit

inefficiently, by the carboxylase), in a total reaction volume of 125μl. 14CO, incorporation into FLEEL was quantitated in a LS1801 liquid scintillation counter (Beckman, Fullerton,

CA). A blank value of 200 to 500 cpm, obtained by carrying out the reaction in the absence of peptide substrate or in the absence of enzyme, was substracted from reported data.

Although the in vitro carboxylation of FLEEL is stimulated by the addition of low (~1μM) concentrations of proPTlδ, the high concentration (10mM) of proPT18 necessary to elute the carboxylase from affinity resin-A (or -B) completely inhibits measurable carboxylation of FLEEL. As evidenced by the failure of dialysis, which greatly reduced the amount of proPTlδ present in the eluate, to restore the lost carboxylase activity, the loss of activity appears to be irreversible.

Analysis of the eluate by SDS-polyacrylamide gel

electrophoresis, however, revealed a homogeneous protein with a molecular weight of 77,000 (Fig. 2, lane A).

This purified protein, after dialysis, was employed as an antigen to raise anti-carboxylase antibodies in

hyperimmunized rabbits, using standard immunological

techniques. Any anti-proPT18 antibodies present in the

antiserum were removed by passing the antiserum over a column of affinity resin-B and collecting the antibodies which failed to bind. Even though prepared against an inactive form of the enzyme, these antibodies were capable of inhibiting

carboxylation of the synthetic substrates proPT28 and FLEEL using partially purified (detergent-solubilized and ammonium sulfate-fractionated) carboxylase (Fig. 3). Using this

antiserum, Western blot analysis of (A) ptopeptide-eluted carboxylase eluted from affinity resin-A, (B) the DTT-eluted carboxylase described in Example 2, and (C) partially purified carboxylase, demonstrated reactivity with the 77,000 dalton band in all carboxylase fractions (Fig. 4), indicating the presence of the carboxylase antigen in all three preparations. Although the propeptide elution technique was therefore deemed useful for the preparation of purified carboxylase where activity of the enzyme is not essential, a method of affinity column purification which preserved the enzymatic activity of the carboxylase was sought. Example 2 Affinity purification of carboxylase by reductive cleavage of the enzyme-propeptide complex from the resin.

A. Preparation of affinity column.

A synthetic polypeptide ("affinity peptide-C") consisting of an amino terminal cysteine residue, a dipeptide linker segment, and proPT18, was synthesized by the method described in Example 1, except that the cleavage reaction was performed in HF:anisole:dimethylsulfide:resin(10:2:2:1). The sequence of the peptide was verified as CGGHVFLAPQQARSLLQRVRR.

All buffer solutions used for preparing the affinity resin were extensively degassed prior to use. 2-Thiopyridyl-activated thiol-Sepharose 4B (Pharmacia), containing 1μmols of activated sites per ml of packed resin, was swelled in

100 mM Tris, pH 7.0/1 mM EDTA, and washed with buffer T (100 mM Tris, pH 7.5/lmM EDTA/500 mM NaCl). The resin was suspended in buffer T.

10 μmols of affinity peptide-C were dissolved in

6 ml of buffer T and added to 4 ml of activated resin,

resulting in a molar ratio of 2.5 peptide-thiol groups to each activated site on the resin. The disulfide-bond-forming reaction was allowed to proceed for 3.5 hr at 25ºC under nitrogen with gentle mixing. After filtering, the resin

("affinity resin-C", illustrated in Fig. 1C) was washed with 6 ml of buffer T. The coupling efficiency was measured by quantitation of the release of 2-thiopyridone, using a molar extinction coefficient of 8080 at 343 nm, according to the method of Stuchbury et al., Biochem. 151:417-432, 1975. The coupled resin was washed extensively with 100 mM ammonium acetate, pH 4.5, and the unreacted 2-thiopyridyl groups were displaced by β-mercaptoethanol in 100 mM ammonium acetate, pH 4.5, added in a 3:1 ratio of β-mercaptoethanol molecules to the original number of activated sites on the resin. After 30 min under nitrogen at 25ºC, the resin was washed sequentially with 100 mM ammonium acetate (pH 4.5), PBS, and 0.02% NaN₃ in PBS; the affinity resin-C was then stored at 4°C until use.

B. Purification of carboxylase.

Carboxylase was detergent-solubilized and ammonium sulfate-precipitated in order to purify it partially

(approximately 100-fold) from a crude microsomal preparation, as described by Soute et al. (Table I). Because affinity resin-C contains some reactive sulfhydryl groups that are not coupled to the affinity peptide, any proteins in the

carboxylase preparation which have reactive sulfhydryl groups could bind to the affinity resin-C column and contaminate the carboxylase fraction. In order to remove these nonspecific proteins from the preparation prior to passing it over the affinity resin-C column, the preparation was first applied to a column of 2-thiopyridyl-activated thio-Sepharose 4B without a coupled peptide (Fig. 1D), as follows:

2.0 ml of the partially purified carboxylase preparation (41.8mg protein/ml) was applied to a 2-thiopyridyl- activated thio-Sepharose 4B column (6 ml of resin in a 1 ×

10 cm column) previously equilibrated in buffer B [20 mM sodium phosphate, pH 7.4/0.15 MNaCl/0.1% (w/v) CHAPS/15% (v/v) glycerol/1 mM EDTA/1.34 mM phospholipid {91% chicken

egg L-α-phosphatidylcholine (Type V-E, Sigma), 4.5% Folch

Fraction III bovine brain extract (Sigma), 4.5% bovine

heart L-α-phosphatidylethanolamine (Type VII, Sigma)}] at

4°C, and the flow stopped for 3 hr. The column was then washed with buffer B at a flow rate of 1 ml/hr. The carboxylase activity was recovered only in the unbound fraction, albeit with a 90% loss of carboxylase activity due to the

time-dependent instability of the impure enzyme. (A parallel loss of carboxylase activity was observed in an aliquot of partially purified carboxylase maintained at 4ºC for 3 hr but not exposed to activated thiol-Sepharose.) The carboxylase fraction was applied to a 1 × 10 cm column containing 3.75 ml of affinity resin-C previously equilibrated in buffer B, and the flowthrough was recycled through the column for 3 hr at a flow rate of 2 ml/hr. The affinity resin was then washed at a flow rate of 20 ml/hr with, in sequence: 50 ml buffer B, 50 ml buffer B containing NaCl at a final concentration of 1M, 10 ml of buffer B, and 100 ml buffer B/0.25% (w/v) CHAPS, all at 4ºC. Approximately 40% of the applied carboxylase activity remained bound to the column. The column was then equilibrated with a reducing buffer

(buffer B/0.25% (w/v) CHAPS/35 mM DTT) at 4ºC, at which

temperature the reducing buffer does not cleave the disulfide bond linking the cysteine of the affinity peptide and the thiol group of the resin. The column containing the reducing buffer was then warmed to 25ºC, and reductive cleavage of the affinity peptide-resin disulfide bond was allowed to proceed for

45 min. The carboxylase-affinity peptide-C complex, thus cleaved from the resin, was eluted with an additional 8 ml of reducing buffer at 25ºC, flowing at 20 ml/hr. Carboxylase activity was quantitatively recovered in the eluate, with an overall 10,000-fold purification from the crude microsomal preparation (Table I). SDS-polyacrylamide gel electrophoresis revealed a single homogeneous protein band corresponding to a molecular weight of 77,000 (Fig. 2, lane B). This band

co-migrated with the purified proteins obtained with propeptide elution of affinity resins-A and -B, and was similarly reactive with anticarboxylase antiserum prepared as described in

Example 1 (Fig. 4, lane B). The purified carboxylase was stable for at least one month at 4ºC.

Example 3. Cloning and expressing the carboxylase gene.

The amino acid sequence at the amino terminal of the 77,000 dalton protein purified by the affinity chromatography technique described in Example 1 was determined by standard techniques, utilizing automated Edman degradation. Beginning at the amino-terminal of the protein, the first eleven amino acids are Trp-Glu-Glu-Asp-Lys-Lys-Glu-Asp-Val(?)-Gly-Thr(?);

a portion of this peptide corresponds to a DNA coding sequence of

T T A T T

5' TC_CTT_CTT_GTC_CTC_CTCCCA 3

A family of synthetic DNA oligonucleotides having the above sequences was synthesized by standard techniques using an

Applied Biosyεtems Model 380 DNA synthesizer. These

oligonucleotides have been labelled with γ-³²P-dATP and

used as probes to screen a randomly-primed bovine liver cDNA library cloned into the vector λgtll. Other DNA probes based on different or longer portions of the carboxylase amino acid sequence will be developed by similar methods if necessary in order to locate the carboxylase coding sequence. Since the library in λgtll is a protein expression library, clones which hybridize with the oligonucleotide probes will be

rescreened with the anti-carboxylase antibodies as an

additional confirmation of the origin of the clones. In this regard, a positive result will be informative while a negative result will not. The inserts deemed interesting from the experiments above will be cloned into the filamentous

bacteriophage M13 mp18 and mp19, and the sequence determined by the dideoxy nucleotide chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463, 1977). The derived protein sequences will be compared with partial amino acid sequence data obtained as described above. Before abandoning isolated cDNA inserts that do not coincide with polypeptide-derived sequence data, we will determine whether the peptide sequence predicted by the cDNA has any unique features that would allow us to design and implement a strategy for identifying such a polypeptide should it be present in the carboxylase. Peptides rich in a particular amino acid could be identified in a protein digest by incorporation of labeled side chain derivatizing agents (for example, cystemes with 14_C

acetamide or tyrosines with ¹²⁵I)., and then isolated and sequenced by Edman degradation.

It is likely that the cDNA inserts we isolate by the above procedures will not represent a full length clone. The inserts will be labeled by nick-translation and used to

rescreen, by plaque hybridization, the λgtll library as well as a bovine liver library in pBR322. The relationships of the inserts to one another will be determined by restriction enzyme analysis and Southern blot analyses. Orientation of the fragments in an insert will be determined by establishing where linker sequences- are found, and by digestion with alternate restriction enzymes. The relationship of the length of the largest insert to the length of the carboxylase transcript will be determined by blot analysis of poly(A)+ RNA from bovine liver cells.

If possible the nucleotide sequence of the most complete insert will be determined on two independent clones. The cDNA insert and restriction fragments derived from the insert will be cloned into M13 mp18 and mp19 for sequencing (both strands). Sequencing will be performed by the dideoxy chain termination method (Sanger et al, 1977). Modification of the basic method may be made to sequence through difficult regions (e.g. regions of high GC content). Computer analysis of the of the sequence data will be performed using the Protein Identification Resource at the National Biomedical Research Foundation. The amino acid sequence will be deduced from the nucleotide sequence. We will search for regions of homology with other proteins, in particular those that are known to be localized to the endoplasmic reticulum (e.g. proline

hydroxylase, protein disulfide isomerase). We will look for amino acid sequences which are likely membrane binding sites (Yost et al, Cell 34: 759-766, 1983), for the KDEL sequence implicated in retention of proteins in the endoplasmic reticulum (Munro and Pelham, Cell 48:899, 1987) and for other homologies which may yield insight into the subcellular

localization and mechanism of action of the enzyme.

Once a full-length cDNA clone of the carboxylase is obtained, it will be cloned into the mammalian expression vector pMT2 and expressed in Chinese hamster ovary (CHO) cells (Jorgensen, et al., J. Biol. Chem 262:6729, 1987b). Expression of carboxylase will be monitored by immunofluroesence.

Polyclonal anti-carboxylase antibodies will be bound to

permeabilized transfected cells and the polyclonal antibody visualized with a fluorescently-labeled second antibody. We expect to see the vitamin K-dependent γ-carboxylase localized in the rough endoplasmic reticulum of the CHO cells if the protein is expressed and properly targeted within the cell.

Cells expressing the carboxylase will be co-transfected with the cDNA for human Factor IX, a protein which is generally only partially carboxylated in normal CHO cells. The supernanant from the co-transfected cells will be assayed for Factor IX and the level of carboxylation determined by immunologic and direct amino acid analysis (Kaufman et al., J. Biol. Chem. 261:9622, 1987; Jorgensen et al., 1987b). The level of expression of Factor IX will be amplified using methatrexate and the assays repeated (Kaufman et al., 1987). We anticipate that we will be able to produce high levels of fully carboxylated Factor IX by this approach.

In an alternative approach, the cDNA for the vitamin K-dependent carboxylase will be altered to remove sequences coding for a putative transmembrane sequence or for a KDEL sequence which would localize the enzyme to the rough

endoplasmic reticulum. When the altered DNA is transfected into CHO cells we expect a secreted form of the carboxylase to be expressed. A soluble form of the enzyme could be used for in vitro carboxylation of VKD protein precursors. Other Embodiments

Other embodiments are within the following claims.

For example, a resin other than cyanogen bromide-activated Sepharose 4B or 2-thiopyridyl-activated thiol-Sepharose 4B may be utilized as the matrix to which the propeptide is bound.

The size of the column could be varied, or the resin could be utilized in a form other than a column, as, for example, a beaker or test tube from which unbound material is simply decanted. Instead of the entire propeptide, a portion capable of binding carboxylase may be used. The preparation of

carboxylase need not be partially purified prior to passage over the affinity column, or it could be further fractionated, if necessary, after passage over the affinity column. Methods other than those described above may be used for removing the purified carboxylase from the affinity resin: for example, reducing agents other than DTT, such as β-mercaptoethanol, or peptides other than proPT18, such as proPT28 or a portion of a propeptide from a VKD protein other than prothrombin, may be utilized to separate carboxylase from the affinity resin.

Methods other than screening a cDNA library may be used to isolate the carboxylase coding sequence: for example, a genomic DNA library may be screened for the carboxylase gene, using the synthetic DNA oligonucleotide of the invention. This gene, when transfected into a eukaryotic cell along with the gene for a VKD protein precursor, could be expected to express a carboxylase capable of γ-carboxylating the VKD protein.

The affinity chromatography method of the invention would be generally applicable to the purification of proteins other than carboxylase. By substituting an appropriate ligand or portion of a ligand for the propeptide on the affinity resin, a polypeptide other than a γ-carboxylase could be bound to the resin and then released by treating the resin with free ligand, or with a reducing agent to cleave the disulfide bond between the affinity peptide and the matrix material. For example, the PADGEM receptor protein could be isolated from a mixture of proteins by the use of an affinity resin containing a peptide from PADGEM.

Claims

1. Substantially purified vitamin K-dependent γ- carboxylase.

2. The substantially purif ied carboxylase of claim 1 , wherein said carboxylase is liver microsomal vitamin K-dependent γ-carboxylase.

3. The substantially purified carboxylase of claim 2, wherein said carboxylase is bovine liver microsomal vitamin K-dependent γ-carboxylase.

4. An affinity resin for separating, from a mixture of polypeptides, a first polypeptide which is not an antibody, and which is capable of binding to a ligand which is not an antibody, said affinity resin comprising an insoluble matrix material linked to a second polypeptide comprising a portion, but not all, of said ligand, said portion being capable of binding to said first polypeptide.

5. The affinity resin of claim 4, wherein said firs polypeptide is a vitamin K-dependent γ-carboxylase and said ligand portion comprises a γ-carboxylase-binding segment of the propeptide region of a substrate for said carboxylase.

6. The affinity resin of claim 5, wherein said ligand portion comprises part or all of the 18-amino acid sequence HVFLAPQQARSLLQRVRR.

7. The affinity resin of claim 4, wherein said matrix material is cyanogen bromide-activated agarose.

8. The affinity resin of claim 4, wherein said linkage comprises a disulfide bond.

9. The affinity resin of claim 8, wherein said matrix material comprises 2-thiopyridyl-activated thiol-agarose.

10. An affinity column for separating a vitamin K-dependent γ-carboxylase from a mixture of polypeptides, said affinity column comprising a column containing the affinity resin of claim 5.

11. A method of separating vitamin K-dependent γ-carboxylase from a mixture of polypeptides, said method comprising

(1) applying said mixture to the affinity column of claim 10 under conditions permitting the binding of said

carboxylase to said affinity resin,

(2) washing unbound constituents of said mixture from said affinity resin, and

(3) eluting said carboxylase from said affinity resin.

12. The method of claim 11, wherein said carboxylase is eluted from said affinity column by application to said affinity column of a solution containing a molecule capable of binding to said carboxylase's ligand-binding site.

13. The method of claim 11, wherein said carboxylase is eluted from said affinity resin by subjecting said affinity resin to conditions capable of cleaving the linkage between said second polypeptide and said matrix material of said

affinity resin.

14. Substantially purified cDNA encoding vitamin K-dependent γ-carboxylase.

15. A vector comprising DNA encoding vitamin K- dependent γ-carboxylase.

16. The vector of claim 15, wherein said carboxylase is taken from the group consisting of bovine vitamin K- dependent γ-carboxylase and human vitamin K-dependent

γ-carboxylase.

17. A cell transfected with the vector of claim 15.

18. The cell of claim 17, wherein said cell is a eukaryotic cell.

19. The cell of claim 17, wherein said cell is a prokaryotic cell.

20. The cell of claim 17, wherein said cell is capable of expressing said carboxylase.

21. The cell of claim 20, wherein said cell is capable of expressing a VKD protein precursor.

22. The cell of claim 21, wherein said VKD protein is a coagulation protein.

23. The cell of claim 22, wherein said coagulation protein is taken from the group consisting of prothrombin, Factor VII, Factor IX, Factor X, protein C and protein S.

24. The cell of claim 21, wherein said cell is capable of γ-carboxylating glutamic acid residues on said VKD protein.

25. An affinity resin for separating, from a mixture of polypeptides, a first polypeptide which is capable of binding to a second polypeptide, said resin comprising an insoluble matrix material linked via a disulfide bond to said second polypeptide.

26. An affinity column comprising the affinity resin of claim 25.

27. A method of separating, from a mixture of polypeptides, a first polypeptide which is capable of binding to a second polypeptide, said method comprising

(1) applying said mixture to the column of claim 26 under conditions permitting the binding of said first

polypeptide to said affinity resin,

(2) washing unbound constituents of said mixture from said affinity resin,

(3) subjecting said affinity resin to conditions under which said disulfide bond is cleaved, and

(4) eluting said first polypeptide from said affinity resin.

Table 1. Purification of the vitamin K-dependent carboxylase.

Total protein Specific Activity Total Activity Purification (mg) com × 10^-4 Com × 10^-4

mg protein × hr hr

1. Bovine liver N.D. N.D.

microsomes

2. Microsomes, 83.7 12.5 1043 95

detergent-solubilized

ammonium eulfate

fractionated

3. Activated thiol- 22.6 4.8 108 36

Sepharose

4. CGGproPT18-thiol- 0.035 1342.4 47 10,205

Sepharofe

Values for the purification of carboxylase from crude microsomes to

detergent-solubilized microsomes fractionated with ammonium sulfate are from

Soute et al , 1987 .