CA2087970A1 - Gamma-carboxylase and methods of use - Google Patents

Abstract

Protein compositions having gamma-carboxylase activity enriched at least 20,000-fold as compared to liver microsomes are provided.
Also provided are DNA molecules encoding gamma-carboxylase. The protein compositions and DNA molecules are useful within methods of producing gamma-carboxylated vitamin K-dependent proteins.

Description

~ ~o 92/01~95 2 0 8 7 9 7 0 PCT/U591/05177 Descri~tion GAMMA-CARBOXYLASE AND METHODS OF USE

Technical Field The present invention relates generally to isolated proteins and methods of making proteins. More specifically, the invention relates to isolated gamma-carboxylase, DNA sequences encoding gamma-carboxylase, and methods of using the DNA sequences in the production 4f gamma-carboxylated proteins.

Backaround of the Invention Gamma-carboxyglutamic acid (abbreviated "gla") 15 i8 an amino acid found in certain calcium-binding proteins. These proteins include factor VII, factor IX, factor X, prothrombin, protein C and protein s, plasma proteins that are components of the coagulation system;
protein Z, also found in plasma; pulmonary surfactant-associated proteins (Rannels et al., Proc. Natl. Acad.Sci. USA 84: 5952-5956, 1987); and the bone proteins ost~ocalcin (also known as bone gla-protein) and matrix gla-protein. Prot-in~ containing this amino acid are variously re~erred to as "vitamin K-dependent proteins,"
"gla prot-in~," or "gamma-carboxylated proteins." The plasma vitamin X-dependent proteins are dependent on gla-mediated binding to calcium and membrane phospholipids for their biological activity. Gla-containing proteins show a high degre- o~ amino-terminal amino acid ~eguence homology.
Gamma-carboxyglutamic acid is formed by the post-translational modification of specific glutamic acid re~idues in a reaction requiring vitamin K hydroquinone (KHa) as a cofactor. The carboxylation reaction requires the oxidation of KH2 to vitamin K epoxide ~KO), however carboxylation and KO formation are not strictly coupled.
The reaction is believed to proceed by removal of a ~WO92/017~ PCT/US91/~1 ~
~2087~7~ 2 ~

hydrogen ion from the ~-carbon of glutamic acid, followed by the addition of C02. KH2 is regenerated by the action of reductases. Gamma-carboxylase is the subject of a recent review by Vermeer (Biochem. J. 266: 625-636, 1990).
In recent years a number of vitamin K-dependent proteins have been produced by genetic engineering.
Production levels of active protein are, however, limited by the ability of host cells to properly carboxylate glutamic acid residues in the nascent proteins. It has lo not heretofore been possible to produce biologically active vitamin K-dependent proteins in prokaryotic or lower eukaryotic (e.g. fungal) host cells, necessitating a reliance on higher eukaryotic (e.g. mammalian) cell lines, whlch are generally more difficult and expensive to culture. Even so, production levels of biologically active vitamin K-dependent proteins have been quite low when compared to levels of other proteins produced using genetically engineered mammalian cell lines. For example, Hagen et al. (U.S. Patent No. 4,784,950) reported the expression of recombinant human factor VII at up to 200 ng/ml in a baby hamster kidney cell line; Kaufmann et al.
~ iol. Chem. ~~: 9622-9628, 1986) produced 100 ~g/ml o~ ~a¢tor IX u~ing th- CH0 cell line, but only 1% of the ~t-rial wa- blologically active; and Walls et al. (Gene 2S ~: 139-149, 1989~ produced protein C at up to 25 ~g/ml following amplification. In contrast, other proteins are typically produced in biologically active form at levels Or 100-200 ~g/ml. Further studies have shown that ecretion of vitamin K-dependent proteins by cultured mammalian cells is reduced up to 20-fold if vitamin K i6 omitted from the culture media, and factor IX and protein C ar- poorly secreted if their propeptides are deleted.
Together, these experimental results suggest that vitamin X-dependent carboxylation is required for efficient 3S production of these proteins, and that the inability of cell linQs to efficiently carboxylate may limit expression levels. These data further indicate that improperly ,,: . ,- . :~ : -~ -- ~

D Wo 92/01795 ~0 8 7 9 7 0 /U591/~177 ,- :
processed (i.e. uncarboxylated or undercarboxylated) vitamin K-dependent proteins are not efficiently secreted and may be unstable in the host cells.
Previous attempts to isolate gamma-carboxylase have not resulted in useful levels of purification.
Can~ield et al. (Arch. Biochem. Biophvs. 202: 515-524, 1980) reported a 100- to lS0-fold enrichment of carboxylase activity in a rat liver microsomal preparation. Girardot (J. Biol. Chem. 257: 15008-15011, 1982) reported a 400-fold purification of a carboxylase-sub6trate complex from rat liver microsomes. Van Haarlem et al. (Biochem. J. 245: 251-255, 1987) prepared a crude microsomal fraction enriched for carboxlase activity from bovine aortae. This preparation also exhibited vitamin K
reductase activity. Hubbard et al. (Proc. Natl. Acad.
Sc~l USA 86: 6893-6897, 1989) reported the purification of a 77 kDa carboxylase from bovine liver. This protein was later determined to be BiP (also known as GRP78) (Furie, 1990 Gordon Research Conference, Hemostasis and Thrombosis; Walsh, 1990 Gordon Research Conference, H-mo~tasis and Thrombosis).
There remain~ a need in the art ~or puri~ied pr-paration~ Or gamma-carboxylase to permit the oharact-rization o~ thi~ enzym-. The i~olated enzyme may b- u~-d to dir-ct the ~n vitro carboxylation of vitamin K-dap-ndent prot-ins. Carboxylase-encoding DNA molecules may be used in produoing biologically active vitamin K-dop-nd-nt proteins in low-r euXaryotes and proXaryotes.
In addition, DNA molecul-s ncoding the enzyme may be used within gen-tic engin-ering proc-ss-s to incr-ase production o~ vitamin K-dependent proteins in cultured m~cmalian cell lines. The present invention provides such enzyme pr-parations and DNA molecules, as well as methods ror producing vitamin K-dependent proteins that employ the nzyme preparations or DNA molecules. In addition, the pr-s-nt inv ntion provides other, related advantages.

~ W092/017~ O PCT/US91/~l ~
~ 208797~ 4 Summary of the Invention In one aspect, the present invention provides protein compo6itions having gamma-carboxylase activity enr~ehed at least 20,000-fold as compared to liver microsomes In one embodiment, the protein is selected from the group consisting of bovine,~ human and rat gamma-carboxylases In another embodiment, the protein is liver gamma-carboxylase In yet another embodiment, the gamma-carboxylase activity of the protein composition is enriched 100,000-fold as compared to liver microsomes In another aspect, protein compositions as described above are provided wherein the protein is af~ixed to a solid support These compositions are use~ul, ror example, in carboxylating vitamin K-dependent proteins in vitro In yet another aspect, the present invention provides DNA molecules encoding gamma-carboxylase, including bovine, human and rat gamma-carboxylases In ono embodiment, the gamma-earboxylase is liver gamma-earboxylase In another embodiment, the DNA moleeule is aeDNA molecule A r-lat-d aspeet o~ the present invention provid-- a eultur-d eell trans~eeted or transformed to xpr--8 ~ DNA ~-qU~ne- eneoding gamma-earboxylase Within 2~ eertaln embodim nts, the eultured eell is a eukaryotie e~ll, sùeh as a yeast eell or a mammalian eell In another embodiment, the eultured eell i5 further trans~ected or transrormed to express a DNA seguence oneoding a vitamin X-dependent protein, sueh as raetor VII, raetor IX, faetor X, prothrombin, protein C, aetivated protein C, protein S, protein Z, osteoealein, matrix gla-protein or pulmonary surraetant-assoeiated protoin~ Within another embodiment, the vitamin X-dop ndent protein is a human protein The eultured eellc may be used within methods of produeing vitamin K-dependent proteins by eulturing the cells and isolating the vitamin X-dependent protein Within these methods, - - . . -~ ~ 92/01795 2 0 8 7 9 7 0 PCT/US9l/05177 ~ 5 - , :
.
- the cells may be cultured in the presence of o.l-lO ~g/ml vitamin K.
In another aspect, the present invention provides methods of producing a vitamin K-dependent protein comprising the steps of (a) introducing into the germline of a non-human animal a first DNA sequence encoding gamma-carboxylase and a second DNA sequence encoding a vitamin K-dependent protein, (b) growing the animal and (c) isolating from the animal the vitamin K-lo dependent protein encoded by the second DNA sequence. Inone embodiment, the vitamin K-dependent protein is selected from the group consisting of factor VII, factor IX, factor X, prothrombin, protein C, activated protein C, protein S, protein Z, osteocalcin, matrix gla-protein and pulmonary surfactant-associated proteins. In another embodiment, the vitamin K-dependent protein is a human protein.
A related aspect of the present invention provides non-human animals into which have been introduced a cloned DNA molecule encoding gamma-carboxylase, wherein i; the DNA molecule i8 expressed in the animals. The animals may further have introduced into them a second DNA
molecule encoding a vitamin K-dependent protein, wherein th- -oond DNA molecule is also expressed. In one 2S mbodim-nt, the vitamin K-dependent protein is selected from the group consisting of ~actor VII, factor IX, factor X, prothrombin, protein C, activated protein C, protein S, protein Z, osteocalcin, matrix qla-protein and pulmonary surfactant-associated proteins. In another embodiment, the vitamin K-dependent protein is a human protein.
These and other aspects of the invention will become evident upon reference to the accompanying detailed description and drawings.

,. . . .. . . . .

: : : :.~ - ,. ;, : ~ . .
. .

~ W092/017~ PCT/VS91/~l ~ 2087970 Brief Description of the Drawin~s Figure 1 shows a polyacrylamide gel of fractions from a gamma-carboxylase purification procedure Molecular weight markers (200, 93, 68 and 43 kD) are indicated Figure 2 illustrates thë construction of leader-deleted IX-pD5 Symbols used are o-l, tke adenovirus 5 o-1 map unit sequence; E, the SV40 enhancer; MLP, the adenovirus 2 major late promoter; L1-3, the adenovirus 2 tripartite leader; 5~, 5' splice site; 3', 3' splice site;
FIX, factor IX cDNA; and pA, the sV40 early polyadenylation signal Figure 3 shows three samples of purified bovine gamma-carboxylase (lanes 1-3) Lane 4, molecular weight markers (205, 117, 106, 80 and 50 kd); lane 5, molecular weight markers as shown Figure 4 shows the results of a Western blot of purified bovine gamma-carboxylase probed with anitsera to bovine gamma-carboxyla~e Positions of molecular weight markers are shown ; Figure 5 shows a polyacrylamide gel of proteins purified from G3 and D30 microsomes using antibody ~ffinity chromatrography, cation exchange chromatography and l-ntll l-ctin chromatography Lane 1, molecular 2S w-lght mark-r~ ~200, 93, 68, 43 and 26 kD ); lane 2, D30 mlcro~om-s; lane 3, G3 microsomes; lane 4, markers ~l~g1l9~LL~cripti~On Q~ Invention Prior to settlng forth the invention, it may be u-eful to define certain terms to be used hereinafter Transfection or trans~ormation The proce~s of stably and hereditably altering the genotype of a r-cipi-nt cell or microorganism by the introduction of purifi-d DNA This is typically detected by a change in 3S th- ph-notype of the recipient organism The term ~transformation" is generally applied to microorganisms, : ' ' ` ' `7 ' '' `" . . .: ".' ' '' ,'' ',;.'` ' ' .. . .~ ,,, ' ' . , : ' : :, .

, ~ ' . ; ' ' ! ' . , ' ; . . ' ~ ~

~ ~Og2/017~ 208797~ PCT/US91/~177 while "transfection~ is used to describe this process in cells derived from multicellular organisms.
Cultured cell: A cell capable of being grown in liquid or solid media over a number of generations. In the case of cells derived from multicellulàr organisms, a cultured cell is a cell isolated from the organism as a single cell, a tissue, or a portion of a tissue.

As noted above, carboxylation of specific glutamic acid residues is necessary for the biological activity of certain proteins. The present invention provides protein compositions that are highly enriched for gamma-carboxylase activity, as well as DNA molecules encoding gamma carboxylase. These compositions and molecules are useful within methods of making vitamin X-dependent proteins.
Within the present invention, gamma-carboxylase was isolated from microsomes prepared from rat and bovine liver and from a cultured human cell line producing recombinant factor IX. Gamma-carbaxylase may also be isolated from other tissues or cells Xnown to produce the protein. The isolated carboxylase may be used for ln vit~g carboxyl~tion of vitamin X-dependQnt proteins. The c~rboxyl~se ~l-o provides a useful tool for the isolation Or g-nomic and cDNA clon-~ encoding it. These clones are u--d to transr-ct or transform cultured eukaryotic or prokaryotic cells to produce ho~t cells suitable for the hlgh l-vel production Or vitamin X-dependent proteins, lnoluding vit~min K-dependent blood coagulation prot-in~
and vitamin K-d-pondent bone protein~.
A prererred source o~ gamma-carboxylase is liver microsomes, such a8 microsomes prepared from rodent (e.g.
rat or mouse), bovine or human liver, although liver microsomes from other species may also be u~ed. The 3S production of biologically active, recombinant human vlt~min K-dep-ndent proteins in host cells derived from a variety of mammalian species ~Hagen et al., U.S. Patent No. 4,784,950; Foster et al., U.s. Patent No. 4,959,318) demonstrates that gamma-carboxylase is active across ~pecies lines, thus a gamma-carboxylase derived from one mammalian species will be useful in carboxylating proteins from a second mammalian species, although the syngeneic carboxylase may be preferred. Other suitable sources include microsomes from cell lines known to be capable of producing active vitamin X-dependent proteins, such as the transformed human kidney 293 cell line (available from American Type Culture Collection under accession number CRL 1573). It will be understood by those skilled in the art that, in principle, nearly any tissue or cell type is a candidate source of gamma-carboxylase. Gamma-carboxylase activity has been detected in most tissues examined, and carboxylase activity may be detected in a candidate tissue or cell line by assaying the ability of that tissue or cell line to incorporate radiolabeled Co2 into protein in a carboxylation reaction as described herein. Typically, microsomes are prepared from 1-2 g of tissue and incubated in a carboxylase reaction with NaH14CO3. The reaction is run in the presence and absence of vitamin K hydroguinone, and the reaction products are analyzed by g~l el-ctrophoresis and autoradioqraphy. The pr---no- of one or more labeled protein bands specific to 2S th- vitamin X-containing reaction i8 indicative of gamma-carboxyla~e in the tested tissue.
Microoomes are prepared from cells or tissues according to procedures known in the art. One ~uitable m thod iB that de~cribed by Swan~on and 8uttie ~l5GhQml~LY ~: 6011-6018, 1982), which is incorporated h-roin by re~erence. Briefly, the cells or tissue are homogenized in a slightly acidic to slightly basic buffer containing sucrose and a protease inhibitor. Microsomes ar- harvested from this preparation by ultracentrifugation. Preparation of microsomes is also disclosed by Rannels et al. (ibid) and Soute et al.
(Thromb. Naemostasis 57: 77-81, 1987J.

:: . . .. . . . .. :.- ..

:- . - : . . - , . : . , . .. : .. ~ .. . . . , .. : ... ..... .
, - : : . . ~ - :-.- ..
, . . - - ~: - - . - , ~ -~ ~ 092/0~795 2 0 8 79 7 0 ~ PCT/US91/05177 A protein extract is then prepared from the microsomes. A suitable extract may be prepared by disrupting the membranes in a tissue homogenizer, solubilizing in detergent, removing insoluble membrane material, and precipitating detergent-solubilized proteins by adjusting the (NH4)2S04 concentration of the solution to about 60~ of saturation. The precipitated proteins are recovered by centrifugation.
Within the present invention, several different methods were used to fractionate the microsome extracts and purify gamma-carboxylase. Certain of these methods take advantage of the ability of gamma-carboxylase to bind to a vitamin K-dependent protein such as factor IX, prothrombin, or protein C.
In a preferred embodiment, gamma-carboxylase is isolated from extracts of microsomes from vitamin K-treated animals by affinity chromatography on a propeptide of a vitamin K-dependent protein. To maximize recovery, it is preferred to incubate the extract and propeptide for at least 16 hours. Bound proteins are eluted from the propeptide and electrophoresed. Comparison to the pattern o~ protein6 bound to a non-specific peptide is used to identiry gamma-carboxyla~e. Propeptides of vitamin K-d-p-nd-nt protein~ may be synthesized on the basis of 2S Xnown amino acid sequence data. See, for example, the di-clo~ures o~ Kurachi et al. ~Proc. Natl. Acad. Sci. USA
79: 6461-6464, 1982), Foster et al. ~Proc. Natl. Acad.
Scl. USA ~2: 4673-4677, 1985), Hagen et al. ~Proc- Natl-Acaq~- Sci. USA 83: 2412-2416, 1986) and Leytus et al.
(BiQc~h~mlstry ~: 5098-5102, 1986), which are incorporated h-rein by reference. Propeptides may be synthesized by the solid phase method of Merrifield ~Fe~k Proc. ~: 412, 1962; J. Amer. Chem. Soc. 85: 2149, 1963) or by use of an automated peptide synthesizer. To isolate gamma-carboxylase, a propeptide is covalently bound to a ~uitable solid support, such as a derivatized agarose gel ~e.g. cyanogen bromide-activated agarose gels or activated .
- - ~
- , , . :
- - . .... . .
- ~. . .
- ., -:, ` . -. . .. ' :: ...... ', ., -, ,: t ; W092/01795 2 ~ 8 7 9 7 ~ PCT/USg~
' 10 thiol agarose gels). A preferred such support is activated thiol Sepharose 4BTM, available from Pharmacia, Piscataway, NJ. Methods for coupling peptides to solid supports are known in the art, and specific instructions are generally provided by the suppiier. When using an activated thiol support, the coupled propeptide and gamma-carboxylase are eluted with a reducing agent such as dithiothreitol. In the alternative, a complete proprotein (e.g. pro-factor IX or pro-prothrombin) is attached to the support and bound gamma-carboxylase is eluted using an isolated propeptide of a vitamin K-dependent protein.
Chromatography can be carried out in either batch processing or on a column. This step typically provides about a 500-fold enrichment of gamma-carboxylase activity.
15 An alternative embodiment utilizes antibody affinity chromatography. An antibody, preferably a monoclonal antibody, to a vitamin K-dependent protein is coupled to a solid support (e.g. activated thiol Sepharose or CNBr-activated Sepharose). Antibodies to vitamin K-d~pendent proteins are disclosed by, for example, Wakabayashi t al. ~. Bi~l. Chem. ~~: 11097-11105, 1986), Kau man t al. (ibid.) and Busby et al. (Na5ure 316: 271-273, 1985). In on~ embodiment, a microsome xtract, pr-pared from warfarin-treated animals in a 2S manner similar to that described above, is applied to the immobilized antibody. Gamma-carboxylase is eluted from th- antibody using a propeptide o~ a vitamin K-dependent prot-in or by incubating the antibody-enzyme complex in a carboxylation reaction mix. In the alternative, calcium-d~p~ndent antibodies are used, and the carboxylase isolutod by altering the calcium concentration. The eluant, containing gamma-carboxylaso, i8 reCOVerQd.
Antibody affinity chromatography is also used to id~nti~y gamma-carboxylase. Cultured cells are ~5 transfected to express a vitamin K-dependent protein lac~ing its propeptide. An extract of these cells is exposed to the immobilized antibody. Bound protein is ~ , . . . . ,. . ..... - ... ... .. .. . . . .
.. . , .... ~ . ... ,. , ~ . " . . . , - ~ - . . .

092/01795 2 08 79 70 ~ PCT/US91/~177 ' 11 eluted and electrophoresed. The protein pattern is compared to a parallel preparation made from cells transfected to express the corresponding native vitamin K-dependent protein (i.e. including the propeptide). Gamma-carboxylase i8 found specifically in the preparation fromcells expressing the native protein.
Further purification is achieved using ion exchange chromatography and/or lectin affinity chromatography. Ion exchange chromatography may be carried out using either anion exchange or cation exchange media. Bovine gamma-carboxylase has been found to bind to cation exchange media, while human and rat gamma-carboxylases have been found to bind to anion exchange media. Suitable anion exchange media include derivatized, cro~s-linked agaroses and dextrans, including DEAE and QAE
derivatives. Particularly preferred anion exchange media include DEAE Sepharose CL-6BTM and Q-SepharoseTM, available from Pharmacia. Suitable cation exchange media include carboxymethyl and sulfopropyl derivatized resins.
Suitable lectins include mannose-specific lectins, with lentil lectin particularly preferred. Additional puri~ication may be obtained by u~ing lectins with other ~p-cl~icitie~ to remove contaminating glycoprotein~.
L-ct~n- are coupled to support matrices according to 2S conv-ntional ~ethods (e.g. CNBr activation), or lectin matrice~ may be obtained ~rom commercial suppliers.
In a typical anion exchange chromatography step, a partially puri~ied carboxylase preparation is dialyzed in a neutral to slightly ba~ic bu~er containing phospholipids and a non-ionic detergent. Preferred bu~fers includ~ 20 mM Tricine pH 8.5 or 20 mM Bis-Tris pH
6.8 containing 1 mg/ml phosphatidyl choline type VE, 50 pg/ml pho~phatidyl serine, 50 ~g/ml phosphatidyl thanolamine and 0.1% 3-~3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate [CHAPS~. The dialyzed solution i8 then combined with an anion exchange medium, allowed to incubate for about 16 hours, and eluted with . . - : . , ~ .

~ 2 0 8 7 9 7 O PCT/US9l/05~ ~

the same buffer containing salt (e.g. 0.2 M to 1.0 M
NaCl), preferably about 0.5 M NaCl. The eluant, which contains the carboxylase, is recovered.
It has been found by the inventor that gamma-carboxylase contains core glycosylation, allowing it tobind to mannose-specific lectins. However, gamma-carboxylase lacks more complex carbohydrate additions.
Based on these observations, lectins of different specificities may be used in purifying gamma-carboxylase.
Lectin affinity chromatography is typically carried out using lentil lectin bound to a derivatized agarose support that has been washed in phosphate buffered saline. A
partially purified carboxylase preparation (e.g. purified by propeptide affinity chromatography or propeptide a~finity chromatography followed by anion exchange chromatography) i8 combined with the washed lectin resin.
It is preferred to allow the mixture to incubate for at least about 16 hours. The resin is then washed to remove unbound material, and the bound carboxylase is eluted with a suitable sugar, such as mannose or ~-methyl mannoside.
The typical level of enrichment at this point is about 20,000 x ovor the initial microsome preparation. The lev-l of enrichment can be increased to about 100,000 x by u-ing a ~econd lectin to remove contaminating 2S glycoprot-in~. Pre~erred second lectins include Ervthrina s~la3~9~ lectin, Tetraaonolobus PurDureas lectin, P~-udomonas aeruainosa PA-I lectin and Limulus polyphemus l-ctin, which do not bind gamma-carboxylase. These and other l-ctins are available from commerical suppliers (-.g. Sigma Chomical Co., St. Louis, M0). Lectins are bound to solid supports using conventional chemical methods a8 generally described above.
Cation exchange chromatography of a partially puri~ied gamma-carboxylase preparation is carried out 3S using a neutral to slightly acidic buffer containing phospholipids and a nonionic detergent.

.. :: ~ . .-. . - . . . .
. , ;,: ~ ..................... ~.:, -... ~. .................... .

' ~ .. . . .

~ ~092/01795 2 0 8 7 ~ 7 0 PCT/US91/05177 ... .
13 ~

An alternative purification protocol employs, in order, propep~de or antibody affinity chromatography, lectin affinity chromatography and ion exchange chromatography.
Throughout purification, it is preferred to perform all manipulations involving gamma-carboxylase in the cold (e.g. 4OC) and in the presence of a l:l ratio of detergent to phospholipid.
Purification of gamma-carboxylase is monitored by assaying the biological activity of samples using the carboxylation assay described in more detail below.
Protein samples may be electrophoresed on non-denaturing polyacrylamide gels in the presence of phosphatidyl choline and CHAPS, and protein bands cut from the gels and tested for activity in the carboxylation assay. Total protein concentration of samples is monitored according to routine procedures, such as methods utilizing bicinchoninic acid ~e.g. BCATM protein assay reagent, available from Pierce Chemical Co., Rockford, IL) or by gel scanning.
The above-described methods were used to id-ntify prot-in~ that speci~ically bound to a prothrombin prop-ptid-. With$n one a~pect Or the invention, further puririoation of rat gamma-carboxylase by lentil lectin a~lnity chromatography was used to identify proteins having molecular weights of approximately 90 and 150 kDa on denaturing gels. Gamma-carboxylase is identified ~rom th--- candidate proteins, for example by amino acid equ-nc- analysi8. Picomole guantities of protein are s-quenced essentially as described by Aebersold et al.
~proc. Natl. Acad. Sci. USA 84: 6970-6974, 1987), incorporated herein by reference. Peptide fragments are g-n-rated by gel electrophore6is of the protein and tran~-r to nitrocellulose, followed by proteolytic 3S dige-tion. The resulting enzymatic cleavage fragments are ~eparated by HPLC and seguenced on an automated gas-phase sequenator. The amino acid seguences are compared to .
:, . , -- - , ~ :, . .
.... .: :~ ........

~W092/01795 - PCT/US91/~17~7 -2087970 ~

known sequences, such as the sequences contained in the GenBankTM database of the U.S. Department of Health and Human Services or the Protein Sequence Database of the National Biomedical Research Foundation. In the alternative, gamma-carboxylase is~identified by preparing peptides corresponding to the amino acid sequences of the candidate proteins, preparing antibodies to the peptides, and using the antibodies to identify the gamma-carboxylase by immunodepletion of biological activity.
Gamma-carboxylase purified as described above may be used to generate polyclonal or monoclonal antibodies according to conventional procedures. These antibodies are useful for large-scale affinity purification of the protein.
Information obtained from the isolated protein i8 then used to clone DNA molecules encoding gamma-carboxylase. Standard cloning methods are employed.
Although most tissues are believed to produce gamma-carboxylase, ~or cloning it is preferred to use a library derived from a tissue known to produce substantial amounts o~ vitamin X-dependent proteins or known to contain gamma-oarboxylase activity, such as liver, kideny, testis, heart, bone or lung. ~iver tissue is a particularly pr-~-rr-d ~ourc-.
2S For example, a DNA molecule encoding gamma-carboxylase can be isolated using oligonucleotide probes d-~igned from the amino acid seguence o~ the protein. It i8 pre~erred to use an amino acid sequence having a low l-V-l o~ degeneracy. These probes are used to screen genomic or cDNA libraries according to known procedures.
In general, it is preferred to use a cDNA library.
In the alternative, oligonucleotide primers de~igned on the basis o~ the amino acid sequence are used within the polymerase chain reaction cloning method 3S ~disclosed by Mullis et al., U.S. Patent No. 4,683,195 and Mullla, U.S. Patent No. 4,683,202, which are incorporated herein by reference).

r;, 'Ç~ -~ ~ 92/01795 2 0 8 7 9 7 a PCT/US9~ 77 ,; 15 The primers will qenerally comprise a restriction site at the 5' end to facilitate subsequent cloning of the fragment and about 14 to 30, preferably about 18-20, nucleotides corresponding to the predicted DNA sequence. For highly degenerate sequences, shorter primers (e.g. those comprising about 14 nucleotides corresponding to the DNA sequence) are preferred. Primer synthesis is simplified by substituting inosine at positions of four-fold degeneracy. When working with primers of high degeneracy, the initial PCR reactions will generally be run at a low stringency (low annealing temperature). In this event, it is advantageous to repeat the PCR procedure at slightly higher stringencies to reduce the background (non-speci~ic sequences). ~he PCR
reaction products are separated by gel electrophoresis, and bands of the predicted size for the fragment of interest are sequenced to confirm their identity. PCR-generated clones are used as probes to screen cDNA or genomic libraries or to generate longer clones by the RACE
(rapid ampllfication of cDNA ends) procedure of Frohman et al. ~Proc. Nat~,_Acad. Sci. USA 85: 8998-9002, 1988). The RACE procedur- can be run using combinations o~ PCR-g-n-rat-d prim r~, PCR-generated primers with mixed oligonucl-otid- prim-rs or oligo~dT) or mixed with 2S ollgonucleotid- prlm-r~ d-signed from amino acid sequence having low degen-racy at the nucleotide level. PCR
methods are disclosed by Innis et al., eds. PCR Protocols:
~ Lh ~ and App~ ons, Academic Press, Inc., 8an Diego, l990 and by Erlich., ed- P~R TeChnolQqy:
principles and_ App~ ions ~or DNA Ampli~ication, 8tockton Press, NY, 1989.
In a third alternative, antibodies raised against purifi-d gamma-carboxylase or fragments of gamma-carboxyla~e are used to screen expression libraries, such a~ cDNA libraries prepared in the ~gtll vector ~ATCC
37194)-- . . . - . ~ : .: , .. , . , , .- . . . . . . .
- . . - .,, , . .,, ~

. , " . ' ~ - . :`' .,' ' . - . ~. , ,; . . ' ' .

The identity of cloned sequences may be confirmed in an expression cloning system, such as the ~gtll system of Young and Davis (Proc. Natl. Acad. Sci.
~E~ ~Q: 1194-1198, 1983). Using standard immunization S protocols, antibodies (preferably monoclonal antibodies) are prepared against peptides synthesized from the gamma-carboxylase sequence. These antibodies are used to screen the expression library. Methods for fusing lymphocytes and immortalized cells and generating monoclonal antibodies from the resultant hybridomas are disclosed by Kohler and Milstein (Nature 256: 495-497, 1975; Eur. J.
Immunol. 6: 511-519, 1976). Alternatively, the antibodies are used to purify larger quantities of carboxylase, and the purified protein i8 used to generate polyclonal~
antisera, which are in turn used to screen the library.
The present invention enables the production of vitamin X-dependent proteins in a wide variety of cultured host cells, including prokaryotic (e.g. bacterial), lower eukaryotic (e.g. fungal) and higher eukaryotic (e.g.
mammalian) cells. Methods for transforming or tran~fQcting these cell types to expres~ exogenous DNA
~egu-nces are known in the art and are disclosed, for x~mpl-, by Itakur~ et al. (U.S. Patent No. 4,704,362), Go-dd-l t al. ~U.S. P~tent No. 4,766,075), Hinnen et al.
~P~Qc. Natl. Ac~. Sci. USA 75: 1929-1933, 1978), Murray t al. (U.S. Patent No. 4,801,542), Upshall et al., (U.S.
P~tent No. 4,935,349), Hagen et al. (U.S. Patent No.
4,784,950) ~nd Axel et al. (U.S. Patent No. 4,399,216), which are inoorporat-d herein by re~erence.
A DNA molecule encoding gamma-carboxylase is expr-ssed in a cultured cell to provide or enhance the ~bility to gamma-carboxylate proteins. Cells expressing clon-d gamma-carboxylase sequences are thus particularly u-erul in making vitamin K-dependent protein~i, including 3S factor VII, factor IX, factor X, prothrombin, protein C, activat-d protein C, protein S, protein Z, osteocalcin, ~ 2087~70 ~ ~ 92/01795 ~ ` PCT/US9l/05177 , ,, matrix gla-protein and lung surfactant-associated proteins.
DNA sequences encoding plasma vitamin R-dependent proteins and the expression of these DNA
sequences are known in the art. See, for example, the disclosures of Kurachi et al. (Proc. Natl. Acad. Sci. USA
79: 6461-6464, 1982), Foster et al. (European Patent Office Publication EP 266,190; U.S. Patent No. 4,959,318), Bang et al. (U.S. Patent No. 4,775,624), Hagen et al.
(ibid), Degan et al. (Biochemistry 22: 2087-2097, 1983), Leytus et al. (Biochemistry 25: 5098-5102, 1986) and Wydro et al. (European Patent Office Publication EP 255,771), which are incorporated herein by reference. A cDNA
encoding rat matrix gla-protein is disclosed by Price et al. (Proc. Natl. Acad. Sci. USA 84: 8335-8339, 1987), incorporated herein by reference. A cDNA encoding rat bone gla-protein is disclosed by Pan and Price (Proc.
~atl. Acad. Sci. USA 82: 6109-6113, 1985), incorporated herein by reference.
Within the present invention, cells are transfected or transformed to express a DNA sequence encoding gamma-carboxylase and a DNA sequence encoding a vitamin K-depend-nt protein. The DNA sequences are introduc-d into the host cells on ~eparate expression v-ctor- or on ths same exprsssion vector. Cells ~xpr~sslng the introduced sequences are then selected. To facilitate select$on, one or more selectable markers are provid-d on th- expression vector~s). It is preferred to ~ir~t obtain cells xpr-ssing the cloned gamma-carboxylase ~-guence, then transfect or transform these cells to xpres~ a vitamin K-dependent protein.
Expression vectors for use in transfection and tran~formation include an expression unit wherein the DNA
Jegu-nce to be expressed is operably linked to 3S transcript$onal promoter and terminator sequences.
Expression vectors may further include such elements as enAancers, polyadenylation signals, splice sites, wo 92/0~9~8 7 9 7 PCr/US91/0517~

.. .
selectable markers, and one or more origins of replication Selection of these elements and construction of expres6ion vectors is within the level of ordinary skill in the art A preferred group of host cells is cultured mammalian cells, such as the 293 (ATCC CRL 1573), BHK
(ATCC CRL 1632 and CRL 10314) and CH0 (ATCC CCL 61) cell lines Within a preferred embodiment, a cultured mammalian cell line is transfected with an expression vector containing an expression unit for gamma-carboxylase and an expression unit for a selectable marker such as neomycin or hygromycin resistance The selectable marker may also be provided on a separate vector Transfectants are selected on the basi~ of antibiotic resistance Resistant cells are then screened for increased production of gamma-carboxylase, for example by assaying extracts of transfected and untransfected cells Cells producing gamma-carboxylase are transfected with a second expression vector containing expression units for a vitamin K-dependent protein and a second selectable marker It is pre~erred that the second selectable marker be an amplifiable selectable marker, such as a gene encoding dihydro~olate reducta~e Use of an amplifiable selectable mark-r allows for amplification of the exog-nous DNA, 2S l-adlng to nhanc-d expre~sion of the vitamin X-dependent prot-in DNA sequences encoding secreted vitamin K-d-p-ndent proteins, such as coagulation proteins, will g nerally include a secretory signal sequence For intrac-llular proteins, such as bone and matrix gla-proteins, a secretory signal sequence may be omitted In an alt-rnative embodiment, the DNA sequence encoding gamma-carboxylase and a vitamin K-dependent protein are placed on the same expression vector under the control of a ~ingle promoter to produce a polycistronic message Transf-cted c-118 are selected on the basis of a -lectable marker and are screened for gamma-carboxylase activity as described above or by measuring the amount of ~ ~ 92/01795 2 0 8 7 9 7 ~ P(~r/US91/0~177 gamma-carboxylase protein using a gla-dependent antibody (Wakabayashi et al., ibid). Preferred promoters for use in cultured mammalian cells include the SV40 promoter ~Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981), the adenovirus 2 major late promoter (Kaufman and Sharp, Mol.
Cell, Biol. 2: 1304-1319, 1982) and the mouse metallothionein-I gene promoter (Palmiter et al., U.S.
Patent No. 4,579,821).
Media for culturing a variety of prokaryotic and eukaryotic host cells are known in the art. These media will generally contain a carbon source, a nitrogen source, vitamins (including vitamin K) and minerals, as well as growth factors and other nutrients required by the particular host cell. In many instances, the media will further contain a selective agent to insure stability of the exogenous DNA sequence(s). In the case of cultured mammalian cells, many of the required growth factors and other nutrients may be provided as serum, such as fetal bovine serum, although a variety of serum-free media formulations are known in the art. Within a preferred embodiment, cultured mammalian cells transfected to xpr-ss a DNA seguence encoding gamma-carboxylase are cultured in a medium containing 0.1-10 ~g/ml vitamin K, pr-f-rably about 5 ~g/ml vitamin K.
Gamma-carboxylase may also be expressed in non-human transgenic animal~, particularly transgenic warm-blooded animals. Methods for producing transgenic animal~, including mice, rats, rabbits, ~heep and pigs, ar- known in the art and are disclosed, for example, by H~mmer et al. ~N~ure 315: 680-683, 1985), Palmiter et al.
tSc~nce ~ 809-814, 1983), Brinster et al. (Proc. Natl.
Acad. Sci. USA ~: 4438-4442, 1985), Palmiter and Brin~ter ~gLl ~: 343-345, 1985) and U.S. Patent No. 4,736,866, wh~ch are incorporated herein by reference. Briefly, an 3S xpre~sion unit, including a DNA sequence to be expressed together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized - .; ~: ~- . . ..

:. , ., , ,. . , :. .. : . - , `~ ' `.:~' ' ' ".''`'`,' "''': .' .` `- " : ' .- ' ': '`
.: , : . . , - .,- : - . -.

~ W092/01795 ` ` PCT/US91/~17~

eggs. Introduction of DNA is commonly done by microinjection. Integration of the injected DNA is detected by blot analysis of DNA from tissue samples, typically samples of tail tissue. It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the animal's progeny. Although systemic expression of gamma-carboxylase and vitamin K-dependent proteins is generally preferred, proteins showing toxicity to the host animal are best expressed in a regulated and/or tissue-specific manner. Tissue-specific expression is achieved through the use of a tissue-specific promoter, such as an insulin gene promoter. Use of an inducible promoter, such as a metallothionein gene promoter (Palmiter et al., 1983, ibid), allows regulated expression of the transgene. In this way, animals are made transgenic for gamma-carboxylase and a vitamin K-dependent protein. The animals are grown and the vitamin K-dependent protein is isolated from cells or body fluids (e.g. milk, blood or urine) of the animal.
Gamma-carboxylase puri~ied as disclosed above or pr-par-d by gen-tic engineering methods (e.g. cell culture or tran~genic animals) is also useful within in vitro carboxylation sy~t-ms. One such system is disclosed by 2S V-rm--r t al. (International Patent Application Publication WO 87/04719). Brierly, the purified enzyme is a~rixed to a solid support (e.g. derivatized dextran or agarose boads), and an un- or undercarboxylated vitamin X-d-pendent prot-in i~ passed over the ~upport. The reaction is carri-d out in the presence o~ vitamin K-hydroquinone. Such a system is particularly use~ul for activating vitamin K-dependent proteins produced in prokaryotic or lower eukaryotic host cells. ~f the protein lack~ a propeptide, exogenous propeptide is added 3S to the reaction mixture to drive carboxylation (Knobloch and Suttie, J. ~iol. Chem. 262: 15334-15337, 1987).

~0 92/01795 2 0 8 7 9 7 0 Pcr/usgl/o~l77 Proteins containing propeptides are proteolytically processed after carboxylation to remove the propeptide The following examples are offered by way of illustration, not by way of limitation Examples Vitamin K was obtained from Sigma Chemical Co , St Louis, MO and was reduced by the method of Sadowski et al (J Biol Chem 251 2770-2776, 1976) PSN
antibiotic mix was obtained from GIBCO BRL Life Technologies, Inc (Gaithersburg, MD) Phospholipids and lectins were obtained from Sigma Gamma carboxylation was assayed by measuring the incorporation o~ radiolabeled CO2 into the synthetic peptide substrate NBoc-L-glu-L-glu-L-leu-methylester (EEL?
(obtained from Bachem Bioscience, Philadelphia, PA) using essentially the method o~ Vermeer (ibid ) The reaction mixture contained 950 ~l of 3 8 M (NH4)2SO4, 150 ~l 1%
CHAPS, 150 ~l 10 mg/ml phosphatidyl choline type III, 1%
Na cholate, 75 ~l 0 2 M dithiothreitol, 750 ~l 1 mM EEL and 150 ~l NaH14CO3 (50 mCi/mmol) One hundred twenty-eight mi¢roliters Or this mixture was oombined with a 100 ~l t--t ample plu~ 5 ~g/ml vitamin X hydroquinone Control raaction- l~ck-d vitamin K The reaction was allowed to proo--d ~or on- hour in the dark at room temperature The r-actlon was stopped by the addition of 1 ml of 10%
trlchloroacetic acid, placed on ice ror 30 minutes, and c-ntrifuged 15 minutes at 4C one milliliter o~ the r-sulting supernatant was boiled rOr rive minutos and combin-d with 10 ml o~ ~cintillant (E3ioSare II, obtained from Researoh Products International Corp , Prospect, IL) ~nd countod Incorporation Or label was compared to a control reaction lacking vitamin K
293 cells were metabolically labeled as ~ollows The 293 cells were seeded into 125 mm plates to a confluenoy of 10% and were grown in Dulbecco~s Modified Eagle medium (Hazelton Biologics, ~exena, KA) supplemented . ~, . . . . . , . . .. . .. .. .. . . . .

.. . . . ~ . . . . , -' ~ . ~.. ., . ~ . - . .. , - . . - , 2 0 87 9 7 22 PCT/US91/~7 ' - with 1% G418 + 1% PSN (GIBCO BRL, Gaithersburg, MD) + 10%
dialyzed, fetal bovine serum (HyClone Laboratories, Inc , Logan, UT) for two days to 60-70% confluency The media was removed from one set of 293 cell cultures by aspiration and the cells were washed with 10 ml of PBS
(Sigma, St Louis, MO) that had been prewarmed to 37OC
The PBS was removed and 25 ml of prewarmed pulse media (100 ml Dulbe~co's Modified Eagle medium -cys/-met tHazelton Biologics], 1 ml 100 mM sodium pyruvate [Irvine Scientific, Santa Ana, CA], 1 ml of 200 ~M L-glutamine tHazelton Biologics], 1 ml of 100x PSN, 10 ml of dialyzed, fetal bovine serum) containing 20 ~Ci/ml of Neg-072 Expre35S35S t35s] Protein Labeling Mix (NEN Research Productfi, Willmington, DE) was added to each plate The cells were incubated at 37C, and the media was changed every 10-12 hours for two days One day after the first set of cultures was processed, the remaining duplicate cultures were labeled as described above and grown at 37C
with media changes every 10-12 hours for one day After labeling, the media from each culture was removed and stored on ice The plates were washed with 2 5 ml lx VQrsene ~GIBCO BRL) and the Versene wa~h wa~ combined with th- pent m-di~ Arter the Versens wa~h, an additional 2 5 ml o~ lx V-rsone wa~ added to each plate, and the 2S plat-- wer- incubated at room temperature for 10-15 minut-s A~ter lncubation, the cells were harvested and ~dded to the spent media ~A~LPLE 1 Peptide~ corresponding to the propeptide of hum~n prothrombin and a portion of the human GM-CSF
r-ceptor (T~ble 1) were synthesizQd using an Applied Biosystem~ Model 431A peptide synthesizer (Applied Biosyst~ms, Inc , Fo~ter City, CA) ... . . . . ... . . . .
. !. . ~ ' . ' ; ' . ' : . '; ' , : .
` ~ ~h~
. . :;, . . .
: : ' :~ : : .. ,~

~ ~ 92/01795 2 ~ 8 7 9 7 0 PCT/US91/~177 pro-prothrombin (Sequence ID Number 1) S C G G H V F L A P Q Q A R S L L Q R v R R
CSF (Sequence ID Number 2) c G G K D K L N D N H E V E D E Y
Thirty-eight mg of pro-prothrombin (pro-PT) peptide (Sequence ID Number 1) or 44 mg of CSF peptide ~Sequence ID Number 2) were dissolved in 7 ml or 6 ml, respectively, of 0 1 M ammonium acetate pH 4 5 The-solutions were each combined with lo ml (-2 5 g) of activated thiol Sepharose 4BTM (Pharmacia, Piscataway, NJ) that had been sequentially washed in phosphate buffered saline (PBS) and 0 1 M ammonium acetate pH 4 5 The mixtures were placed on a rocker overnight at room temperature The mixtures were then filtered on a scintered glass funnel, and the eluants were saved for d-t-rmination of the coupling efficiency (aa described by War- t al , J Bio~ Ch-m 264 11401-11406, 1989) The r--in- w-re ~ch rinssd with 250 ml 0 1 M ammonium acetate 2S pH 4 5 The rin--d re~ins w-re each combined with 10 ml o~ 0 1 M ammonium acetate pH 4 5 containing 4 mM ~-m-rcaptoethanol, rocked for 20 minutes at room t-mp-rature, and filtered again The resins were then r~n~-d with 250 ml 0 1 M ammonium acetate pH 4 5, then with 200 ml PBS, and were istored at 4C until ready for use Just before use, the resins were recovered by filtration.
Rat liver microsomes, prepared essentially as desoribed by Swanson and Suttie (ibid ) ~approximately 800 mg), were isuspended in 40 ml of 0 025 M imidazole pH 7 2 oontaining 0 25 M sucrose, 0 5 M KCl, 0 2% Tritcn X-100 Membranes were disrupted with 8 strokes of a tissue s~

~ W092/017~ - - PCT/US9l/~1 ~

homogenizer (Dura Grind stainless steel Dounce Tissue Grinder, Wheaton Scientific, Millville, NJ), and the solution was placed on ice for 30 minutes The chilled ~olution was centrifuged at 150,000 x g for 1 hour at 4C, and the supernatant was recovered To the supernatant (40 ml volume) was added 60 ml saturated (NH4)2S04 over approximately 30 minutes, and the mixture was stirred for an additional 20 minutes at 4C The solution was centrifuged at 12,000 x g for 20 minutes at 40C
10The (NH4)2So4 pellet was resuspended in 4 ml buffer A (20 mM sodium phosphate pH 7 4 containing 1 mg/ml phosphatidyl choline tvpe VE, 50 ~g/ml phosphatidyl serine, 50 ~g/ml phosphatidyl ethanolamine, 0 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate 15lCHAPS], 0 15 M NaCl, 15% glycerol) to a final volume of approximately 15 ml The resulting solution was dialyzed against 0 5 l of buffer A at 4C for 5 hours, then the buffer was replaced with 0 5 l of fresh buffer A and dialysis was continued at 4C overnight The final volume Or the dialysate was approximately 20 ml The dialy~ed microsome extract was combined with 20 ml or prewashed activated thiol Sepharose 4B, and the mixture was rock-d for 3 S hours at 4C The m~xture was th-n pour-d into a column, and the eluant was recovered 2S Th- column wa- rinsed with additional bu~er A until most of the prot-in was elut-d as determined by a reduction in oluant viscosity The eluant fractions were combined ~total volum- - 30 ml), and two-thirds of this pr-paration wa- combined with 10 ml o~ the pro-PT 8-pharo~e The rema$ning on--third o~ the microsome preparation was combined with 5 ml of the CSF Sepharose, and the mixtures wero rocked overnight at 4C, then centrifuged at 2,000 x g for 5 minutes at 4C The re~ins were recovered, rinsed with 5 ml of buffer A adjusted to 0 5 M NaCl, and contrifug-d The rinse procedure was repeated 4 times, and the resin was resuspended in 5 ml (CSF) or 10 ml (pro-PT) buffer A ad~usted to -50 mM dithiothreitol and placed ~ ~ 92/01795 2 0 8 7 9 7 0 PCT/US9l/~77 , . ; ~ .

on a rocker for 24 hours at 4c The preparations were again centrifuged at 2,000 x g for 5 minutes at 4C, and the supernatants were recovered and assayed for gamma-carboxylase activity and protein content (by BCA) Results, shown in Table 2, indicated that lo times as much activity bound to the pro-PT resin as bound to the CSF
resin Protein Total Fraction Volume concentration CPM
Microsome suspension 40 ml 20 mg/ml 4 x 1o8 Microsome supernatant 40 ml 15 mg/ml 3 x 1o8 Dialyzed microsome 20 ml 19 mg/ml 2 x 1o8 extract Pre-resin eluant 20 ml 11 mg/ml 2 x 103 CSF resin 5 ml N D 1 x 106 Pro-PT resin 10 ml N D 2 x 107 CSF eluant 5 ml N D 3 x 105 Pro-PT eluant 10 ml N D 6 x 106 N D - not determined The eluants from the pro-PT and CSF resins were electrophoresed on an 8% SDS-polyacrylamide gel Figure 1 2S i- a photograph o~ the gel showing equal volumes of the pro-PT luant (lane 1) and the CSF eluant ~lane 2) Ex~L~-z Approximately 800 mg o~ rat liver microsomes ~propared a8 described in Example 1) were ~uspended in 40 ~1 Or 0 025 N imidazole pH 7 2 containing 0 25 M sucrose, 0 5 M XCl, 0 2% Triton X-100 Membranes were disrupted with 10 strokes Or a tissue homogenizer, and the solution was placed on ice ror 30 minutes The chilled solution 3S was centri~uged at 150,000 x g for 1 hour at 4C, and the ~upornatant was recovered To the supernatant was added 65 ~1 saturated (NH4)2S04 over approximately 30 minute~, and the mixture was stirred 30 -minutes at 4C The .. .. . . . ~ : .

W092/U1795 ` ~6 PCT/US91/0517 solution was centrifuged at 12,000 x g for 20 minutes at 4c.
The (NH4)2S04 pellet was resuspended in buffer A
to a final volume of approximately 20 ml. The resulting solution was dialyzed against 0.5 liters of buffer A at 4C for 5 hours, then the buffer was replaced with 0.5 liters of fresh buffer A, and dialysis was continued at 40C overnight.
Ten milliliters of activated thiol Sepharose 4B
was washed in an equal volume of buffer A, and the mixture was rocked at 4C for 3 hours. The mixture was then centrifuged at 2,000 x g for 5 minutes at 4C, the resin was recovered, and the wash procedure was repeated four times. The washed resin was combined with the dialyzed microsomal preparation and placed on a rocker for 3 hours at 4C. The preparation was then centrifuged at 2,000 x g for 5 minutes at 40C, and the supernatant was recovered.
The supernatant fraction was then combined with activated thiol Sepharose that had been coupled to the synthetic prothrombin propeptide as described above. The mixture was placed on a rocker at 4C overnight, then centrifuged at 2,000 x g for 5 minutes at 4C. The resin wa~ recovered, rin~ed with 5 ml of buffer A adjusted to O.S M NaCl, and centri~uged. The rin~e procedure was 2S r-p-~t-d 3 mor- times, and the washed resin was mixed with S ml o~ bu~er A ad~usted to 50 mM dithiothreitol and placed on a rocker for 1 hour at 4C. The preparation was again centrifuged at 2,000 x g for 5 minutes at 4C, and th~ up-rnatant, containing gamma-carboxylase puriried about 200-~old over the initial micro~ome preparation, was recovered.
Purification was monitored by assaying for gamma-carboxylase activity. Of 4 x 108 cpm of activity in th- initlal microsome suspension, 2 x 1o6 cpm was 3S recovered in the pro-PT eluant.
The partially purified carboxylase was further purified by lentil lectin chromatography. Three , ,, ; ,, . - . ,, ,. ,. . : . . - , -, .

..
. :. : . -: , . . .. - ~ : .

~ ~ 92/0179~ 2 ~ 8 7 9 7 ~ PCT/US91/~177 milliliters of lentil lectin Sepharose 4B (Pharmacia) was washed in 4 changes (lO ml each) of PBS. The washed resin was combined with 5 ml of the pro-PT eluant and placed on a rocker at 4C overnight. The resin was washed 6 times (lO ml each) in buffer A adjusted to 0.5 M NaCl as described above, and the supernatant was recovered. The resin was combined with 5 ml of buffer A adjusted to 0.5 M
NaCl, 0.5% CHAPS and 5 mg/ml phosphatidyl choline, and containing 0.5 M mannose, and the mixture was placed on a rocker overnight at 4C. The resin was pelleted by centrifugation as described above, and the supernatant was recovered. Fractions were assayed for gamma-carboxylase activity (Table 3). The two supernatant fractions were electrophore~ed on a polyacrylamide gel. Figure l shows the lentil lectin flow-through (lane 3) and the lentil lectin eluant (lane 4). Equal amounts of carboxylase activity were loaded in lanes l (pro-PT eluant) and 4.
Comparison of the lanes revealed the presence of bands of approximately 90 kD and 150 kD that were specific to the pro-PT and lentil lectin preparations (lane 4). At this point, gamma-carboxylase activity was enriched lO,000- to 20,000-~old over the initial microsome preparation.

TA~E 3 F~tion Volume Total c~m pro-PT eluant 5 ml 2.5 x lO6 ~-ntil lectin resin 3 ml 1.2 x lO6 L-ntil lectin ~low-through S ml 3 x 104 Mannose elution 5 ml 3 x ~05 The partially purified material from the pro-prothrombin purification could also be ~urther purified by anion exchange chromatography. In a test of purification conditions, 5 ml of the propeptide resin eluant was dialyzed against two changes of 20 mM Tricine (Sigma Chemical Co., St. Louis, M0) pH 8.5 containing~0.1% CHAPS

. . . . .. . . . . . . .... . .

.: , , . . ~, . ... . .. . . .

WO92/01795 PCT/US91/~17 (Sigma), 1 mg/ml phosphatidyl choline type VE, 50 ~g/ml phosphatidyl ethanolamine and 50 ~g/ml phosphatidyl serine. One milliliter of the dialyzed solution was then combined with 0.5 ml of DEAE Sepharose (Pharmacia) that had been equilibrated in thë same Tricine buffer or Tricine buffer containing 0.2 M, 0.5 M or 0.8 M NaCl. The mixtures were placed on a rocker overnight at 4C, then centrifuged at 2,000 x g for 5 minutes. The supernatants were recovered and assayed for carboxylase activity. The resins were then rinsed 4 times in 1 ml of Tricine buffer or Tricine buffer containing the appropriate concentration of NaCl with centrifugation at 2,000 x g for 5 minutes.
Each resin sample was recovered and assayed for gamma-carboxylase activity. Results are shown in Table 4.

Sample Total cDm Resin 0 M NaCl 2 x 105 0.2 M NaCl 1 x 105 0.5 N NaCl 0 O . 8 M NaCl 0 Supernatant 0 M NaCl 2 x 104 O. 2 M NaCl 8 x 104 0.5 M NaCl 2 x 105 0.8 M NaCl 2 x 105 In a parallel set of experiments, material from the pro-PT purification step was dialyzed against 2 changes of 20 mM Bis-Tris ~Sigma) pH 6. 8 containing 0.1%
CHAPS, 1 mg/ml phosphatidyl choline type VE, 50 ~g/ml phosphatidyl ethanolamine and 50 ~g/ml phosphatidyl -rine. Four hundred microliters of the dialyzed solution was combined with 0.5 ml of SP SephadexTM (Pharmacia) or CM SepharoseTN (Pharmacia) that had been equilibrated in the same ~is-Tris buffer, and the mixtures were rocked .

.. ., . , . ~ . , .

... ~ .. .
,, , , - , ' :; :
,.,' '`' ~ ' `,-' '.` .. ' . ' ` ' -' .' ' ' :: . ' . ' , ' : . '' ' ~. ; ' ~: .-.':: , . . --. . .. . . ' ~ ~ 092/01795 2 0 8 7 9 7 0 PCT/US91/~177 overnight at 4C Essentially all of the gamma-carboxylase activity was found in the eluant Eighty milligrams of liver microsomes prepared rrom war~arin-treated rats was disrupted with 8 strokes of a tissue homogenizer and placed on ice for 15 minutes The microsome preparation was then combined with 5 ml of CNBr-activated Sepharose to which was coupled 4 mg of mouse anti-proinsulin monoclonal antibody that had been washed 4 times (10 ml) in 025 M imidazole pH 7 2, 0 25 M
sucrose The mixture was rocked for 3 hours at 4C, then centrifuged at 2,000 x g for 5 minutes at 4C The eluant was retained The resin was washed with 5 ml of imidazole-sucrose buffer, centrifuged, and the eluant was recovered and combined with the first eluant The eluant pool was combined with 10 ml (3 g) of CNBr-activated Sepharose coupled to anti-factor X
polyclonal antibody The mixture was rocked overnight at 4C, then washed 6 times with 10 ml of imidazole-sucrose bur~er containing 0 5 M XCl The resin was recovered and incubated in a gamma-carboxylase reaction with cold NaHC03 (10 ml total reaction volume) to release the enzyme A~tor on- hour, th- reaction mixture was centrifuged at 2,000 x g ~or 5 minut-s Th- eluant wa~ recovered, and th- r--ln wa~ wa-h-d with 4 ml or o. oi5 M imid~zole pH
7 2, O S M KCl The wa~h was recovered and combined with th- ~uant The combined eluants were combin-d with 1 ml of w~-h-d l-ntil lectin Sepharose 4B, and the mixture was ro¢k-d ov-rnight at 4C The mixture was centrifuged at 2,000 x g ~or 5 minutes at 4C The resin was washed four tim-s in 3 ml Or buffer A containing 0 5 M NaCl The wa~hed resin was recovered and combined with 2 5 ml of 3S bu~er A ad~usted to 0 5 M NaCl, 5 mg/ml phosphatidyl choline type VE, 0 5% CHAPS and containing 0 5 M manno~e Thi- mixture W~8 rocked overnight at 4C, then centrifuged ! wo 92/0179~ 0 87 ~7 PCT/US91/~7~ ~

at 2,000 x g for 5 minutes at 40c. The eluant fraction was recovered. Recovered fractions were assayed for gamma-carboxylase activity. Results indicated that about 70% to 90% of the carboxylase activity bound to the resin, and about 30% of the initial activity was recovered in the ~inal eluant.

Bovine liver microsomes were prepared as described by Swanson and suttie (ibid.). Ten tubes (each containing 200 mg of protein) of microsomes were pooled in pairs, and each pool was suspended in 4 ml of SI (0.25 M
sucrose, 0.025 M imidazole pH 7.2) containing 0.2% Triton X-lOO. The microsomes were disrupted with lO strokes of a tis~ue homogenizer, pooled, then placed on ice for 30 minutes. one milliliter was removed for subsequent assay.
The mixture was centrifuged at 150,000 x g for l hour at 4-C. The supernatant was recovered and l ml was saved for assay. To the remainder of the supernatant was added 60 ml of saturated (NH4)2S04 over 30 minutes at 4-C, and the mixturo was stirred for an additional 30 minutes at 4-C.
The precipitate was harvested by centrifugation and removal o~ the liguid ~raction. The precip~tate was r-~u-p-nd-d in 5 ml Or bu~fer A' ~buffer A lacking 2S pho-phatidyl thanolamine and phosphatidyl serine) and dlalyzed twice in 600 ml of buffer A'. One milliliter was removed for assay. The dialysate ~-40 ml) was combined wlth 20 ml activated thiol Sepharose ~ATS) and rocked for thr-e hours. The mixture was poured into a column and the ~low-through was collected. one milliliter was removed for assay.
Ten milliliters of the flow-through was combined with 5 ml of CSF Sepharose ~Example l), and 20 ml was oombined with lO ml of pro-PT Sepharose ~prepared as in ~5 Ex~mple l). The mixtures were rocked overnight at 4-C, thon centrifuged at 2,000 x g for five minutes at 4-C.
The resins were washed five times in lO ml of buffer A' .. . . . .
- . . .. . . . . . ..
~. , .: ' : ',:

- , ~ ~ .. . - .. .. . -~ ~ 92/01795 2 0 8 7 9 7 ~ PCT/US91/05177 " 31 adjusted to 0.5 M NaCl, then rocked at 4-c in buffer A' containing 0.5 M NaCl and 50 mM dithiothreitol for between 5 and 24 hours. The mixtures were centrifuged and the supernatants were retained.
Samples removed during the purification procedure were assayed for gamma-carboxylase activity and total protein content (by BCA). ResuLts are shown in Table 5.

Specific Volume Protein Activity Activity ,Fraction (ml) ,(ma/ml~ ,(cpm/ml) (c~m/ma~
TX Suspension 50 40 5 x 106 105 TX Sup. 45 41 4 x 106 105 (NH4)2S04 ppt (dialyzed) 40 30 4 x 1o6 105 ATS Sup. 40 23 3 x 106 105 proPT eluant10 -0.1 1 x 1o6 -107 CSF eluant 5 -0.1 ND

ND- Not d-termined Flve ml each o~ the proPT and CSF eluants were 2S combined with 5 ml of lentil lectin Sepharose 4B that had been prewashed in bufrer A'. The mixtures were rocked overnight at 4-C, then washed 8iX times with 10 ml of buSfer A' ad~usted to 0.5 M NaCl. The wa~hed re~ins were each mixed with 5 ml of buffor A' ad~usted to 0.5 M NaCl, O.S N mannose, 0.5% CHAPS and 5 mg/ml pho~phatidyl choline type VE. The mixtures were rocked overnight at 4-C and spun at 2,000 x g for 5 minutes. The supernatants were ta~en for gamma-carboxylase assay and gel electrophoresis.

; :. : :- . .. . . . .: . -., .. j::; :, ~ , ..- :

.. - ~ . .

3 W092/017~ 32 PCT/U591/~17 Gamma-carboxylase was purified from a 293 cell line transfected to express a factor IX molecule lacking the propeptide (amino acids -18 to -l) An expression vector for a propeptide-deleted human factor IX protein was constructed as shown in Figure 2 A pUCl18 plasmid containing a human factor IX cDNA insert was digested with Eco RV and Hind III, and the 3 3 kb fragment, comprising the vector and 5' factor IX sequences, was recovered The same plasmid was also digested with Hae III and Hind III, and the l 3 kb fragment, comprising the 3' portion of the factor IX cDNA, was recovered The two fragments were ~oined by ligation with oligonucleotides ZC2575 (5' ACA
TTC AGC ACT GAG TAG AT 3'; Sequence ID Number 7) and ZC2576 (5' ATC TAC TCA GTG CTG AAT GT 3'; Sequence ID
Number 8) The DNA was transformed into E ÇQli strain HBlOl Positive colonies were selected by ampicillin resistance one hundred twenty-one positive colonies were replated and screened with 32P-labeled ZC2575 (Sequence ID
Number 7) Twelve positive colonies were selected and ; ~creened by digestion with Hae III 1 Eco RI, Hae III, and Eco RV + Pvu II
Two positive clones were selected on the basis o~ r-~trlction analy~i- and dige~ted with Bam HI to l-olat- th- prop-ptld--delet-d in-erts The in~erts were llg~t-d to Bam HI-digested pD5 (disclosed by Foster et al , U S Pat-nt No 4,959,318, which is incorporated h-r<in by re~erence), a mammalian cell expre~sion vector comprl~ing th- ~denovirus 5 replication origin ~0-l map unlt-)~ 8V40 nhancer, adenovlru~ 2 ma~or late promoter ~nd tripartit- leader, a ~ot o~ splice signal~, the SV40 late polyadenylation signal, and a unique Bam HI cloning ite The re~ulting plasmids were digested with Bam HI, and the ~actor IX in~erts were sequenced A positive 3S pla~mid clone was designated leader-deleted IX-pD5 Th~ propeptide-deleted factor IX expression v-ctor was co-transfected into 293 cell lines by the ,, . , .. . . ... . . ' . - ~ : .

' .
' ,. . ' ' , :, . . : ' .

.. ', ' ' ' ' . ' .: ~, :- ' ' . .:

~i 2 0 8 7 9 7 0 ~/US9l/~177 - calcium phosphate method with a plasmid (pD5neo) containing a neomycin resistance gene. The transfected cell6 were cultured in DMEM supplemented with lx PSN mix (GIBCO BRL) and 10% dialyzed fetal calf serum (FCS).
Randomly selected colonies were screened for factor IX
production by enzyme-linked immunosorbent assay (ELISA).
Positive colonies were selected and screened by radioimmune precipitation with polyclonal antisera to factor IX. This cell line was designated G3.
In a similar manner, a pD5 vector containing a native factor IX cDNA insert was co-transfected into 293 cells with pD5neo. The cells were cultured in DMEM
supplemented with lx PSN mix, 10% FCS and 1% G418 for 36 hours, then split 1:10 into five plates. Six colonies were chosen and plated into 6-well dishes in DMEM
containing 10% FCS, 1% G418 and 1% PSN mix. After seven days, the cultures were split, half into DMEM containing 10% FCS, 1% G418, 5 ~g/ml vitamin K and 1% PSN mix, and half into DMEM eontaining 10% FCS, 1% G418 and 1% PSN mlx.
The six colonie6 grown in the vitamin K-supplemented medium were screened for factor IX produetion by ELISA.
The ELISA-positive eolonies grown in vitamin K-~uppl-mented m~dia w-re sereened by radioimmune pr-eipitation (RIP) with polyelonal anti6era to factor IX.
2S Th- highe~t faetor IX produeing eell line a6 determined by ELISA and RIP was designated D30. D30 eells that had been eultured in DMEM eontaining 10% dialyzed FCS, 1% G418 and 1% PSN were used for the mierosomal preparations.
The D30 and G3 eell lines were eultured in DMEM
eontaining 10~ dialyzed FCS, 1~ G418 and lS PSN in 150 mm diameter plates. Forty plates of eaeh unlabeled eell line were eombined with one or two plates of metabolieally labeled eells. The eells were harve6ted by aspirating off tho medium and rinsing eaeh plate twiee with 2.5 ml of 3S V-rsene (GIBC0 BRL) to remove the eells. The rinse6 from oach set of plates were pooled and eentrifuged at 2,000 x g ~or five minutes at 4-C. The eell pellets (-2 x 109 ., . , . . . - , . ,., , . . . - - , 1 ~
92/2 ~87 97 0 PCT~US91/~17 cells) were each resuspended in 200 ml of PBS, centrifuged at 2,000 x g for five minutes at 4 C and resuspended in 10 ml of cold SI containing Z mM P~SF The resuspended cells were placed on ice and sonicated with four 15-second pulses with 30 seconds between pulses, then disrupted with 7 strokes of a tissue homogenizer These preparations were centrifuged at 4,000 x g for 15 minutes at 4 c The supernatants were removed and centrifuged at 24,600 x g for 1 hour at 4 C The pellets (microsomes) were each resuspended in 5 ml- of SI containing 5~ CHAPS The resuspended microsomes were combined with 3 ml of anti-proinsulin Sepharose (Example 3), and the mixtures were rocked for 3 hours at 4 C The mixtures were centrifuged at 2,000 x g for five minutes at 4 C The supernatants were recovered and placed on ice The pellets were rinsed with 5 ml of SI, mixed, centrifuged as above, and the supernatants were recovered and combined with the first supernatants The microsomes were then combined with anti-factor IX resin (prepared by coupling 5 mg of ESN4antibody [American Diagnostica, New York, NY] with 5 ml of CN8r-activated Sepharose) The resin was washed with 5 ml Or 3 M XSCN, rock-d 1 hour at 4C, wa~hed four times with 5 ml Or SI, th-n combin-d with the microsome preparation 2S and rook-d overnight at 4'C The resin was then washed four times with 10 ml o~ SI containing 0 5 M KCl The carboxylase was eluted from the resin by thr-- di~-rent procedures In the first, the re~in was lncubated with 5 ml of 0 2 N glycine pH 10 containing 50%
thyl-n- glycol on a roc~er for one hour at 4 C The mixture was then rocked one hour at 4 C and spun at 2,000 x g for five minutes at 4 C The supernatant, containing th- factor IX and the bound carboxylase, was recovered This elution method inactivates the carboxylase but is 35 uitable for physical characterization procedures, such as analysis by gel electrophoresis In the second elution procedure, the resin was incubated in 5 ml of SI-.
....
.: . ., - , ~ ~ 92/01795 2 0 8 79 7 0 PC~r/US91/05177 : ,. .

containing 0.1% CHAPS, 1 mg/ml phosphatidyl choline type III and lOo ~M pro-PT peptide. The mixture was rocked overnight at 4C, centrifuged, and the supernatant was recovered and assayed for carboxylase activity. Fifty S percent o~ the carboxylase activity was found in the eluant. In the third elution procedure, the carboxylase was eluted from the resin by incubation in a gamma-carboxylase reaction mixture with cold NaHC03 to release the enzyme, as described above.
10Two to five ml of the carboxylase reaction eluate was mixed with l ml of lentil lectin Sepharose 4B
and rocked overnight at 4C. The mixture was centrifuged for five minutes at 2,000 x g at 4C. Gamma-carboxylase was eluted from the resin by rocking it overnight at 4C
15with 2.5 ml o~ 20 mM Tricine pH 8.5, 0.5% CHAPS, 1 mg/ml phosphatidyl choline type VE, 50 ~g/ml phosphatidyl serine, 50 ~g/ml phosphatidyl ethanolamine, 0.5 M NaCl and 0.5 M mannose. The supernatant was recovered.
In a similar preparation, unlabeled gamma-carboxylase was purified by chromatography on ESN4-Sepharose. The carboxylase was eluted from the resin with SI containing 100 mM CaClz, 0.1 % CHAPS and 1 mg/ml phosphatidyl choline type III. Assay Or ~ractions taken durlng the puri~ication indicated that about 50-70% Or the oarboxyla-- activity wa~ eluted rrom the resin (Table 6).

WO /~ '` PCT/US91/~17 Protein Activity Protein Activity Fraction ~ma/ml) c~m/ml (m~/ml) cpm/ml Cell Extract 10 5 x 105 10 5 x 105 Microsome pellet 10 5 x 105 10 5 x 105 Preresin sup 5 5 x 105 5 5 x 105 ESN4 sup 5 2 x 105 5 2 x 105 CaC12 eluant < ol 4 x 104 < ol ND
ESN4 resin - 1 x 105 - 1 X 104 ND = not determined Rat microsomes ~250,000 cpm total carboxylase aetivity) were electrophoresed on a native polyacrylamide gel The microsomes (-o 5 ml) were combined with an equal volume of 2X sample buffer (0.125 M Tris pH 6 8, 20%
glycerol, o 0125% bromphenol blue) at 4 C The sample was loaded onto an 8% polyacrylamide slab gel containing 0 375 - M Tris, 1~ PC/CHAPS solution (prepared by removing the e~loro~orm rrOm 10 ml o~ phosphatidyl eholine type VE and r-~usp-nding the waxy re~idue in 20 ml Or 100 mg/ml CHAPS), O 1% a~monium per~ul~ate and 0 025t TEMED The g-l wa~ run at 4'C, 40 volts in running bur~er eontaining 3 g/l ~rl~ ba--, 14 4 g/l glyeine and lt PC/CHAPS solution until th- dye front ran o~ the bottom o~ the gel The gel lane eontaining the microsomal pr-paration wa~ eut out and slieed into 21 pieces Eaeh pioeo was ineubated in th3 earboxylase assay rOr one hour at room tomperature one sliee exhibited gamma-earboxylase aetivity 7000 epm above the baekground level EXAMPLE Z
3S To determine whether the earboxylase could be puri~ied away ~rom more extensively glycosylated eontaminants, the partially purified rat pro-PT eluant was ~ ~092/01795 2 0 8 7 9 7 0 PCT/US9l/~177 37 ! . t .

subjected to lectin chromatography on lectins that bind complex oligosaccharide side-chains prior to lentil lectin chromatography These lectins include Ervthrina cris~galli lectin, which binds ~-D-gal(1-4)-DglcNAc moieties, Pçeudomonas aeruginosa PA-I lectin, which binds D-gal moieties, Limulus poly~hemus lectin, which binds ~ialic acid residues and Tetraaonolobus urpureas lectin, which binds ~-L-fuc moieties Partially purified rat carboxylase as a pro-PT
eluant was prepared essentially as described in Example 2 One milliliter of the eluant containing 5 x 1o6 cpm was ~-added to 0 5 ml of either Erythrina cristaaalli lectin coupled to CNBr-activated Sepharose 4B, PseudQmonas aeru~ainosa PA-I lectin coupled to CNBr-activated Sepharose 4B, L~m~ ELolYphemus lectin coupled to CN8r-activated Sepharose 4B or Tetraaonolobus p~rpureas lectin resin The mixtures were rocked overnight at 4C The mixtures were centrifuged at 2,000 x g for five minutes at 4C, and the supernatants were added to 0 5 ml lentil lectin-Sepharose 4B resin The pellets were rinsed four times (0 2 ml each) in buffer A (Example 1) and the washes were combined with their respective supernatants A control containing untreated pro-PT eluant was added to 0 5 ml l-ntil lectin-Sepharose 4B The mixtures were rocked 2S ov-rnlght at 4C rollow-d by centrifugation at 2,000 x g rOr ~iv- minut-~ at 4C recover the resin, and the pellets w-r- rins-d with 1 ml buffer A containing 0 5 M NaCl The rins- procedure was repeated three times The carboxylase wa- luted rrom the resins with 1 ml of bu~er A ad~usted to 0 5 N NaCl, 0 5% CHAPS, 5 mg/ml phosphatidyl choline containing 0 5 M mannose The mixtures were rocked overnight at 4C After incubation, the mixtures were centri~uged at 2,000 x g for five minutes at 4C and the upernatants were removed to fresh tubes The eluants were tested in carboxylase assays and w-re run on an 8% reducing polyacrylamide gel The - eluant was added to lx SDS sample buffer (50 mM Tris-HCl, _.,. ",~,,,, .. ... ... . . . . ; - ' :, `' ; . - i : , -~ WOg2/017g5 PCT/US91/05~7~
20879~ 38 pH 6 8, loO mM dithiothreitol, 2~ SDS, o l~ bromphenol blue, 10~ glycerol) The mixtures were boiled for five minutes and the mixtures were loaded onto an 8%
polyacrylamide gel The results of the carboxylase assay showed that the carboxylase does not ~ind to the Erythrina cristagalli lectin, Pseudomonas ae~ginosa PA-I lectin, L~E~1~ Dolyphemus lectin or Tetragonalobus ~urpureas lectin An autoradiograph of the gel electrophoresis showed that these lectins removed major contaminants from the carboxylase preparation A partially purified rat pro-PT eluant was sequentially chromatographed using Tetraaonolobus urpureas lectin resin and lentil lectin-Sepharose 4B
Partially purified rat carboxylase was prepared e-s-ntially as described in Example 1 using the pro-PT
peptide Ten milliliters of pro-PT eluant containing 4 x 106 cpm was dialyzed into 20 mM Tricine, pH 8 5, 0 1%
CNAPS, 1 mg/ml phosphatidyl choline type VE, 50 ~g/ml phosphatidyl ethanolamine and 50 ~g/ml phosphatidyl -rine The dialyzed eluant waC added to 6 ml of pr-wa~hed Tetr~Qnglobus ~Uxpureas lectin resin The ample was rocked overnight at 4C followed by ¢-ntrirugation at 2,000 x g ror ri~e minutos at 4 C The up-rnatant wa- r mov d into 5 ml o~ prewashed lentil 2S l-ctln-8-pharo-- 4B, th- p~ t wa~ rinsed with the dlaly-1- burr-r, and the second supernatant was combined wlt~ th- sup-rnatant-lectin ~ixture The supernatant-l-ctin mixtur- wa~ rocked ov rnight at 4C, and the ~ixtur- wa~ c-ntrirug-d at 2,000 x g rOr five minute- at 4 C The up-rnatant was discarded and the pell-t was wa h-d ix times with 10 ml o~ bur~er A ad~usted to 0 5 M
NaCl Th- carboxylase was eluted rrom the lectin resin w~th burrer A ad~usted to 0 5 M NaCl, 0 5% CHAPS, 5 mg/ml pho-phatidyl choline containing 0 5 M mannose overnight ~t 4 C The sample was centrifuged at 2,000 x g ~or five ~lnut-s, and th- supernatant was sub~ected to a ~ ~ 92/0l7~ 2087970 ~ PCT/VS91/~177 , . .. .
39 A ~

carboxylase assay and was TCA precipitated and run on a polyacrylamide gel as described above EXAMPL~_8 Bovine liver microsomes, prepared essentially as described by Harbeck et al (Thrombosis Res 56 317-323, 1989), were resuspended to 30 mg/ml in 70 ml of 20 mM Tris pH 7 3, 0 1 M NaCl and centrifuged at 105,000 x g for sixty minutes at 4C The supernatant was ~iscarded, and the pellets were resuspended in 70 ml of ~0 mM Tris pH
7 3, 0 1 M NaCl and solubilized with 0 5% CHAPS The solution was incubated at 4C for 30 minutes, then csntrifuged at 150,000 x g for 60 minutes at 4C The supernatant was discarded, and the pellet wa~ resuspended in 20 mM Tris pH 7 3, 1 M NaCl, 1% CHAPS The suspension was incubated 30 minutes at 4C, then combined with anti-prothrombin resin (prepared by coupling CNBr-activated Sepharose-4B and affinity purified rabbit antisera to bovine prothrombin) The mixture was incubated for 16 hours at 4C, then loaded into a column (35 ml volume), and the resin was washed with five column volumes Or 20 mM
Tris p~ 7 3, 0 1 M NaCl, 0 5% CHAPS, 0 5% phosphatidyl cholin- (typ~ E), 5 mM dithiothreitol ~burrer C), then With riv- column volumes Or burSer C containing 1 mM ATP
~S and ~ ~M MgC12 at 20C Aliquots Or unbound material and Or antl-prothrombin r~sin were assayed as described above In g-n-ral, 50% o~ the carboxylase activity could be bound to th- r~sin Carboxylase was eluted rrom the r-sin with burr-r C containing 100 ~M Or human ractor X propeptido (conslsting o~ the -18 to -1 sequence o~ human factor X
~ ytus et ~1 , ibid ; incorporated herein by r-ference)) Elution was performed in several batches, with 3-hour incubations at 20C for each batch Aliquot~ Or po~t-3S lution anti-prothrombin resin and of propeptide eluant w r- assayed, and approximiately 70% of the carboxyla~e ~1 ZO ~ 9 40 ~CT/US9l/~l~

was found to be eluted from the column by the propeptide (Table 7) The propeptide eluant (ZOO ml) was adsorbed onto 0 5 ml of S-Sepharose (Pharmacia) at 4C for 16-20 hours, and the resin wa6 washed with ten volumes of 50 mM Tris pH
7 4, 100 mM NaCl, 0 25% phosphatidyl choline type VE, O 25% CHAPS The resin was then rocked for 30 minutes in 0 5 ml of 50 mM Tris pH 7 4, 200 mM NaCl, 0 25~
phosphatidyl choline VE, 0 25~ CHAPS, then incubated for an additional 30 minutes in 1 ml of the same buffer ad~usted to 0 5 M NaCl The resin was pelleted by low speed centrifugation Both adsorption of propeptide eluant and salt elution were guantitative, giving a recovery of 100% ~or this step ~Table 7) Material eluted from S-Sepharose was then adsorbed onto 200 al lentil lectin Sepharose (Sigma Chemical Co ) The mixture was adjusted to 5 mM MnCl2, 5 mM CaCl2, and incubated at 4C for sixteen hours The resin was washed with a 100-fold sxcess (100 volumes) of 50 mM Tris pH ~ 4, 100 mM NaCl, 0 25% phosphatidyl choline VE, 0 25% CHAPS, and carboxylase was eluted in 1 ml o~ the ~ame bur~er containing 0 5 M ~-methyl-mannoside and 10 mM
EDTA Overall r-covery at thi~ step was 50% (Table 7) Neith-r prothrombln nor any other vitamin X-dependent 2S prot-in oould be d-t-ct-d in the eluant, indicating that ad-orptlon Or th- carboxylase to lentil lectin occurs via a dir-ct interaction .. ~ . ................... , ~ , ................. .. . . .
- ~ . - : . . . .

. -~ ~ 092/0l795 2 0 8 7 9 7 0 PCT/US91/05177 Specific ActivityProtein Activity Fold Purifi-Sam~le (cpm~ (ma~* (cpm/mq) cation Solubilized 6x107 5Soo l x 104 microsomes detergent-extracted microsomes 9 x 107 2000 5 x 104 5 Propeptide eluant 2 x 107 16 1 x 1o8 104 S-Sepharose eluant 1 6 x 107 08 2 x 1o8 2 x 104 lentil lectin eluant 4 x 1o6 002 2 x 109 2 x 105 *determined by scanning Coomassie blue or silver stained gels using a BSA standard , The eluant from the lentil lectin Sepharose was lsctrophoresed, and the gel was stained with Coomassie blu- A ingl- 90 kD band was observed ~Figure 3) When th- g-l wa- ilv-r ~tain-d or when radio-iodinated protein wa- analyz-d, this 90 ~D ~and was the major band, although ~-v-ral minor protein bands were also present The propeptide-eluted bovine carboxylase ~6 x 3S ~OC cpm activity in 100 ml buffer C containing 100 yM
propeptide) was batch adsorbed onto l ml S-Sepharose for 16 bours at 4C Bound material was washed with 10 ~olum 8 of 50 mM Tris pH 7 4, 100 mM NaCl, 0 25%
phosphatidyl choline VE, 0 25~ CHAPS, and a small aliquot ~0 o~ the resin was assayed for carboxylase activity The ro~aining resin was mixed with an equal volume of Freund~s ad~uvant ~obtained from ICN Biochemicals, Costa Mesa, CA) ~ .

:- . - ~; , ~ . - - . -~ WO92/01795 ! PCT/US91/05 ~

and injected intraperitoneally into five Balb/c mice. The mice were boosted at 2-week intervals with carboxylase prepared in the same manner, except incomplete Freund's adjuvant was used.
Following the third injection of antigen, serum was collected and tested using an activity immunocapture assay. Serum from a non-immunized mouse was used as a control. Increasing amounts (0-20 ~1) of sera were incubated with bovine microsomes (1.5 mg in 50 ~1) for 8 hours at 40C followed by the addition of 50 ~1 of a 1:1 mixture of protein A Sepharose (sigma Chemical co~) in 50 mM Tris pH 7.4, 100 mM NaCl. Samples were rocked at 4C
for 12 hours, and the resins were then washed 1000-fold with 50 mM Tris pH 7.4, 100 mM NaCl, 0.25% phosphatidyl choline VE, 0.25% CHAPS. The resins were then incubated in 128 ~1 of the carboxylase reaction for four hours at 20C, precipitated with 10%-TCA (1 ml), then counted after removal of 14Co2 by boiling. Test samples were run to show that carboxylaæe activity is linear over this period.
Samples were run in duplicate and an average was taken for ach. With sera isolated from carboxylase-in~ected mice a linear increase in carboxylase activity occured wlth an incr-ase in antisera. With the highest amount o~ antisera u--d t20 ~1), 20% Or the carboxylasQ activity bound to the ~5 r--ln.
W-stern blot (Towbin et al., Proc. Natl. Acad.
S~i. USA 76: 4350-4358, 1979; U.S. Patent No. 4,452,901) analysis of carboxylase wa~ carred out in parallel with ith-r non-immune or anti-carboxylase antisera. Lentil lectin--luted carboxylase (105 cpm Or activity) was electrophores-d on an 8% denaturing gel and transferred to nitrocellulose tBiotrace NT, Gelman Sciences, Ann Arbor, MI) at 60 volts over 24 hours at 4C in 1.4% glycine, 25 mM Tris pH 8.8, 20% methanol and .005% SDS.
3S Nitrocelluloso was washed in Western buffer A (50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25%
- ~elatin), then incubated in 20 ml Western buffer A

- . . .. . .. . . . . .
. ~ :, ; . , , , "

.: .: i . . , , . .. . . ::

:, ~ - . . . . . . . . . . ~ :- ... . . .. . . .. . .

~ ~ 092/01795 2 0 8 7 9 7 0 - ;- . PCT/US91/~177 suppleme~ted with a 1:1000 dilution of sera. After 16 hours rocking at 40C, the nitrocellulose was washed in Western buffer A (three times, 100 ml each), then lncubated in 20 ml Western buffer A containing 125I-labeled rabbit anti-mouse IgG (2 x 106 cpm) for one hour at 4C. The nitrocellulose was washed three times in 100 ml of Western buffer B (50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 1 M NaCl, 0.4% N-laurylsarcosine, 0.25%
gelatin), air dried and exposed to film. The only immunoreactive protein observed with the anti-carboxylase antisera was a 90 kD band (Figure 4). No reactivity was observed with control sera (data not shown).
The starting microsomal preparations and S-Sepharose eluants were also analyzed by Western blot.
Equal amounts of carboxylase activity from each stage of purification were analyzed, and equal amounts of carboxylase reactivity were observed. These results indicate that there was no significant 1088 of activity during purification.

X~m~le g Mlcrosomal preparations were prepared rrom 80 150-mm plat-~ (ca. 2 x 109 cell~) or D30 and G3 cells ~Ex~mpl- 5). The cells were taken orf the plates with 2S V-r--n- ~2.5 ml per 150 mm plate to rinse; then 2.5 ml to take ofr cells) and concentrated by centrifugation at 2000 x g ror 5 minutes. The cells were rinsed in cold PBS
ttotal volu~e 400 ~1) and spun at 2000 x g ror 5 minute~.
The cells were resuspended in SIP ~0.25 M ~ucrose, 0.25 M
imidazole pH 7.2, 2 mM PMSF) to a final volume o~ 20 ml and sonicated in rour 15-second bursts with 30 seconds betw--n bUrsts. The cells were then disrupted with seven ~troke~ Or a tissue homogenizer and centrifuged rOr 15 ~inutes at 4000 x g, 4C. Microsomes were then prepared ~rom the supernatants by spinning at 45,000 RPM for 1 hour at 4C in a Beckman ultracentrifuge. The resulting supernatants were discarded, and the pellets were ~' W092/o~ 0 ~ PCT/U59 disrupted in 10 ml SIP using 7 strokes of a tissue homogenizer, then adjusted to 0.2% CHAPS. After 30 minute6 on ice the samples were combined with an anti-factor IX resin (prepared from polyclonal anti-factor IX
IgG, purified over a protein A-Sepharose column, then coupled to CNBr-activated Sepharose at S mg/ml).
The anti-factor IX resin was rocked at 4~C
overnight, then washed with 0.1 M NaCl in 0.05 M Tris pH
7.4, then eluted with O.OS M Tris pH 7.4, 0.1 M NaCl, 100.25% phosphatidyl choline, 0.25% CHAPS plus 100 ~m propeptide (either the -18 to -1 sequence of human prothrombin or of human factor X). Elution was effected by rocking the resin in this buffer overnight at 4C, then collecting the ~upernatant and assaying it.
15The propeptide eluants were then incubated with 0.2-0.5 ml Q-Sepharose (Pharmacia) overnight at 4C. The resin was rinsed in 0.05 M Tris pH 7.4, 0.1 M NaCl, 0.25%
phosphatidyl choline, 0.25% CHAPS, then incubated for 30 ~econds at 4C with the same buffer adjusted to 0.2 M
NaCl. The buffer was then adjusted to 0.5 M NaCl, incubated for another 30 ~econds, and the eluant was coll-cted and a--ay-d. Protein content was determined by g-l canning. R-sults are shown in Table 8.

, . : .. . . , ~ ~ . ,. ,.................. ,- , . .

.. ... ,, , .. : ~ . .. , .. ., , ,... -. .. .. . .

~ ~ 92/01795 208 79 70 PCT/US91/~177 , . . .

Specific Activity Protein Activity Fold Purifi-S~am~le ~cDm) (mq) * (cDm/m~) cation microsomes D30 1 x 1o6 100 1 x 104 G3 3 x 105 100 3 x 103 propeptide eluant D30 3 x 105 02 1 5 x 107 1500 G3 2 x 104 02 1 x 1o6 0-SeDharose eluant D30 2 x 105 01 2 x 107 2000 G3 1 x 104 01 1 x 106 Eluants from microsomes prepared from metabollically labeled D30 and G3 cells were further ~ractionated on lentil lectin resin Purified fractions were gel electrophoresed and expo~ed for autoradiography As shown in Figure 5, the lentil lectin eluant from D30 c~ contain~ an approximately 65kD band not present in the C3 cell ample Whol- c-ll extracts were prepared from D30 o~ Eighty 150 mm platos of cells were harvested with V-r--ne, concentrated, washed and disrupted as described above 0 5 ml o~ CHAPS was added, and the lysate was plac-d on ice ~or 30 minutes The lysate wa~ then c-ntrifug~d at 4000 x g for 15 minutes, and the upernatant wa8 retained The re~ulting extract was adsorbed to Sepharose-coupled ESN4, and carboxylase was 3S ~lut-d using 0 05 M Tris pH 7 4, 0 1 M NaCl, 0 25%
pho-phatidyl choline, 0 25% CHAPS, 100 mN CaC12 ~ESN4 i8 Ca+~-dependent) Elution was performed for l hour at 4C
Th- eluant was then adsorbed onto concanavalin A Sepharose - . : : . : -. . - -.

- .

W092/01795 ' `~ PCT/US91/~

20 8~ ~ 46 (300 ~1) by incubating at 40C overnight The resin was washed with 0 05 M Tris pH 7 4, 0 1 M NaCl, 0 25%
phosphatidyl choline, 0 25% CHAPS, a small aliquot of re~in was assayed to monitor the amount of activity, and the rest of the resin was injected intraperitoneally into Balb/c ~ice Approximately 3-5 x 105 cpm of activity was used per mouse per boost A sample of D30 whole cell extract was run out for Western blot analysis The sample was boiled in SDS
sample buffer for five minutes, centrifuged one minute at room temperature in a Beckman Microfuge 12 at a setting of 4, and electrophoresed on an 8% discontinuous denaturing polyacrylamide gel at 40 volts for 15 hours at room temperature Protein was transferred to nitrocellulose at lS 60 volts rOr 24 hours The nitrocellulose was removed and washed three times, 20 minutes per wash, in 100 ml Western buffer A at 4C, roc~ing The gel was stained to verify transfer The washed nitrocellulose was placed in 20 ml rresh Western burfer A, and 20 ~1 of anti-carboxylase antiserum (Example 8) was added The blot was rocked for 18 hours at 4C, then washed three times as above The blot was plac-d in 20 ml rresh Western buffer A containing 10 pl (O S ~g; 2 0 x 106 cpm) iodinatad rabbit anti-~ouse lgC ~organon T knika Corp , W-~t Ch--ter, PA; iodinated wlth ~2SI obt~in-d from Am-r8ham, Arlington Heights, IL) ~nd rock-d for on- hour at 4 C The nltrocellulose was th~n wa~hed thre- times as above, air dried, wrapped in aran and xpo~-d to X-ray tilm ~XaMEL~ 10 Total RNA was prepared from bovine liver using guanldine i~othiocyanate (Chirgwin et al Biochemistry S2-94, 1979) and CsCl centrifugation (Gilsin et al ~ }~ 2633-2637, 1974) Poly(A)+ RNA wa~
3S elect-d from the total RNA using oligod(T) cellulose ~hro~atography (Aviv and Leder, p~oc Natl Acad Sci USA
69 1408, 19~2) . . - . . . . . ..

~ ~092/0l7~ 2 0 8 7 9 7 0 PCT/US9~ 77 .
First strand cDNA was synthesized from one time poly d(T)-selected bovine liver poly(A)+ RNA in two separate reactions One reaction, containing radiolabeled dATP, wa6 used to as6ess the quality of first strand synthe6i6 The second reaction was carried out in the absence of radiolabeled dA~P and was used, in part, to assess the quality of second strand synthesis Superscript reverse transcriptase (GIBCO BRL) was used specifically as described below A 2 5x reaction mix was prepared at room temperature by mixing, in order, 8 ~1 of 5x reverse transcriptase buffer (GIBCO BRL; 250 mM Tris-HCl, pH 8 3, 375 mM KCl, and 15 mM MgC12), 2 0 ~1 200 mM
dithiothreitol (made fresh or stored in aliquots at -70C) and 2 0 ~1 of a deoxynucleotide triphosphate solution containing 10 mM each of dATP, dGTP, dTTP and 5-methyl dCTP (Pharmacia) The reaction mix was aliquoted into two tubes of 6 ~1 each To the first tube, 1 0 ~1 of 10 ~Ci/~l ~32P-dATP (Amersham) was added and 1 0 ~1 of water was added to the second reaction tube Fourteen microliters of a solution containing 10 ~g of bovine liver poly~At+
RNA diluted in 14 pl of 5 mM Tris-HCl, pH 7 4, 50 ~M EDTA
wa~ mixed with 2 ~1 of 1 ~g/~l first strand primer, ZC3747 (GAC AGA GCA CAG AAT TCA CTA CTC GAG TTT TTT m TTT TTT;
8-qu-nc- ID Number 10), and the primer was annealed to the RNA ~y h-ating th- mixture to 65C for 4 minutes, followed by chilling in ice water Eight microliters of the RNA-prim-r mixture was added to each of the two reaction tubes ~ollow-d ~y 5 ~g o~ 200 U/~l Superscript reverse tr~n-cripta~- ~GI~CO ~RL) The reactions were mixed gently, and the tu~es were incubated at 45C for 30 minute~ A~ter incubation, 80 ~1 of 10 mM Tris-HCl, pH
7 4, 1 mN EDTA was added to each tube, the samples were vortex-d and centrifuged briefly Two microliters of each r-action was removed to determine total counts and TCA
pr-cipitable counts (incorporated counts) Two microliters of each sample were analyzed by alkaline gel electrophoresis to assess the quality of f rst strand .. , , . . . .. : . : - .
.

.. .. :. .. .
. . , , .. . . .:. ~ . :
. .. . . - , - .. ~, . . - ~ . . .

W092/U17~ 4a PCT/US9l/~ ~

synthesis. The remainder of each sample was ethanol precipitated. The nucleic acids were pelleted by centrifugation, washed with 80% ethanol and air dried for ~, ten minutes. The first strand synthesis yielded 1.0 ~g of liver cDNA or a 20% conversion of RNA into DNA.
Second strand cDNA synthesis was performed on the RNA-DNA hybrid from the first strand reactions under conditions which encouraged first strand priming of second strand synthesis resulting in DNA hairpin formation. The nucleic acid pellets containing the first strand cDNA were resuspended in 71 ~1 of water. To assess the guality of second strand synthesis, ~32P-dATP was added to the unlabeled first strand cDNA. To encourage formation of the hairpin structure, all reagents except the enzymes were brought to room temperature, and the reaction mixtures were set up at room temperature. (Alternatively, the reagents can be on ice and the reaction mixture set up at room temperature and allowed to eguilibrate at room temperature ~or a short time prior to incubation at 16C.) Two reaction tubes were set up for each synthesis. One reaction tube contained the unlabeled first strand cDNA
and the other reaction tube contained the radiolabeled first Jtrand oDNA. To each reaction tube, 10 pl of 5x -cond atrand bu~f-r (100 mM Tri~, pH 7.4, 450 mM KCl, 23 mM MgCla, 50 mM ~NH4)2(804), 3 pl o~ beta-NAD and 1 ~1 of a d-oxynucl-otide triphosphate solution containing 10 mM
ach o~ dATP, dGTP, dTTP and dCTP (Pharmacia), 1 ~1 ~32p_ dATP or 1 pl of wator (the radiolabeled dATP was added to th- tube containing tho unlabeled first ~trand cDNA), 0.6 pl of 7 Ulpl ~. 9Qll DNA ligase (New England Biolabs, B-verly, MA), 3.1 pl o~ 8 U/pl ~. ÇQLi DNA polymerase I
(Am~rsham), and 1 pl of 2 U/pl of RNase H (GIBCO BRL). The r-actions were incubated at 16C for 2 hours. A~ter incubation, 3 pl was ta~en from each reaction tube to 3S determine total and TCA precipitable counts. Two ~lcrollters of each sample were analyzed by alkaline gel oloctrophoresis to assess the guality of second strand ~ .

.... : , . . . .. .

.; . ~ ~. . .. ,. ... ., . . . .. - ~.. - .:

~ ~ 92/0l795 2 ~ 8 7 9 7 0 PCT/US91/~177 synthesis by the presence of a band of approximately twice unit length. To the remainder of each sample, 2 ~1 of 2.5 lg/~l oyster glyco~on, 5 ~1 of 0.5 M EDTA and 200 ~1 of 10 mM Tri~-HCl, pH 7.4, 1 mM EDTA were added, the samples were phenol-chloroform extracted, and isopropanol precipitated. The nucleic acids were pelleted by centrifugation, washed with 80% ethanol and air dried.
The yield of double stranded cDNA in each of the reactions was approximately 2 ~g.
10The single-stranded DNA in the hairpin structure was clipped using mung bean nuclease. The double stranded cDNA samples were resuspended and combined in 30 ~1 of water. Five microliters of 10x mung bean buffer (0.3 M
NaOAC, pH 4.6, 3 M NaCl, 10 mM ZnS04), 5 ~1 of 10 mM
15dithiothreitol, 5 ~1 of 50% glycerol, and 5 ~1 of 10 U/~l mung bean nuclease (Promega Corp, Madison, WI) were added and the reactions were incubated at 30C for 30 minutes.
After incubation, 50 ~1 of 10 mM Tris-HCl, pH 7.4, 1 mM
EDTA was added to each tube, and 2 ~1 of each sample was sub~ected to alkaline gel electrophoresis to assess the cl-avage of the second strand product into unit length cDNA. on~ hundred microliter~ of 1 M Tris-HCl, pH 7.4 w-r- ~dded to ach sample, and the samples were twice xtr~cted with phenol-chloro~orm. Following the final 2S ph-nol-chloro~orm extraction, the DNA was isopropanol pr-cipltated. The DNA was pelleted by centrifugation, w~shed with 80% ethanol and air dried. Approximately 2 ~g o~ DNA w~s obtaln~d ~rom each reaction.
The cDNA w~s blunt-ended with T4 DNA polymerase ~tor the cDNA pellet was resuspended in 30 ~1 o~ water.
Fivo microliters of 10x T4 DNA polymerase buffer (330 mM
Tris-~cetate, pH 7.9, 670 mM KAc, 100 mM MgAc, 1 mg/ml gelatin), 5 ~1 of 1 mM dNTP, 5 ~1 50 mM dithiothreitol, 5 ~1 of 1 V/~l T4 DNA polymerase ~oehringer-Mannheim) were 3S added to each tube. After an incubation at 15C for 1 hour, 150 ~1 of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA was added and the sample was phenol-chloroform extracted .

. :, ::, . . : , . . . ~ . . .

: ' '- ~ ' . ' ~ - . ;
: . . . ;, . ~
.. .

20879~ 50 PCI/USgl/nsl~

- followed by isopropanol precipitation. The cDNA was pelleted by centrifugation, washed with 80% ethanol, air dried and resuspended in 4 ~l water. Eco RI adapters (Invitrogen, Cat. # N409-20) were ligated to the blunted cDNA.
The first strand primer encoded an Xho I cloning site to allow the cDNA to be directionalIy cloned into an expression vector. The cDNA was digested with Xho I
followed by phenol-chloroform extraction and isopropanol precipitation. After digestion, the cDNA was electrophoresed in a 0.8% low melt agarose gel, and the cDNA over 2.0 ~b was electroeluted using an Elutrap (Schleicher and Shuell, Xeene, NH). The electroeluted cDNA in 500 ~l of buffer was isopropanol precipitated and the cDNA was pelleted by centrifugation. The cDNA pellet was washed with 80% ethanol.
The double-stranded, linkered, and Xho I-cut cDNA was resuspended in 34 al of distilled water. Five microliters of lOX kinase buffer (500 mM Tris, pH 7 . 8, 100 mM MgCl2, 1 mM EDTA), 5 ~l of 10 mM ATP, 1 ~l 200 mM
dithiothreitol and 5 ~l of T4 polynucleotide kinase (1 U/~l, GIBCO BRL) were added to the DNA. The reaction mixturo wa~ gently mlxed, and incubated at 37C for one hour. Aft-r lncubation, the mixture was phenol-chloroform xtr~ct~d, chloroform extracted and isopropanol pr-olpitat~d using 5 ~g of mussel glycogen (Boehringer Mannheim, Indianapolis, IN) as a carrier.
One hundred and twenty nanograms (1.5 ~l) of the CDNA was combined with 0.5 ~l of distilled water, 2 ~l o~
Lambda Uni-ZAP (1 ~g/~l, Stratagene Cloning Sy~tems, La Jolla, CA), which had been digested with Xho I and Eco RI
and treated with calf intestinal phosphatase, and 2 ~l of 3X ligation mix (7 ~l lOX ligation buffer (500 mM Tris, pH
7.8, lOO mM NgCl2, 10 mM ATP , 500 ~g/ml BSA), 7 ~l 100 mM
3S DTT, 7 ~l T4 DNA ligase (1 U/~l, Boehringer Mannheim, Indianapolis, IN)). The ligation mixture was incubated for nine hours at room temperature. Following the .. . , ., . ~ - .... . .
; - . , . . . . .

- .. ..
. . ~

, ~ 92/0179~ 2 0 8 7 9 7 0 PCT/US91/05177 51 ;` `- ?'~ `

ligation, the DNA was packaged into phage heads using Gigapack Plus II (Stratagene Cloning Systems) according to the manufacturer supplied protocol. The packaging reaction was plated out onto PLXF' host cells (Stratagene Cloning Systems) on 59 plates at approximately 250,000 plague ~orming units/plate for a yield of approximately 15 x 106 independent plaques. The plates were overlaid with TM buffer (10 mM Tris, pH 7.8, lo mM MgS04) and allowed to elute for six hours at room temperature. The liquid lysate was removed, pooled and stored at 4C. Fifty millilers of chloroform was added to the lysate to prevent bacterial growth. As a test of quality, the library was screened with a radiolabeled human plasminogen DNA
fragment as a probe. Positive clones were seen at a-frequence o~ 0.53% and greater than 50% of the positives were full length when DNA prepared from the clones was analyzed.

Nucleotide sequences encoding gamma-carboxylase were obtained using polymerase chain reactions (PCR) and oligonucleotides designed from amino acid sequences d-termined by amino acid microsequencing of the partially puri~ied material described in Example 8. Partially 2S purlri-d S-Sopharos- luant ~Example 8) was concentrated lth-r u~lng a Centricon concentrator (Amicon, Danvers, NA) or by extracting the phospholipids from the S-S~pharose eluant and eluting the protein with ~-thanol/chloroform as described by Wessel and Flugge ~r9LL.~kiQihUm~ 138: 141-143, 1984, which is incorporated by re~erence herein). The concentrated material was electrophoresed in an 8t SDS-polyacrylamide gel and tr~nsferred to nitrocellulose essentially as described in Ex~mple 8. The nitrocellulose was stained with amido black ~Sigma, St. Louis, M0) essentially as described by A-b-rsold et al. ~Proc. Natl. Acad. Sci. USA 84: 6970-6974, 1987) and Schaffner and Weismann ~Anal. Biochem. 56:

. . .. ', . ' .
.
. ', ''' ' , . ,' , ,' ' . ', s W092/017~ - PCT/US91/OS ~
20879~ 52 502-514, 1973), which are incorporated herein by reference, and the 90 Kd band was cut out The band was rinsed with distilled water and stored at -20C The procedure was repeated four times to collect a sufficient ~ample ~or microsequencing The 90 kD bands on nitrocellulose were pooled and then was microsequenced by Harvard Microsequencing (Cambridge, MA) The amino terminal sequences of three peptides were determined as shown in Table 9 TABLE g Peptide 1 ~Sequence ID Number 3) F T L L A P T S P G D T T P [K~

Peptide 2 (Seguence ID Number 4) G R D P A L P T L L N P K

Peptide 3 (Sequence ID Number 5) D D tR] G P S G Q G Q G Q G Q F L I Q Q V T
Peptide 4 (Sequence ID Number 6) F L W D E G F ~ Q L V I Q R

P-ptid- S ~Seguence ID Number 19) ~ P Q P L L T tG]

Peptide 6 (Seguonce ID Number 20) T L P S G L D D Y X

P-ptide 7 (S-guence ID Number 21) L A E Q L G E A E A A A E L G P L A A S L G A E E X [L] D
lE~

3S where residues enclosed in brackets ~] indicate prob~ble/reasonable residues at the designated position , .-, ..... ...

~92/01795 208737~ PCI`/US~1/05177 ; 53 j Families of degenerate oligonucleotides, ZC4135 (Table 10; Sequence ID Number 11) and ZC4136 (Table 10;
Sequence ID Number 12) were designed to correspond to the terminal portions of the amino acid sequences of Peptide 1 5 (Table 9; Sequence ID Number 3) and in addition, Eco RI
restriction sites were added to the 5' termini of the oligonucleotides to facilitate subcloning. The oligonucleotides were synthesized on an Applied Biosystems 394 RNA/DNA Synthesizer (Applied Biosystems, Foster City, 10 CA). The antisense oligonucleotide family, ZC4135 (Table 10; Sequence ID Number 11), had a 128-fold degeneracy.
The sense oligonucleotide family, ZC4136 (Table 10;
Sequence ID Number 12), had a 512-fold degeneracy.

TABh~ 10 D~erate Oliqonucleotide Primer Families ZC4135 (Sequence ID Number 11) A A A A
C C C
T T T
ATA GAA TTC TTG GGG GTG GTG TC

2S ZC4136 ~S-quence ID Nu~ber 12) A A A
C C C
T TT TT T
ATT AGA ATT CTT CAC GCT GCT CGC

ZC4138 (Sequence ID Number 13) A A A A

T T T T T
CCG ACG AGG CCG GG

:-' : -~ :
: , ; . .
. - - . .: ,. - ' :': , ,,,' . -: : .- .

WO92/017~ PCT/US91/05 ~

208~ 97 54 TABLE 10 continued ZC4204 (Sequence ID Number 14) A A
S C C C
T T
TAT AGA ATT CGT GAC TTG TTG GAT

ZC4205 (Sequence ID Number 15) A A A
c c c c G G
ATA AGA ATT CGA TGA TCG TGG TCC --ZC4217 (Sequence ID Number 16) A A A A
C C C C C
T TT T T
ATA AGA ATT CGG GCC GAG GGG GCA

ZC4241 (S-quenc~ ID Numb~r 17) A A A A
C
T T
TGG TGG AAG CCC TCG TCC CA

First strand bovine cDNA was prepared and used a- the template cDNA for the PCR reactions. First strand cDNA was synthesized from one time poly d(T)-selected bovine llver poly(A)+ RNA in two separate reactions. One r~aotion, containing radiolabeled dATP, was used to assess 3S the guality of first strand synthesis. The second roaction was carried out in the absence of radiolabeled dATP and was used, in part, to assess the quali-ty of ~ 092/01795 2 0 ~ 79 7 0 : ~

second strand synthesis. - superscript reverse transcriptase (GIBCO BRL) was used specifically as described below. A 2.5x reaction mix was prepared at room temperature by mixing, in order, 8 ~l of 5x reverse transcriptase buffer (GIBcO BRL; 250 mM Tris-HCl, pH 8.3, 375 mM KCl, and 15 mM MgCl2), 2.0 ~l 200 mM dithiothreitol (made fresh or stored in aliquots at -70OC) and 2.0 ~l of a deoxynucleotide triphosphate solution containing 10 mM
each of dATP, dGTP, dTTP and s-methyl dCTP (Pharmacia).
The reaction mix was aliquoted into two tubes of 6 ~l each. To the first tube, 1.0 ~l of 10 ~Ci/~l ~32P-dATP
(Amersham) was added and 1.0 ~l of water was added to the second reaction tube. Fourteen microliters of a solution containing 10 ~g of Bovine liver poly(A)+ RNA diluted in 14 ~1 of 5 mM Tri~-HCl pH 7.4, 50 ~M EDTA was mixed with 2 ~1 of 1 ~g/~l fir6t strand primer, ZC2938 (GAC AGA GCA CAG
AAT TCA CTA GTG AGC TCT m TTT TTT TTT TT; Sequence ID
Number 9), and the primer was annealed to the RNA by heating the mixture to 65C for 4 minutes, followed by chilling in ice water. Eight microliter~ of the RNA-primer mixture was added to each of the two reaction tubes followed by 5 ~g of 200 U/~l Superscript reverse transcriptase ~GIBCO BR~). The reactions were mixed gently, and the tubes were incubated at 45C for 30 2S minut--. A~t-r incubation, 80 ~l of 10 mM Tris-HCl, pH
7.4, 1 mM EDTA w-re added to each tube, the samples were vort-x-d and centrifuged briefly. Two microliters of each r-action was removed to determine total counts and TCA
pr-clpitable counts (incorporated counts). Two mlcroliters Or each sample was analyzed by alkaline gel lectrophoresis to assess the quality of first strand ynthQsis. Five microliters of 0.5 M EDTA and 10 ~l of 5 M ROH were added to each tube, and the reactions were pl~c-d at 65C for five minutes. Following the 3S incub~tion, the reaction was terminated by the addition of 3S ~l of 8 M NH4AC and 135 ~l of isopropanol. The mixture - was chilled on ice for 15 minutes and centrifuged for 20 , 20~9~ PCT/~59~

minutes at lO,OOo rpm. The pellet was resuspended in S ~1 of distilled water. A yield of l.S ~g of cDNA was obtained.
A PCR reaction mixture was prepared which contained 50 ng of first strand bovine cDNA, S al of 2.SmM
dNTPs (containing all four deoxynucleotide triphosphates, Cetus, Emeryville, CA), S ~1 of lOx PCR buffer (Perkins-Elmer Cetus, Norwalk, CN) and S ~1 of each oligonucleotide (ZC4135 and ZC4136; Table 10; Sequence ID Nos. 11 and 12) at a starting concentration of 20 pmole/~l. The total volume was 50 ~1. One microliter of Taq I polymerase (Perkins-Elmer Cetus) was added, and the PCR reaction was carried out under the conditions shown in Table 11.

~recvcle - 3 cycles ! 94C rOr 1 minute 32C for 1 minute 72C for l.S minute ~ - 35 cycles 94-C ~or 1 m~nut- -SSC tor 1 mlnute 72C ~or 1.5 minute A~ter the last cycle o~ the Ampli~ication cycle, the reaction product~ were separated by electrophore~is in a Nu-ieve gol (FMC Bioproduct~, Rockland, ME). The DNA of pproximately 65 baso pairs was isolated by melting the gel at 65C in S ml Or 10 mM Tri~ pH 8, 1 mM EDTA and S ml o~ phenol. T~e mixture was ~pun at 4000 rpm ~or five 3S minute~ at ~oom temperature (Beckman GPR centrifuge, ~-ckman Instruments, Carlsbad, CA) and the agueous layer was transferred to a fresh tube. The amplified DNA in the .. , , . ,. . .. , -.: -,,: , .: - . .

, . . .. . .. .. . . . ..

~ 092/01795 2 0 8 7 9 7 ~ PCT/US91/~177 aqueous layer was ethanol precipitated and pelleted by centrifugation The PCR reaction was repeated on the pellet by ~irst resuspending the pellet in 30 ~1 of distilled water PCR reagents were added as described above, and the reaction mixture was brought to a final volume of 50 ~1 The PCR cycles were repeated as described in Table 11 except that the precycle was omitted and the amplification cycle was run twice The resulting reaction products were 6eparated on a gel as described above Several bands of approximately 65 base pairs in length were seen including a unigue band not seen in control reactions using each oligonucleotide primer family independently The region around 65 base pairs was cut from the gel, and the DNA was extracted as described above The PCR reaction was repeated a third time using the second round reaction products The reactions were run as described in Table 11 except that the precycle was omitted The reaction products were gel purified as described above and the resulting DNA was digested with Eco RI The Eco RI-digested DNA was phenol extracted and ethanol precipitated Ths ampliried DNA was ligated into pUC118 or pUCll9 which had b--n linearized by digestion with Eco RI
dlg--t-d and tr-at-d with cal~ alkaline phosphatase to pr-v-nt r-circularization The ligation mixture was transrormed into DHlOB competent j~ coli cells (GI~C0 BRL) uJing a Biorad Gene Pul~er ~Biorad Richmond~ CA), a¢cording to th- high efriciency electro-transformation protocol speci~ied by the manufacturer Plasmid DNA wa8 prepared from selected transrormants, and the DNA was sequenced for positive ldentification of the gamma-carboxylase insert A clone was isolated having the sequence shown in Table 12 ~S-quenc- ID Number 18) In addition, Southern analysis was carried out on aliquots of the plasmid DNA using the degenerate oligonucleotide ZC4138 ~Table 10; Sequence ID

. , . .. :; ... : . . -wo92/nl795 ~ 58 PCT/US91/051 Number 13), corresponding to an internal amino acid sequence of Peptide 1 (Table 9; Sequence ID Number 3) flanked by the oligonucleotide primers ZC4135 and ZC4136 (Table 10; Sequence ID Nos 11 and 12) PCR Clone Sequence (Sequence ID Number 18) GAA TTC TTT ACG CTT TTG GCA CCA ACC AGT CCA GGA GAT ACC
ACT CCC AAG AAT TC

Nucleotide sequences were also obtained using ~amilies of degenerate oligonucleotide probes which were designed to correspond to the terminal portions of the amino acid sequences o~ Peptide 3 (Table 9; Sequence ID
Number 5) and contain Eco RI restriction sites at the 5' termini of the oligonucleotides to facilitate subcloning The oligonucleotide family ZC4204 (Table 10; Sequence ID
Number 14) corresponds to the amino terminus of Peptide 3 ~Table 9; Sequence ID Number 5) The oligonucleotide ~amily ZC4205 tTable 10; Sequence ID Number 15) corr-~ponds to the carboxyterminus or Peptide 3 (Table 9;
8-qu-n¢e ID Numb~r 5), The oligonucleotide family ZC4217 2S ~T~bl- 10; S-qu-nc- ID Numb~r 16) corresponds to internal ~mlno t-rmlnal r-~iduc,s o~ Peptide 3 ~Table 9; Sequence ID
Numb r 5) ~ ovine rirst strand cDNA, prepared as described in Exampl- 10, was used as a template in two PCR
r-actions In the rirst r-action oligonucleotide ramily ZC4205 (Table 10; S-quence ID Number 15) was combined with oligonucl-otide ra~ily ZC4204 ~Table 10; Sequence ID
Nu~b r 14) In the second reaction, oligonucleotide ~mily ZC4205 (Table 10; Sequence ID Number 15) was 3~ ¢ombined with oligonucleotide family ZC4217 (Table 10;
Saqu-no~ ID Number 16) The PCR reactions were carried out e-sentially a8 described above using the protocol ~ ~ 92/017~ 2~87970 PCT/USg~ 77 ss - .

-described above and the conditions described in Table 13 except that the PCR products were gel purified using a crush and soak method The PCR products were isolated ~rom the gel by cutting the appropriate band from the gel, crushlng the band with a pasteur pipette in TE (lo mM
Tris, pH 8 0, 1 mM EDTA) and allowing the crushed gel to soak in the TE overnight at room temperature After the overnight incubation, the gel-TE mixture is phenol extracted and the aqueous layer containing the DNA is ethanol precipitated ,TABLE 13 ,Precycle - 4 cycles 94C ~or 1 minute 36C ~or 1 minute 72C for 1 5 minute First Amplification cycle - 25 cycles 94C ~or 1 minute 60C ~or 1 minute 72C ~or l S m~nut0 2S 8-con~ Ameliricatlon cvcle - 25 cycles 94 C ~or 1 minute 60 C ~or 1 minute 72 C ~or 1 5 minute Th- sequ-nces of the PCR product shown in Table 12 ~S-quence ID Number 18) was used to generate an oligonucleotide probe corresponding to the twenty nucleotide sequence from nucleotide 19 through 38 of Table 12 tsoquenc- ID Number 18) The oligonucleotide probe is u~ed to probe a bovine liver cDNA library, prepared as . . ~ ......... . : . . ~ ......... . . - -,, . . : .. . . .;. . .. . , , , :. . . .

~! 20~9~ 60 PCT/US41/051 ~

described in Example lO and probed according to standard techniques essentially as described by Sambrook et al (Moleçul~ L~lL~ b~bor~to~y ManualA Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), which is incorporated herein by reference) Oligonucleotide probes designed from the PCR product sequences are also used in a RACE protocol essentially as described by Frohman et al (Proc Natl Acad Sci USA
85 8998-9002, 1988, which is incorporated herein by reference) to obtain gamma-carboxylase sequences The amino acid sequence of Peptide 4 (Table 9;
Sequence ID Number 6) contains a sequence from which a family of degenerate oligonucleotides may be designed containing a degeneracy Or 64 over 20 base pairs The family of degenerate oligonucleotides, ZC4241 (Seguence ID
Number 17) is used as a probe of the bovine lambda gtll cDNA library described in Example lO The library is screened using standard techniques described Sambrook et al (ibid ) Positive clones are plaque purified and analyzed by restriction and sequence analysis ~2 Monoclonal antibodi-s were prepared essentially a~ d--crlb-d by H~rt (U S Pat-nt Application, Serial No 2S 07/139,960, whioh i- incorporat-d h-rein by reference) Brlerly, partially purified bovine gamma-c~rboxyla~e wa~ bound to S-S-pharose, as described in Ex~mpl- 8 or to sulro-propyl S-pharose The bound ~terl~l w~ mix-d with Complete Freund's Ad~uvant (ICN
Bioch-mic~l~, Costa Me~a, CA ), and l50 ~l Or the solution p-r mou-! wa~ in~ected intraperitoneally into ~even-week-old Balb/c temale mice (Simonsen Labs, Gilroy, CA) Boo-ter in~ection material was prepared by absorbing parti~lly pur$fied g~mma-carboxyla~e to ~ul~o-propyl or S-80ph~ro~ harmacia), and the bound material was mixed with Incomplete Freund's Ad~uvant Each ~ouse was boosted - with l50 ~l of booster injection material at approximately -~ 2087970 '' ~';~92/01795 PCT/US91/05177 two week intervals for the first two to three months and then at approximately monthly intervals thereafter. Mice were identlfied as expressing anti-carboxylase antibodies after the first three injections as described in Example 8.
Two three- to four-week-old Balb/c mice, which were shown to express anti-carboxylase antibodies, were sacrificed and the spleens and lymph nodes were removed from the immunized mice and minced with scissors on top of fine-mesh stainless steel screens. The minced tissues were washed through the fine screens into petri dishes with 10 ml RPMI 1640 (GIBC0 BRL). The remai~der of the minced tissues were pressed through the screens with a spatula and the screens were washed with 5 ml RPMI 1640.
To remove any remaining cell material, the bottom of the screens were scraped and the material was added to the petri dishes.
The strained tissues were transferred into 50 ml centrifuge tubes, and the petri dishes were washed with 10 ml RPMI 1640 to remove any remaining material. The cell ~uspensions were centrifuged for 10 minutes at 200 x g.
The supernatants were discarded and the pellets were resuspended in 4 ml RPNI 1640. After resuspension 1 ml f-tal calf ~-rum ~BioCell, Carson, CA) was added to each tub-.
The red blood cell contaminants were lysed by adding 18 ml sterile distilled water to the cell susp-nsions. The mixtures were swirlQd quickly and 5 ml 4.2S% NaCl was added to each tube. The cell suspensions were centriruged ~or 10 minutes at 200 x g. The sup-rnatants were discarded, and the pellets resuspended in 10 ml RPMI 1640.
To remove any remaining tissue material, the suspensions were filtered through two layers of sterile 3S gauz- into 50 ml tubes. The centrifuge tubes and gauze were rinsed wlth an additional 10 ml RPMI 1640. Dilutions of the resultant cell suspensions were counted with a - : . .
. , . : ; . - :
- -, ~.................... . .............. . .
' ' , ' ''' ' ' ' .. ,' ,. . ~ ~ . .' :

W092/017~ PCT/US91/~ ~

20879~ 62 hemacytometer to determine the yield of lymphocytes. The prepared cells were kept at room temperature for approximately one hour until ready for use.
Thymus glands obtained from baby mice are the S ~ource o~ the thymocytes which act as the feeder layer for the cell ~usions. Thymocytes were prepared from thymus glands obtained from two three- to four-week old Balb/c mice. The thymus glands were rinsed with NS-1 medium (Table 14) and minced on a fine-mesh stainless steel screen with scissors. The minced tissues were rinsed through the screen with 10 ml NS-l medium into a petri dish. The thymus tissue was pressed through the screen with a spatula into the petri dish, and the screen was washed with 10 ml NS-1 medium. The bottom of the screen was ~craped to remove any adhered material and the materlal was pooled in the petri dish. The cells were transferred to a 50 ml centrifuge tube through two layers of sterile gauze. The petri dish and gauze were rinsed with an additional 10 ml NS-1 medium. The cells were ¢entrifuged for 10 minutes at 200 x g. The supernatants were discarded and the pellets resuspended in 10 ml NS-1 m-dium. Dilutions sf the cell suspensions were counted u~ing hemacytometer. The yield should be about 400 million cells. The c-lls were stored at room temperature 2S until r-~dy ror U8-.

,., ., .,: . : ' : ` ''- ': - ' , ' - ,: . :
: . . .: -: , ::
, :- ` . ,: -.. . .
. , :~ .. .. .

~ ~ 92/01795 2 08 79 7 0PCT/US9l/~77 ; 63 ; i ~-1 Mç~1~m For a 500 ml solution 5 ml 10 mM non-~ssential amino acids (GIBCO BRL, Lawrence, MA) S ml 100 mM sodium pyruvate (Irvine, santa Ana, CA) 5 ml 200 mM L-glutamine (GIBCO BRL) 5 ml 100x Penicillin/Streptomycin/Neomycin (GIBCO
BRL) 75 ml inactivated fetal calf serum (Hyclone, Logan, UT) 1 gm NaHCO3 100x HT Stock 38 5 mg thymidine 136 1 mg hypoxanthine (Sigma, St Loui~, MO ) 1000x A Stock 17 6 ng aminoptorin 8teril- di~tilled water Wa8 added to the aminopterin to a 2S ~olume of 50 ml The aminopterin wa~ dissolved by the drop-wi~e addition of 1 N NaOH, and sterile distilled wat-r wa8 add-d to a final volum- of 100 ml Th- solution wa- t-riliz-d by filtration t~rough a 0 22 ~m filter and tored frozen at -20C
~Q~ HAT
50 ml 100x HT
5 ml 1000x A ~tock 4S ml distilled water Sterilize the solution by filtration through a 0 22 ~m filter Store frozen at -20C

W09~0l795 ~ ' PCT/US9~/0sl ~9 TABLE 13 continued Freezin~ Medium 7 ml NS-1 medium 2 ml fetal calf serum 1 ml DMSO

Mix the ingredients and make fresh for each freezing .' ' '"

The NS-1 mouse myeloma cell line was used for the fusion To optimize the fusion procedure, the NS-1 line wa~ cloned out to isolate a clone with a high fusion e~riciency The NS-l cells were cloned out by limiting dilution into 96-well microtiter plates at an average of rive and ten cells per well in NS-l medium + 2 5 x 1o6 thymocytes/ml (as prepared above) The plates were incubated at 37C with 7~ C02 for ten days On day ten, the cells were examined mieroscopically and screened for wells containing single eolonies On the same day, 100 ~1 of fresh NS-l medium eontaining 2 5 x 106 thymocytes was added to the eells On th- fourt-enth day, eight Or the most vigorou~ly growing ingl- eolonles wer- ehos-n to oxpand ror rusiOn Th- ight eandldat- coloni-s were transrerred to - ~S lndl~ldual 24-w-ll plat-~ oontaining 1 5-2 ml NS-1 medium 1 2 5 X lo6 thymocytes/well The plates were incubated at 37 C with 7% C02 and the eells were ~plit at appropriate lnt-rvals to obtain a surrieient number Or eell- to tran~-r to flask eultur- Ten million eells from ach olon- were inoculated into a 75 em2 tissue eulture rlasks ¢ontaining 50 ml NS-l The rlask~ were ineubated at 37C, w~th 7% C02, until the eell~ reaehed a density o~ at least x 105 eells/ml The cells were then harvQsted by o-ntrlfugation and diluted to a concentration of 3S approximately 5 x 106 cells/ml with freezing medium (Table 14) The e-lls were divided into 1 ml aliguots and frozen stepwi~e, ~irst at -80C and then at -130C

: .. . . ... . . . . . . . ..

~ ~ 92/01795 2 ~ 8 79 7 o PCT/US91/~177 6s , The clones were assayed by quick thawing one vial of each clone in water held at 37c. The cells were inoculated into flasks containing NS-l medium to a concentration of 2 x 105 cells/ml. The cells were grown at 37C with 7% C02. The cells were cut back to 2 x 105 cells/ml daily. One day before fusion, two 75 cm2 flasks containing 50 ml of cell cultere were set up. Each candidate NS-1 clone was mixed with immunized mouse spleen cells and fused as described below. The results of the fusion showed that one clone, designated clone F, had an increased fusion efficiency.
For fusion, 2.5 x 107 NS-l clone F cells were quickly thawed, as described above, and were added to the prepared immunized mouse spleen and lymph node cells. The mixed cells were centrifuged ~or 10 minutes at 200 x g, and the supernatant was removed. The cell pellet was re~uspended in 100 ~1 RPMI 1640 and warmed in a 37C water bath.
one milliliter of a 50% polyethylene glycol (PEG) solution in RPMI 1640 was ad~usted to within the range o~ pH 7.0 to pH 8.0 using 1% sodium bicarbonate.
Th- PEG solution was added to the cell suspension over a p-riod of one minute with gentle stirring. The solution wa- tirr-d for one additional minute. One milliliter of 2S th- N8-1 m-dium wa- add-d ov-r a period of 1 minute with ~-ntly tirring. An additional milliliter was added to th- ~usup-nsion over a period of one minute. Eight milliliters of NS-1 media was added over a period of two minut-s with gentle stirring and the su~pension was then p-llet-d by centri~ugation at 125 x g ~or 10 minutes at room te~perature. The supernatant was discarded, and the Cell8 were gontly resuspended in 25 ml o~ NS-l medium.
Th- cells were transferred into a 175 cm2 flask.
Four hundred million thymocytes (prepared a~
3S above) were added to the flask. The volume was ad~u~ted to 160 ml with NS-l medium, and the mixture was incubated at 37-C with 7% C02 for two to four hours. - -WO ~/~17~ 0 ~ :~ 66 pCT/US91/~S

After incubation, 3.2 ml of 50x HAT (Table 14) was added. The cell suspension was transferred to eight 96-well plates at 200 ~1 per well., and the plates were incubated at 37C with 7% CO2. The plates were examined S microscopically after three days to determine fusion e~iciency with the expectation of approximately five hybridoma colonies per well. The cells were fed after seven days by replacing 100 ~1 of the medium with fresh NS-1 medium containing lx HAT and 2.5 x 1o6 thymocytes per ml. The hybridomas were tested between day nine and fourteen for the production of specific monoclonal antibodies.
The monoclonal antibodies were tested both for the ability to selectively bind 125I-gamma-carboxylase and ~or the ability to bind to material which exhibited carboxylase activity, as measured by CO2 incorporation into a peptide substrate.
Iodination of gamma-carboxylase was performed using Iodobeads (Pierce, RocXford, IL) and the standard protocol supplied by the manufacturer. One hundred microliters of gamma-carboxylase was added to one Iodobead wlth 0.5 mCi Or 125Iodine, and the mixture was placed on la- or 15 minut-~. Arter incubation, l.S ml o~ TNC/P
bur~-r (Tabl- lS) wa- added, and the mixture was passed ov-r a G2S ¢olumn ~Pharmacia). Eight rrac~ions were coll-cted with a rirst rractiOn volume o~ 1.5 ml and aub--quent rractions volumes Or 0.5 ml. All eight ~ractlon~ were counted on the gamma counter.

TA~L~ 15 TCN~P
0.1 M NaCl SOmM Tris, pH7.4 0.2S% Phosphatidyl Choline Type V E (Sigma) 0.2S% CHAPS (Sigma) , , . ,:
'''..... ''' .' ' - : , -, - : , ., ' . , . - . .. .:

. --, -, , . ,' . '-- . ~ :
. ' - : .. ,. :

~ 92/0l795 2 0 8 7 9 7 ~ PCT/US91/05177 TABLE 15 continued RIp buffer 10 ml 2 M Tris-base, pH 8 25 ml 4 M NaCl 5 ml Nonidet P-40 (Sigma) 5.0 gm Sodium deoxycholate 1.5 gm Sodium Iodine 10.0 gm BSA fraction V 1~

Total volume brought to 1 liter and adjusted to pH 8.

ca.~boxvlase reaction m x 950 ~1 3.8 M Ammonium sulfate 750 ~1 10mM EEL (BOC-Glu-Glu-Leu-OME) (Bachem Bioscience, Philadelphia, PA) 150 ~1 1% CHAPS (C32HsgN2O7S) (Sigma) 150 ~1 1% Phosphatidyl Choline Type IIIE (Sigma) in 1%
sodium cholate 75 ~1 0.2 M DTT (Dithiothreitol) 150 ~1 NaH14CO3 (tS mCi/2.5 ml] Amersham, Arlington Heights, IL) 80 ~1 Vitamin X hydroquinone Urea/SDS
2S ~iX gra~- Or Ur-a in a ~inal volume o~ 10 ml of water was h-at-d in a 37C water bath until all the urea was dis~olved. one milliliter of 20% SDS was added, and the ~olutlon was stor-d at room temperature.

The ability of the monoclonal antibodies to electively bind 125I-gamma-carboxylase was tested using a radioimmunoprecipitation assay (RIPA). Further id-nti~ication or the binding complex was made using gel eparation. Five microliters of 125I-gamma-carboxylase (prepared as described above) was added to each well of a microtiter plate. Fifty microliters of TWC/P buffer was ,- .~ -: . ....... : , , .. , ., . . .- :
: . , - . .- , , i ,, , , ", " , ,, ,~ ,, , , , , : ,,, ", . . , . : . . ~ , , ~.. . .. ,. . , -. .. . . . . . -, .

WO 9Z/01795 68 PCr~US91/OSI~

added to each well, and 1 M sodium iodine was added to each well to achieve a final concentration of 10 mM.
Finally, 50 ~1 of each monoclonal hybridoma-conditioned medium was added. The plate was incubated shaking for one hour at 40C. One microliter of 0.1 mg/ml rabbit anti-mouse IgG (Cappel Laboratories) which had been diluted in PBS was added to each well, and the plate was incubated shaking at 40C for one hour.
Precleared Pansorbin cells (Calbiochem, La Jolla, CA) were prepared by centrifuging 1 ml of throughly mixed Pansorbin for 40 seconds at maximum speed in a microfuge at 4C. The supernatant was removed and the pellet was resuspended in 1 ml RIP buffer. The cells were incubated on ice for 20 minutes. After incubation the cells were pelleted and resuspended, as described above, two more times. The cells were stored at 4C.
Following the incubation, 25 ~1 of precleared Pansorbin Staphylococcus aureus cells in RIP buffer (Table 15) were added to each well. The plates were incubated at 4C, on a shaker at high speed. The cells were pelleted by centrifuging the plate in a Beckman TJ-6 c~ntrifuge with a TH-4 rotor ~Beckman, Carlsbad, CA.) at 2000 rpm at 4C ~or 5 minutes, and the supernatants were removed using a b-nt m~nirold ~Fi-ch-r 21-169-lOE, Fi~cher Scientific ~5 Group, 8anta Clara, CA). Th- pollet~ were resuspended in 150 pl RIP bufr-r per well. This procedure was repeated two moro times.
After th- ~inal rinse, each resuspended pellet wa~ trans~-rrod to a plastic tube and counted on a Packard Cobra Auto-Gamma counter ~Downer~ Grove, IL).
The monoclonal antibodies were tested for the ~b~lity to bind a protein having carboxylase activity.
Carboxyla~e activity was measured as a function o2 the lncorporation o~ 14C-C02 in a carboxylase reaction. Fi~ty miorolit-rs of hybridoma-conditioned medium from each clon- was added to the wells of a 96-well Linbro Titertek multiwell plate (Flow Laboratory). Fifty microliters ~of : ,, - ~ ` . , . ~ .; , .

.:

, ~ 92/0l795 2 0 8 7 9 7 0 PCT/US91/~177 bovine microsomes were added to each well. After mixing, the plate was covered with a Costar Serocluster Plate Sealer (Costar, Cambridge, MA) and incubated with gentle ~haking for one hour at 4C.
Ten microliters per well of a 1:10 dilution of 1 mg/ml purified rabbit antimouse IgG (Cappel, West Chester, PA) was added to each well, and the plates were incubated at 4C for one hour. Following the incubation, 25 ~1 of TNC/P-precleared Pansorbin Staphlyococcus aureus cells was added to each well and the plates were incubated for one hour at 4C. Precleared cells were prepared as described above except that TNC/P buffer (Table 15) was substituted for RIP buffer. After incubation, the plates were spun in a Beckman Model TJ-6 centri~uge with a TH-4 rotor at 2,000 rpm for five minutes at 4C. The supernatants were aspirated and the pellets were resuspended in 150 ~1 of TNC/P buffer. The plates were spun as described above and the supernatants were again aspirated. The pellets were resuspended in 150 ~1 of TNC/P buffer and spun as de~cribed above. The rinses were repeated a total of three times. Following the final rinse the pellet was resuspended in 100 ~1 per well TNC/P buffer and 133 ~1 carbo~ylase reaction mix (Table 15) was added to each ~ample.
- 2S Th- sa~pl-s w-r- tran-ferred to microfuge tubes and w-r- lncubat-d from one hour to overnight at room t mp-rature. After incubation, 1 ml o~ 10%
trichloroacetic acid was added to each sample. After incubatlon, the samples were spun fifteen mlnutes at maximum speed in an Eppendor~ microfuge at 4C. One milllllter of supernatant ~rom each sample was trans~erred to scintlllation vials, and 1 ml of sterile water was added to each vial. The samples were boiled with one bolllng chip each for five minutes, until only several microliters of liguid remained. The vials were cooled to room temp-rature and 10 ml of Bio-Safe II (Research ~ Products International, Mt. Prospect, IL) was added to ~" : : ' ' " ' ' ' -. . .

W092/0179S PCT/US91/~l ~
~,o8~9~ 70 each vial The samples were counted in a Beckman LS-1800 Liquid Scintillation counter ~
Wells from the first` fusion were screened on the fir~t day ~or the ability to selectively bind 125I-gamma-carboxyla~e using the RIPA assay described above Wells ~ound to produce antibodies capable of selectively binding 125I-gamma-carboxylase were screened on the second day to detect wells producing antibodies capable of binding a protein having carboxylase activity in the carboxylase activity assay described above From the first fusion, two wells, designated 170 1 1 and 170 3 1, were found to be positive in both assays Wells from the second fusion were screened in parallel using both the RIPA assay and the carboxylase aetivity assay described above Of the wells, two clones, de~ignated 171 2 1 and 171 4 4, were shown to produce antibodies capable of both selectively binding 125I-gamma-carboxylase and binding a protein having carboxylase aetivity Approximately sixty wells were identified as produeing antibodies eapable of binding a protein having ¢arboxylase activity but incapable of binding 125I-gamma-earboxylase OS these latter elones, two, de~ignated 171 S l and 171 4 1, wero ehoson ~or further use Tho 8ix elones 170 1 1, 170 3 1, 171 2 1, 2S 171 4 1, 171 4 4, and 171.5.1 were sorially diluted into 96-W ll mierotit-r plates to isolate elones arising from ingle e-lls Tho elones arising from single eells were r---er-en-d and the elones w-re expanded The media from tho monoelonal lines woro passed over a Protein A eolumn ror puri~ieation essentially as deserib-d in A~fin~y SbrQ~D~99~hY Prin~ and Mothods (Pharmaeia Fine Ch ml¢al~, Uppsala, Swedon, 1983, whieh is incorporated h-r-in by roferenee) Clone 170 3 1 eells were in~eeted lntraperitoneally into miee to obtain aseites fluid whieh 3S w~- puriried over a protein A eolumn as deseribed above to purl~y mono¢lonal antibodies for use in subsequent experiments .. , . . ........... ,, " .. .....

:

' ~ ~ 92/01795 2 0 8 ~ 9 7 ~ PCT/US91/~177 71 ` ~

The monoclonal antibodies described above were used individually to screen the bovine lambda gtll cDNA
library prepared as described in Example 10 or were combined at a concentration of 1 ~g/ml each to screen the cDNA library. Polyclonal serum from mice immunized as generally described in Example 12 was also utilized to screen the bovine lambda gtll cDNA library.
One to three million phage from the bovine lambda gtll cDNA library described in Example 10 were absorbed to a limiting number of PLKF~ cells. The cells were then plated with E. coli Y1090 cells to obtain 50,000 plaques per 150 mm plate. Alternatively, one to three million phage were plated with PLKF' cells to obtain 50,000 plaques per 150 mm plate. The plates were grown at 37C or 42C for 3.5 hours. Nitrocellulose filters, previously soaked in 10 mM IPTG, were placed on the lawn and the plates were incubated overnight at 37C or 42C.
The filters were removed from the lawn and a second pre-~oaked nitrocellulose filter was laid on the lawn and incubated at 37C or 42C for an additional 8iX hours.
me ~-cond ~ilter wa~ removed ~rom the lawn.
The duplicate ~ilters were washed two times in TB8 ~50 mM Tri~, pH 8.0, 150 mM NaCl). Following the -cond wa~h, th- rilt-rs were blocked for one hour in TBS
+ 3~ BSA. The blocked filters were transfered to a olution containing 1-2 ~g/ml o~ antibody (170.3.1.1, 171.2.1, the monoclonal antibody pool or polyclonal ~erum) S0 in TBS + 3% BSA. The ~ilters were incubated for 4-20 hours at room temperature. Following incubation, exce~s antibody was removed with two washes with TBS, one wash with TBS + 0.1% NP-40, two additional washe~ with TBS and a ~inal wash in TBS + 3% BSA. The filters were incubated in TBS I 3% BSA + 125I-Protein A (obtained ~rom Amersham or iodinated using the Iodobead iodination protocol '. ' ,, . . . , ' ' :,, . ` . ' , ` ,, , ', ,' ` - . ': . ` ,- ' W092/0l79~
~0~ 9~ 72 PCT/US9l/~1 ~

described in Example 13 using Protein A obtained from Sigma) for 4-20 hours at room temperature.
Excess label was removed by two washes in TBS, 1 wash with ~BS + 0.1% NP-40 and three washes with TBS. The ~ilters were dried and exposed to X-ray film. Positive plaques were picked for plaque purification.
Although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

: - . . . . . -. ~, . . ........... . . . ~ , ' '` ' , . : .... .
' ., ` . .' , , ';.' ~ ' . ' ' ~ ' ' ` ' " j ~092/01795 208 79 7~ Pcr/usgl/o~l77 , . . .

SEQUENCE LISTING

(I) GENERAL INFORMATION:
(i) APPLICANT: Berkner, Kathy L
(11) TITLE OF INVENTION: GAMMA-CARBOXYLASE AND METHODS OF USE
(lii) NUMBER OF SEQUENCES: 21 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Seed and Berry (B) STREET: 6300 Columbia Center, 701 Fifth Avenue (C) CITY: Seattle (D) STATE: WA
(E) COUNTRY: USA
(F) ZIP: 98104-7092 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatlble (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFT~ARE: PatentIn Release Jl.O, Version #1.25 (v1) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vll) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/557,220 (B) FILING DATE: 23-JUL-1990 (v111) ATTORNEY/AGENT INFORMATION:
At NAME: Mak1, Dav1d J
B REGISTRATION NUMBER: 31,392 C REFERENCE/DOCKET NUMBER: 990008.544PC
(1x) TELECOMMUNICATION INFORMATION:
A) TELEPHONE: 206-622-4900 B) TELEFAX: 206-682-6031 ~2) INFORMATION FOR SEQ ID NO:I:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 am1no aclds (B) TYPE: am1no ac1d (D) TOPOLOGY: 11near .
(v) FRAGMENT TYPE: 1nternal (v1) ORIGINAL SOURCE:
(A) ORGANI~M: Homo sapiens W092/01795 PCr~U59WSI~

(xt) SEQUENCE DESCRIPTION: SEQ ID NO:I:
Cys Gly Gly Hls Val Phe Leu Ala Pro Gln Gln Ala Arg Ser Leu Leu l 5 l0 15 Gln Arg Val Arg Arg (2) INFORMATION FOR SEQ ID NO:2:
(t) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Cys Gly Gly Lys Asp Lys Leu Asn Asp Asn Hts Glu Val Glu Asp Glu . 5 10 15 Tyr (2) INFORMATION FOR SEQ ID NO:3:
(1) SEQUENCE CHARACTERISTICS:
A) LENGTH: 15 am1no ac1ds B TYPE: amlno acld D TOPOLOGY: 11near (v) FRAGMENT TYPE: 1nternal (xl) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Phe Thr Leu Leu Ala Pro Thr Ser Pro Gly Asp Thr Thr Pro Lys l 5 lO 15 (2) IHFORMATION FOR SEQ ID NO:4:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 am1no ac1ds (B~ TYPE: am1no ac1d (D) TOPOLO6Y: 11near (v) FRAGMENT TYPE: 1nternal . ~ . , ., ; . .

.., .. ~... .

~0 92/0179~ 2 0 8 7 9 7 0 PCI/US91/05177 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Gly Arg Asp Pro Ala Leu Pro Thr Leu Leu Asn Pro Lys (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Asp Asp Arg Gly Pro Ser Gly Gln Gly Gln Gly Gln Gly Gln Phe Leu Ile Gln Gln Val Thr (2) INFORMATION FOR SEQ ID NO:6:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amlno ac~ds (B) TYPE: amlno acld (D) TOPOLOGY: 11near (v) FRAGMENT TYPE: 1nternal (xl) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Phe Leu Trp Asp Glu Gly Phe His Gln Leu Val Ile Gln Arg .

(2) INFORMATION FOR SEQ ID NO:7:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pa1rs (B) TYPE: nucle1c ac1d (C) STRANDEDNESS: s1ngle (D) TOPOLOGY: l~near (v11) IMMEDIATE SOURCE:
(B) CLONE: ZC2575 . , . . . . ~ . . . . . . ....... .. .. .

W092/01795 ~ ~ PCI~/US91/0517 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE:
(B) CLONE: ZC2576 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (v11) IMMEDIATE SOURCE:
(B) CLONE: ZC2938 (X~) SEQUENCE DESCRIPTION: SEQ ID NO:9:

~2) INFORMATION FOR SEQ ID NO:10:
(1) SEQUENCE CHARACTERISTICS:
A LENGTH: 42 base pairs B TYPE: nuclelc acld (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (vll) IMMEDIATE SOURCE:
(B) CLONE: ZC3747 (xl) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GACAGAGCAC AGAATTCACT ACT~GAG m ~ IIIIII TT42 . .. . ., . ......... .. ~ . ~.; ~: , -. : .
.. . - . . . . . ..
,. . .. . .. . .

.

~092/0179S 208797l1 PCl/Us9l/osl77 .- 77 ;:

(2) INFORMATION FOR SEQ ID NO:11:
(1) sEquENcE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE:
(B) CLONE: ZC4135 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHAMCTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE:
(B) CLONE: ZC4136 - (x1) SEQUENCE DESCRIPTION: SEQ ID NO:12:

t2) INFORMATION FOR SEQ ID NO:13:
(l) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 14 base pairs (B) TYPE: nuclelc acid ~C STRANDEDNESS: stngle (D TOPOLOGY: l~near (vt~) IMMEDIATE SOURCE:
(B) CLONE: ZC4138 (x~) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CCNACN~SNC CNGG 14 WO 92/017 ~ ~ 78 PCI~/US91/0517 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (vli) IMMEDIATE SOURCE:
(B) CLONE: ZC4204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: llnear (v~i) IMMEDIATE SOURCE:
(B) CLONE: ZC4205 (xl) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
~1) SEQUENCE CHARACTERISTICS:
A LENGTH: 24 base palrs B TYPE: nucle1c acld C STRANDEDNESS: slngle D TOPOLOGY: llnear (vll) IMMEDIATE SOURCE:
(P) CLONE: ZC4217 (xl) SEQUENCE DESCRIPTION: SEQ ID NO:16:

.. . . ~ . . - - ~ . -,' .: : ':
. ~ : .............................. . . ..

.' ~ ' - . .: ' - . . '. '. . ' :. ' ' ` : : , :

o 92/o179s 208797 o ~ PCT/U591iO5177 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l1near (vii) IMMEDIATE SOURCE:
(B) CLONE: ZC4241 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
T6RTGRAANC CYTCRTCCCA . 20 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs -B) TYPE: nucleic acld C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GAATTC m A CGCTTTTGGC ACCMCCAGT CCAGGAGATA CCACTCCCM G MTTC 56 (2) INFORMATION FOR SEQ ID NO:19:
(1) SEQUENCE CHARACTERISTICS:
(A) EENGTH: 8 amlno acids B TYPE: am1no ac1d D TOPOLOGY: llnear (v) F MGMENT TYPE: 1nternal (xl) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Thr Pro Gln Pro Leu Leu Thr Gly (2) INFORMATION FOR SEQ ID NO:20:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 am1no ac~ds ~B) TYPE: am1no ac1d (D) TOPOLOGY: l1nQar (v) FRAGMENT TYPE: ~nternal ' .

... . . . . . .. . . . .

WO 92/017~ 9r~ PCI/US91/OSt7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Thr Leu Pro Ser Gly Leu Asp Asp Tyr Lys (2J INFORMATION FOR SEQ ID NO:21:
(1) SEQUENCE CHARACTERISTICS:
(A) LEN6TH: 29 amino acids (BJ TYPE: amino acid (D) TOPOLOGY: linear (v) FRAGMENT TYPE: internal (xl) SEQUENCE DESCRIPTION: SEQ Ib NO:21:
Leu Ala Glu Gln Leu Gly Glu Ala Glu Ala Ala Ala Glu Leu Gly Pro Leu Ala Ala Ser Leu Gly Ala Glu Glu Xaa Leu Asp Glu ' ';'' `'' ~'' ' ' ' ~ '`,''i ,;,` ',,,',," ,, ~ ,, " ,,

Claims (24)

Claims What is claimed is:

1. A protein composition having gamma-carboxylase activity enriched at least 20,000-fold as compared to liver microsomes.

2. The protein composition of claim 1 wherein said protein is selected from the group consisting of bovine, human and rat gamma-carboxylases.

3. The protein composition of claim 1 wherein said protein is liver gamma-carboxylase.

4. The protein composition of claim 1 wherein said protein is affixed to a solid support.

5. The protein composition of claim 1 wherein said activity is enriched 100,000 - fold as compared to liver microsomes.

6. A protein composition according to claim 1, wherein said protein comprises the amino acid sequence of Peptide 1, Peptide 2, Peptide 3, Peptide 4, Peptide 5, Peptide 6 or Peptide 7 of Table 9 (Sequence ID Nos. 3, 4, 5, 6, 19, 20 and 21).

7. An isolated DNA sequence encoding gamma-carboxylase.

8. A DNA sequence according to claim 7 wherein said sequence comprises the nucleotide sequence from nucleotide 19 to nucleotide 38 of Table 12 (Sequence ID Number 18).

9. A DNA sequence according to claim 7 wherein said gamma-carboxylase comprise the amino acid sequence of Peptide 1, Peptide 2, Peptide 3, Peptide 4, Peptide 5, Peptide 6 or Peptide 7 of table 9 (Sequence ID Nos. 3, 4, 5, 6, 19, 20 and 21).

10. A DNA sequence according to claim 7 wherein said gamma-carboxylase is selected from the group consisting of bovine, human and rat gamma-carboxylases.

11. A DNA sequence according to claim 7 wherein said gamma-carboxylase is liver gamma-carboxylase.

12. A cDNA sequence according to any of claims 7-11.

13. A cultured cell transfected or transformed to express a DNA sequence according to any one of claims 7-12.

14. A cultured cell according to claim 13 wherein said gamma-carboxylase comprises the amino acid sequence of Peptide 1, Peptide 2, Peptide 3, Peptide 4, Peptide 5, Peptide 6 or Peptide 7 of Table 9 (Sequence ID Nos 3, 4, 5, 6, 19, 20 and 21).

15. A cultured cell according to claim 13 wherein said cell is a mammalian cell.

16. A cultured cell according to claim 13 wherein said cell is further transfected or transformed to express a DNA sequence encoding a vitamin K-dependent protein.

17. A method of producing a vitamin K-dependent protein, comprising the steps of:
culturing a cell transfected or transformed to express a first DNA sequence encoding gamma-carboxylase and a second DNA sequence encoding a vitamin K-dependent protein;
and isolating the vitamin K-dependent protein encoded by said second DNA sequence.

18. A method according to claim 17 wherein said first DNA sequence comprises the nucleotide sequence from nucleotide 19 to nucleotide 38 of Table 12 (Sequence ID Number 18).

19 A method according to claim 17 wherein said gamma-carboxylase comprises the amino acid sequence of Peptide 1, Peptide 2 Peptide 3, Peptide 4, Peptide 5, Peptide 6 or Peptide 7 of Table 9 (Sequence ID Nos. 3, 4, 5, 6, 19, 20 and 21).

20. A method according to claim 17 wherein said cell is a cultured eukaryotic cell.

21. A method according to claim 17 wherein said cell is a cultured mammalian cell.

22. A method according to claim 17 wherein said cell is cultured in the presence of 0.1 - 10 µg/ml vitamin K.

23. A method according to claim 17 wherein said gamma-carboxylase is selected from the group consisting of human, bovine and rat gamma-carboxylases.

24. A method according to claim 17 wherein said gamma-carboxylase is liver gamma-carboxylase.