JPH01104175A - Recombinant purified protease nexin - Google Patents

Abstract

PURPOSE: To relate to a DNA coding mammalian protease nexin (PN-1) and efficiently recover PN-1 by preparing a recombinant DNA that does not include the DNA sequence relating the DNA.

CONSTITUTION: This is a recombinant DNA coding mammalian protease nexin and a recombinant DNA that does not include the DNA sequence relating to the DNA coding this mammalian protease nexin is transfected into host cells. This transfected host cells are cultured and the objective mammalian protease nexin 1 (PN-1) is recovered from the culture mixture. This PN-1 is free of impurities linking thereto, has a molecular weight of 43kD, when glycosylated and the N-terminal sequence represented by formula I.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the recombinant production of proteins that affect the cardiovascular system. In particular, one invention involves the cloning and expression of genes encoding various forms of protease nexin I (PN-1).

BACKGROUND OF THE INVENTION Connective tissue cells secrete protease inhibitors specific for serine proteases. Since serine proteases are involved in cell development and migration, control of the activity of these enzymes is necessary to control tissue reconstruction or destruction.
Control (1975), Re1ch+E
,l etal, eds,,Cold Sprin
The inhibitor, which is specific for the protease nexin, irreversibly binds the serine protease at its catalytic site (37-45) and binds the bound protease by receptor-mediated endocytosis or sosomal degradation. Cleavage occurs (Low
, D, A, et al+ Proc Natl 8ca Sci (USA) (1981)
E8: 2340-2344; Barker
.

J, B, et al, in The Rece
tors 3 (1985) + Conn.

P.M., ed., Academic Press,
(in press).

Three protease nexins have been identified.

Protease nexin I (PN-1) is purified from serum-free medium prepared with human foreskin cells (Sc
ott, R.W., et al., J. Biol.
Chem (1983) 58:10439-1044
4). It is a 43 kd glycoprotein that is released by fibroblasts, tubules, cardiomyocytes, and vascular smooth muscle cells. Its release is stimulated by phorbol esters and by mitogens, with the release of plasminogen activators (B
aton, D, L, + etal, J Ce1l
Biol (1983) 123: 128),
Natural PN-I contains approximately 40% carbohydrates, approximately 6%
It is a protein with 0 amino acids. This is obvious since natural PN-1 exists only at trace levels in serum.

1 functions at or near the surface of stromal cells. P
N-1 is urokinase proenzyme, plasmin.

Inhibits all known activators of trypsin, thrombin and factor Xa (Eaton, D.L.,
etal, JOI Chem (198-I)
259: 6241). It also inhibits one tissue plasminogen activator and urokinase.

A protein called neurite promoting factor (NPF)
It has been reported that it can be isolated from glioma cells, has a molecular weight of 43 kd, and inhibits proteolysis catalyzed by urokinase or plasminogen activator (Guenther).

J., et al. [EMBOJournal
(1985) 4: 1963-1966).

The protein was first reported to induce neurite derivatives in neuroblastoma (Barde, Y.A.,
etat, Nature (1978) 274:
81B). The amino acid sequence of this protein (but not the sequence of the cDNA encoding the protein)
Gloor, S., et al.

It is disclosed in Chodan (1986) 47: 687-693. Any relationship between this DN8 and the facts reported here is uncertain. This is because the restriction map for the Dahlia cDNA is clearly different from the restriction enzyme map for the cDNA disclosed herein. This NPF protein is a 379 amino acid sequence generated by a signal following an 18 amino acid Met. At amino acid position 241 of the mature protein, the PN-
It is different from 1β.

The practical amount of purified PN4 required is several times higher.

First, PN-T has obvious uses as a drug for conditions characterized by excessive amounts of urokinase and tissue plasminogen activator, or as an antidote to excessive doses of these enzymes as reagents in solutions for blood clotting. There is. Indicators clearly susceptible to PN-r treatment include the autoimmune disease Penfigus (p.
enphigus), and dryness, which is thought to be due to overproduction of plasminogen activators. Second, the role of PN-I in regulating various developmental stages of tissue formation and remodeling is relatively complex;
It would be desirable to be able to use a model system to identify in more detail the role played by 'N-I. This can only be done effectively if practical quantification is available. Finally, PN-1 is useful as an analytical reagent in immunological assays for the analysis of immune levels in serum or other tissues, or other biological assays.

Examples of conditions where further investigation of the role of PN-1 is desirable are tumor metastasis, wound healing, and ulcers.

In III tumor metastasis, malignant cells must penetrate the extracellular matrix lined with vascular smooth muscle cells. The process is mediated by two secreted plasminogen activators. Jones.

P, 8, et al, Cancer
Res (1980) 40: 3222
In a model system (which is an in vitro system based on extracellular matrix invasion by human fibroblasts), 0.1μ of PN-1 was shown to almost completely inhibit that invasion. (Ilergman, B.L.
+et at, Proc Natl 8c
ad Sci USA (1986))
, the proteolytic activity of thrombin, a fibroblast mitogen important in wound healing, is only effective when thrombin is added to the culture at a concentration greater than or equal to the concentration of secreted PN-I (Barker + J. B,+
et al, J Cetan” Tatsudan o1 (1982)
■2: 291: Low.

D,Δ,, et al., Nature (198
2) 298:2476), it has been suggested that PN-I has anti-inflammatory functions.

The secretion of PN-1 by lamellar fibroblasts is significantly increased when the cells are treated with interleukin-1 (Krane, S., + 8rth Rheum).
worship → 27:52-I). PN-1 may also have neurological functions. Similar protease inhibitors mentioned above stimulate neurite outgrowth (Mo
nard etat, Pro Brain Res
(1983) 58:359).

In any of the foregoing, elucidation of the exact function of PN-1 would be greatly facilitated if the required amounts of pure material were available. Such amounts are also necessary for use in PN-I as a drug and in diagnosis and analysis. The present invention provides a solution to the problem of obtaining sufficient amounts of PN-I and a mechanism for modifying the structure of l'N-r to make that solution more effective.

SUMMARY OF THE INVENTION The present invention provides highly purified PN-1 proteins, including recombinant forms, DNA encoding sequences, expression systems, and methods for producing recombinant mammalian PN-I.

Two representative forms of r'N-1 are disclosed.

The recombinant DNA of the present invention is a recombinant DNA encoding protease nexin I (PN-1),
Original DNA associated with the DNA encoding said PN-I
Contains no arrays.

The method for preparing PN-1 of the present invention involves the use of a recombinant DNA encoding the breast cervical protease nexin I (PN-1), in which the original DNA sequence related to the DNA encoding the PN-1 is culturing recombinant host cells transfected with free recombinant DNA and recovering PN-I from the culture.

PN-1 of the present invention is prepared by the above method.

The protease nexin of the present invention is generally free of bound impurities, has a molecular weight of 43 kd when glycosylated, and has an N-terminal sequence: 5er-11is
-Phe-Asn -Pro-Leu-5er-Le
u-G 1u-G lu-Leu-Gly-5er-
85n-Thr-Gly-11e-Gin-Val-P
he-8sn-Gln-11e-Val-Lys-3e
r-Arg-Pro-old 5-A5p-Asn-11e-
Val-11e and contains, by weight, about 2.3% amino sugars, about 1.1% neutral sugars, and about 3.0% sialic acid.

The method for purifying natural PN-1 of the present invention involves subjecting a medium containing PN-1 to affinity chromatography using heparin bound to a solid support.

Then apply gel filtration chromatography.

includes.

The antibodies of the present invention are immunoreactive with PN-1 prepared according to the above-described method for preparing PN-I or the above-described method for purifying natural PN-1, and are formed in response to immunization of a mammal.

By using these materials and methods.

The desired amount of this PN-1 protein can be produced either in IJiylated form or in non-glycosylated form. This amount depends on the expression system used. This gene can be modified, if desired, to change the precise amino acid sequence in order to enhance the desired properties of the protein.

This is entirely possible through obtaining the gene encoding human PN-I. This gene is useful both directly to produce the corresponding PN-1 and as a probe to obtain cDNA sequences encoding these genes in one species.

The human genes encoding two closely related PN-1 proteins are illustrated below. Searching for DNA encoding PN-1 in other species is also desirable. DNA encoding murine proteins is particularly desirable. Many model systems for providing a detailed explanation of the role of such factors are well based on murine cell 9 tissues, or whole tissues.

Therefore, in one aspect, the present invention relates to DNA sequences encoding lactiferous cervix PN-1, and derivatives thereof. Derivatives thereof can be expressed to obtain proteins with PN-I activity. In other aspects, the present invention provides two aspects.

Regarding cells transformed with these DNA sequences, and regarding the I'N-1 protein produced by these cells. Furthermore, the present invention relates to a purified protein having the N-terminal sequence of the native protein disclosed herein, and relates to antibodies prepared by administration of the recombinant or purified native protein, and which can probe the PN-1 cDNA. Regarding DNA probes.

Zero B to A,n As used herein, "protease nexin I
J (PN-1) indicates a protein active in a standard diagnostic assay for r'N-1. This assay is based on four criteria: (1) the protein forms a complex with thrombin; (2) this complex is facilitated by heparin; (3) the protein forms a complex with thrombin; , binds to cells of its origin 2, such as fibroblasts as exemplified below; and (-I) heparin must inhibit this binding.

PN-1 is the other two protease nexin factors.

PN-II and PN-11 (Knauer+ D, J
,, et al., JBiol Chem (19
82) 257: 15098-1510-I). These are also major thrombin inhibitors, but they bind less strongly to this protease.

"Control sequence" refers to a DN^ sequence that, when successfully linked to a desired coding sequence, is capable of effecting its expression in a host compatible with such sequence. Such control sequences include at least promoters in prokaryotic and eukaryotic hosts.

Preferably, a transcription termination signal is included. Additional factors necessary or helpful in bringing about expression may also be identified. As used here.

"Control sequences" simply refers to all DNA sequences necessary to effect expression in the particular host used.

"Cell" or "cell culture" or "recombinant host cell" or "host cell" are often used interchangeably, as is clear from the context.

These terms include the cell of immediate interest and, of course, its progeny. Not all progeny are exactly the same as the parent cell, due to encountering mutations or changes in the environment.

it is obvious. However, such altered progeny are included within these terms so long as the progeny retain characteristics associated with those conferred on the naturally transformed cell. In this case, for example, 9.

Such a property would be the ability to generate two recombinant PN-1s.

"Purified" or "pure" refers to a material free of the substances normally associated with it when found in its native state. Therefore, "pure"r'N-r-encoding DNA is defined as one found isolated from its native environment and free of other proteins normally produced by cells that naturally produce PN4. Refers to DNA that is not attached to coded DNA 8. “Pure JPN-1 is defined as pure JPN-1 in human or other mammalian tissue.

PN-I free of its original environment and normally associated materials
refers to Of course, "pure JPN4 may contain glycosidic residues or 2 covalently linked materials, such as materials incorporated into therapeutic formulations."
"Pure" refers to the substance being referred to.

It simply refers to the state in which it can be or has been isolated from its native environment and the materials normally associated with that environment.

Although of course purified and free of the materials normally associated therewith, the DNA claimed herein so as to encode PN-1 includes, for example, the 5′ and/or 3′ ends of the following coding sequence: Additional sequences may be included. This coding sequence may result from reverse transcription of the non-coding portion of Messenger RNA, when this DN8 is derived from a cDN library, or may include not only the sequence encoding the mature protein (reverse transcription for the signal sequence). will include products.

As stated in DNA sequences, "degenerate with" refers to a nucleotide sequence that encodes the same amino acid sequence as the referenced amino acid sequence.

"Operably linked" refers to a juxtaposition in which the components are arranged so as to accomplish their normal functions. Therefore, a control sequence or promoter operably linked to a coding sequence is capable of effecting expression of the coding sequence.

B. From a serum-free medium prepared with human foreskin fibroblasts in microcarrier culture described by Nidonoya, by heparin-agarose affinity chromatography followed by gel filtration chromatography.

PN-I was purified to homogeneity. This means that 5
cott.

R,W, et al., J Biol Chem (19
85) 260:7029-7034, the contents of which are shown here. Of course, other chromatographic supports including heparin for affinity binding can also be used. This purified protein is 42-43 kd based on sedimentation equilibrium analysis or 47 kd as calculated by gel filtration chromatography.
M, is shown. This purified material exhibits the properties of PN4 when included in a prepared medium. For this characteristic.

Formation of sodium dodecyl sulfate stable complexes with thrombin, urakinase and plasmin; inhibition of protease activity: inhibition of thrombin amplified by heparin; and cellular binding of protease-PN complexes in heparin-induced reactions. It will be done. This purified natural protein contains 2.3% amino F, 1.1% neutral sugars, and 3%
．． Contained approximately 6% carbohydrates with 0% sialic acid. The N-terminal amino acid sequence of the isolated and purified protease nexin is that the first 34 amino acids are 5er-
His-Phe-Asn-Pro-Leu-Ser-L
eu-Glu-Glu-Leu-Gly-5er-85
n-Thr-G ly-11e-G ln-Va 1
-Phe-Asn-G1 n-I 1 e-Va l-
Lys-Ser-Arg-r'ro-His-Asp-
It was determined to be Asn-11e-Val-11c.

cDNA encoding the complete human PN-1 protein.

Obtained from the foreskin fibroblast DN^ library.

To search for this clone, we used a probe based on the amino acid sequence determined from the natural protein.

This cloned cDN^ can be obtained by cutting the coding sequence from a carrier vector and placing it into an appropriate expression system in recombinant cells of both prokaryotic and eukaryotic organisms, as described above. 9 expression was obtained by ligating. The mature protein starting with the N-terminal amino acid of Net (which may or may not be processed depending on the choice of the expression system) can be produced as a fusion protein for any additional N-terminal or C-terminal sequences desired, or The signal sequence itself or a heterologous sequence (which may be supplied by one known signal sequence, e.g. associated with a bacterial β-lactamase gene or associated with a secreted human gene such as insulin, growth hormone). PN-1 can be produced directly since it can be secreted as a mature protein. Site-directed mutagenesis provides a means of providing appropriate restriction sites at appropriate positions with respect to the desired coding sequence.

This code sequence is well known and hence.

It can be supplied with a suitable site for linking to a signal sequence or fusion sequence or in an expression vector.

If a bacterial host is chosen, the protein will be produced in an unglycosylated form. if,
If the PN-1 were produced as a "mature" protein within the cell, the N-terminal methionine would be only partially processed or not processed at all. Therefore, the produced protein has an N-terminal M
et. Modifications of proteins produced either intracellularly or secreted by such bacterial hosts may include the provision of polysaccharide substrates, disulfide bond cleavage and regeneration techniques, or other translational techniques. This can be done by regeneration using subsequent ex vivo processing techniques. If the protein is produced in the cervix or other eukaryotic host, the cellular glycosylated form of the protein is produced.

The recombinant cells are cultured under conditions appropriate to the host in question. The protein is then recovered from the cell lysate or culture medium as determined by the two expression modes.

Purification of this protein was carried out by Scott.

R, W, et al+ J Biol Chem
(supra) or by other methods known in the art.

This purified protein is formulated according to its application. For pharmaceutical applications, the protein is formulated into compositions using standard excipients. This is well known to those skilled in the art.

Example, t ha, le' top ■shi's Pha 工hitan ding 1 bei dan Is drama to play 1 atetest edition, M
ack Publishing Company+
Easton+PA. If used in a diagnostic or immunoassay, the protein can be labeled using a radioactive marker (eg, a fluorescent marker).

This protein is used to obtain antibody preparations.

Prepared for injection with appropriate adjuvants.

Methods for modifying the recombinant proteins of the invention according to the desired use will be generally apparent to those skilled in the art.

Two forms of PN-I, PN4α and PN-1β, are illustrated below. They are highly homologous, containing 378 and 379 amino acids in the adult μ) protein, respectively, and differing only at 310 positions (where Arg of PN-1α is replaced by Thr-Gly of PN-Iβ). . Both have a 19 amino acid signal starting with Met. This N-terminal position was deduced from the sequenced native protein, and it appears to be fairly certain that it is correct. However, different processing sites (1
There is a small possibility that one or more of the following may also be useful.

C, JIL; Most of the techniques used to transform cells, construct vectors, extract messenger RNA, type cDNA libraries, etc. are widely known in the art. Most practitioners are familiar with standard materials for specific conditions and procedures.

However, for convenience, the following section may serve as a guideline.

C11, the main and 'configurations of both prokaryotic and eukaryotic systems are two P
It can be used to express sequences encoding N-1. Of course, prokaryotic hosts are the most convenient cloning vehicle. The most commonly used prokaryotic cells are
It is represented by various strains of E. coli. However, other microbial strains can also be used. A plasmid vector containing a replication site 2 selection marker and control sequences derived from a species compatible with the host is used. For example, E. uni is typically derived from pBR322, a plasmid obtained by Bolivar et al. from the species E. uni.
ene (1977) 2:95)). pBR322 contains genes for ampicillin and tetracycline resistance. Therefore, this means that when constructing the desired vector,
This results in a variety of selectable markers that can be retained or destroyed. Commonly used prokaryotic control sequences, including the promoter for transcription initiation, optionally with operator and ribosome binding site sequences;
The beta-lactamase (penicillinase) and lactose (Iac) promoter system (Chang, at al+ Nature (197
7) 198: 1056), tryptophan (
trp) promoter system (Goeddel, et a
l, Nucleic Ac1ds Re5 (1980
) 8:4057), and the PL promoter and N gene ribosome binding site from lambda (Shim
Atake.

et al., Nature (1981) 292
: 128).

In addition to bacteria, also eukaryotic microorganisms such as yeast.

Can be used as a host. Laboratory Strains Saccharomyces cerevisiae
visiae) (baker's yeast) is most commonly used. However, many other strains and species are also commonly available. Examples of vectors include, for example, Broach, J.;

Ro's 2μ replication origin (Meth Enz (198
3) 101:307), or yeast-compatible replication origins (e.g. Stinchcomb, e.g.
tal, Nature (1979) 282
:39, Tschumper, G., et al.
, Gene (1980) 10:157 and C1arke, L. et al., Meth E.
nz (1983) 10:300) can be used.

The control sequences for the yeast vector include promoters for the synthesis of 9 glycolytic enzymes (tress, et al.
J Adv and Mutual” Plate (1968) 7: 149+
Holland, et al.

Biochemistr (1978) 17:
4900) is included. Additional promoters known in the art include:

3-phosphoglycerate kinase promoter (t(
itzeman, et at, J Biol C
hem (1980) 255:2073). Other promoters (these are

have the additional advantage that transcription is regulated by growth conditions and/or genetic background).

Alcohol dehydrogenase2. isocytochrome C2
Located in the promoter region for acid phosphatase, a degrading enzyme associated with nitrogen metabolism, the alpha factor system, and enzymes involved in maltose and galactose assimilation. It is also believed that the 3' end of the coding sequence is desirable as a terminator sequence. Such terminators are present in yeast-derived genes. It is found in the 3'' untranslated region following this coding sequence.

Of course, it is also possible to express genes encoding polypeptides (which are derived from tissues of multicellular organisms) in cultures of eukaryotic host cells.

See, eg, Haxel et al., US Pat. No. 4,339,216. These systems have the additional advantage of being able to splice introns and therefore can be used directly to express genomic fragments. Useful host cell lines include VHRO cells and II e La cells, and Chinese hamster ovary (CIIO) II follicles. For expression vectors for such cells.

Promoter and control sequences compatible with mammalian cells, Simian Virus 40
40+SV 40) commonly used early and late promoters (Fiers, et al.
at, Nature (1978) 273: 1
13), or other viral promoters. to other viral promoters.

For example, polyomavirus, adenovirus 2 (Ad
enovirus 2) + Viruses derived from bovine papilloma virus or avian sarcoma virus. Regulatable promoter hMTII (Karin, Ll
et al, Nature (1982) 2 Atsushi: 7
97-802) may also be used. General aspects for the transformation of mammalian cell host systems are described in Axel
(supra).

It is also now clear that the "enhancer" region is important for optimal expression. These regions are generally sequences found upstream or downstream of the promoter in the non-coding DNA jI region. Yes, if needed, the origin of replication can be obtained from the viral source.

However, this chromosomal integration is a mechanism common to DNA replication in eukaryotes.

C020 dish mutual biography■ Transformation depends on the host cell used.

This is done using standard methods appropriate for such cells. Calcium treatment method using calcium chloride as described by Cohen, S.N.

S, N, Proc Natl Acad Sci
(USA) (1972) 69: 2110) or M31iatis+ et al., Mo1ecula
r C1onin: ALaborator M
annual (19B2) Co1d Spring
HarborPress+ p, 254 and Hanaha
The RbC1z method described in n+o, J Mol Biol (1983) Dishes: 557-580 can be used in prokaryotic cells or other cells that contain a rigid cell wall barrier. Mammalian cells that do not have such cell walls include Graham and Van der Eb.

(1978) 52: 546 or, if necessary, Wigler, M.
, et al., Chodan (1979) 16: 777-7
85 may be used. Transformation into yeast was performed using the Beggs+ J-D-+ method (Natur
e (1978) 275: 104-109) or the former nnen.

A., et al.'s method (Proc Natl Acad Sc
i (USA) (1978) 751929).

Construction of suitable vectors containing the desired coding and control sequences uses standard ligation and restriction methods well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved and tailored.

Recombined into desired shape.

The DNA sequences that make up this vector are available from a number of sources. The backbone vector and control system are constructed using a suitable “
Host°°Vector 2- Found in boat fishing. A typical sequence is above 0.1. states. For suitable coding sequences, the initial construction can be, and usually is, retrieving the appropriate sequences from a cDNA library or a genomic DNA library. However, once this sequence appears, it is possible to synthesize the entire gene in vitro starting from this individual nucleotide derivative. For example, the entire gene sequence for a gene of considerable length between 500 and 1000 bp can be obtained by synthesizing nine individual overlapping complementary oligonucleotides;
and single-stranded standard non-overlapping fill-in using DNA polymerase in the presence of deoxyribonucleotide triphosphates. This method has been successfully used to construct several genes of known sequence. For example, Edge, M, D,,N
ature (1981) 292 Near 56; N
ambajr, K, P, et al., 5science (
198-I) 223:1299; Jay, I
Ernest, J Biol CheIIl (198
-I) 259:6311.

Synthetic oligonucleotides are described by Edge et al., Natu
re (supra), Duckwor th et al., Nucl
eic Ac1ds Res (1981) β-: 1
The phosphotriester method as described in 691.

Or, Beaucage, S, L, and Ca
ruthers+M.

H., Tet Letts (1981) 22:18
59 and MatjeuCC1+M, D, and Ca
ruthers M, H,, J Am CheIIIS
oc (1981) 103:3185, by any of the phosphoramidite methods. This oligonucleotide can be prepared using commercially available automated oligonucleotide synthesizers. Kinaseation of single strands prior to annealing or for labeling was performed using 50mM) Lys (pH 7,6), 10t
anHgc1z+ 5taM dithiothreitol,
1-2mM ATP.

1.7 pmol 1” P-ATP (2,9 mC1/m
mole), 0.1mM spermidine, 0.1mM
In the presence of EDTA, an excess amount (e.g. lnm
This is achieved using approximately 10 units of polynucleotide kinase (polynucleotide kinase) per ol substrate.

Once the desired vector components are available.

The components can be cleaved and ligated using standard restriction and ligation procedures.

Site-specific NA cleavage is accomplished by treatment with appropriate restriction enzymes under conditions generally known in the art and as specified by the manufacturer of the commercially available restriction enzymes. achieved. For example, New Eng.
land Biolabs, Product Cat
See alog. Generally, approximately 1 μg of plasmid or DNA sequence is cleaved with 1 unit of enzyme in approximately 20 μl of buffer; the examples herein typically ensure complete degradation of this DNA substrate. Therefore, an excess of restriction enzyme is used. Incubation times of about 1-2 hours at about 37°C can be used. However, the variation can be ignored. After each incubation, proteins are removed by extraction with phenol/chloroform. Next.

The protein can be subjected to ether extraction and nucleic acids removed from the aqueous fraction by precipitation with ethanol. If desired, size sorting of cleaved fragments can be accomplished by polyacrylamide gel or agarose gel electrophoresis using standard methods. A general description of size sorting can be found in Methods in
Enz molo (1980) 65:499
-560.

The fragments cleaved with restriction enzymes were cleaved with 50mM)
7.6), 50mM NaCl, 6mM MgCl
g, 6mM DTT and 0.1-1. OmMd
in the presence of four deoxynucleotide triphosphates (dNTP') using an incubation time of approximately 15-25 minutes at 20-25°C in NTP.

Blunt ends can be made by treatment with the large fragment of E. eufi DNA polymerase (Klenow). Even if four dNTPs are present, the Klenow fragment fills the 5''-end strand overhang but shaves off the overhanging 3'-strand, if desired, within the limits dictated by the nature of this overhang. Bi-selective remediation can be achieved by providing only one of the dNTPs or selected dNTPs. After Klenow treatment, this mixture is extracted with phenol/chloroform and precipitated with ethanol.S1
By treatment with nuclease or BAL-31 under appropriate conditions.

Any backbone portion is hydrolyzed.

15-50μl under the following standard conditions and temperature:
The connection is made with a capacity of 1. For example, 20 m
M Tris-11cI (pH 7,5), 10mM
MgCh, 10mM DTT, 33μg/InIB
SA, 10-50mM NaCl, and 40μM
ATP, 0.01-0.02 (Weiss) units T, DNA ligase at O″C (“viscous ends°”
(linked) or 1mM ATP, 0.3-0.6 (
Weiss) Unit T, DNA ligase at 14°C
(“blunt end” ligation). Intermolecular “
The viscous end” linkage is usually.

33-100μg lrd total DNA concentration (5-1001M
(all terminal concentrations). Intermolecular blunt end ligation is 1
A total terminal concentration of μN is achieved.

In vector construction using “vector fragments”°,
To remove the 5' phosphate and prevent self-ligation of the vector, this vector fragment is usually 9.

Treated with bacterial alkaline phosphatase (13AP) or calf intestinal alkaline phosphatase (CIP). Approximately 1 unit of BAP or CI per μg of vector
(approximately 10mM) using P.) Lis-IC1, 1mM ED
Decomposition is carried out in TA at pH 8, 60°C for about 1 hour. To recover the nucleic acid fragments, the preparation is extracted with phenol/chloroform and precipitated with ethanol. On the other hand, religation may be prevented in double-cleaved vectors by additional restriction enzyme digestion and separation of unnecessary fragments.

For portions of vectors derived from cDNA or genomic DNA that require sequence modification, site-specific primers can be used to direct mutations (Z o 11 e r
+M, J, and Sm1th, M, Nucle
ic Ac1ds Res (1982) 10:6
4B? -6500 and Adelman, J. P. et al. (1983) 2: 183-193). This is because, except for limited mismatches and expression of desired mutations,
2 is performed using primer synthesis oligonucleotides complementary to the single-stranded phage 11iNA that can be mutated. Briefly, this synthetic oligonucleotide is used as a primer to perform the synthesis of a strand complementary to the phage. The resulting partially or fully double-stranded DN
A is transformed into a phage-supporting host bacterium. This transformed bacterial culture is placed on agar;
A plaque is formed from a single cell containing this phage.

Theoretically, 50% of the new plaques will contain phage with the variant as a single strand: 50% will have the original sequence. After hybridization with the kinased synthetic primers, the resulting plaques are washed at the following wash temperatures. This wash temperature allows for binding with a precise match, but is still high enough that any mismatch with the original strand will interfere with binding. Plaques that hybridized with this probe were then selected and cultured 9 times.

The DNA is then recovered.

C14, Construction ■ To confirm the construction of the plate vector or for other sequencing purposes, DNA is first amplified and isolated. This isolated DNA is analyzed with restriction enzymes and/or sequenced by the dideoxynucleotide method described below. This method is described by Messing et al.
c Ac1ds Res (1981) 9:309
Further described by Sanger, F. et al., P.
roc Natl AcadSci (USA) (1
977) 74: 5463 method or Maxa
m et al., Methods in Enz mono
(1980) 65:499.

(Left below) (Examples) The following examples are for illustrative purposes only and are not intended to limit the present invention. In certain aspects, this embodiment provides the ability to
The method for searching for the A sequence will be described in detail.

However, this process need not be repeated.

The complete NA sequences for the coding regions of the inserts into the PN-Iα and PN-Iβ clones are shown in FIGS. 3 and 4. Standard synthetic methods can be used to construct either these exact sequences or equivalent, degenerate sequences using alternating codons. The synthesis of DNA sequences of this length is now almost routine in technology. For example, Edge et al., Nature
(1981) 292:756. Additionally, on June 4, 1986, Applicants deposited the PN-18 clone of the λg tlO phage with ATCC No. 40238 at the American Type Culture Collection, Rockwill, Maryland. It contains the appropriate coding sequence for PN-1α. This coding sequence can be manipulated starting from biological materials. This PN-Iβ sequence can be easily obtained using single site-directed mutagenesis.

PN-1 is 5cott, R, W, + et al., J
It was prepared from culture medium under serum-free conditions as described in Biol Chem (1985) (supra). Briefly, the collected medium was filtered through a 45μ Millipore filter.

The protein was then concentrated by Amicon hollow fiber filtration. Concentrated medium from a single 312 microparticle carrier culture was passed through a 0.7 x 30 cm heparin-agarose column pre-equilibrated in 0.3 M sodium chloride in phosphate buffer to remove phosphate. Elution was with 1.0M sodium chloride in buffer. Here, both phosphate buffers contained 0.02% sodium azide. Eluates were obtained in 0.55-0.6M sodium chloride. Both fractions containing PN-I were concentrated by dialysis, and then added to a column buffer containing 0.5 M sodium chloride at 1 to 2 mg PN/d.
-1 was dialyzed and subjected to gel filtration chromatography. Then, divide these two parts into 1 x 60 cm of Bt
o-Gel P-100 (Bio-Rad 100-2
00 mesh) column and eluted with column buffer. Both parts of this peak were concentrated to 1 m2,
and stored at -80°C. The amino acid sequence and sugar components were determined on this purified material.

The N-terminal amino acid sequence for the first 34 amino acids was determined using the results described above.

Human foreskin fibroblasts were cultured in 30X 150 mm flasks. 5cott, R, W, J Biol C.
Hem (1983) 258:10439.

Approximately 1-2X101' cells were obtained. Twenty-four hours before harvest, cells were added again to stimulate PN-I mRNA production. Harvest the cells in water cooling.

It was then washed twice with phosphate buffered saline (PBS). The cell pellet was collected and treated with 20mM vanadyl complex and 0.2% Non1det P-
Homogenize in buffer containing 40% detergent.

Centrifugation was performed at 14,000 rpm for 10 minutes. RNA,
It was prepared from this supernatant by phenol/chloroform extraction. The resulting total 12NA was used to obtain mRNA.

Subjected to oligo-dT affinity chromatography.

This isolated metsenger was gel resolved and the first
Probing was performed with a mixture of 24 types of 14-mers having the sequences shown in Figure a. This corresponds to 20 of the determined N-terminal sequence.
Represents degenerate, reverse complementary DNA encoding ~24 amino acids. That probe is.

m of approximately 2500-2700 nucleotides in length
Hybridizes to RNA.

The total mRNA preparation was then cD in λgtlo.
It was used as a template to prepare an NA library.

This means that l1uynh+ T, V-+ et al., DN
A C1onin ni ues war pract
ical A reach (198-I) + Gl
It is well described in over+D, ed, IRL, Oxford.

However, Gubler, V. + et al., Gene
(1983) 25:263-269. The obtained cDNA was cut and analyzed with EcoRI, and λ
The EcoR1 site of gtlO was inserted as described by Ifuynh et al. (supra). Millions of phage plaques were obtained and three filter lifts were prepared. Place the plaque on the 5゛ end of the 14 bodies mentioned above at 32P.
- using a mixture labeled with ATP.

It was screened twice under medium stringency conditions (6X5SC, 30°C). This resulted in a large number of positive clones.

Of the 60 clones selected and cultured.

48 clone also. Hybridization was performed under equally stringent conditions to the sequence of the 36 nucleotide oligomer shown in Figure 1. The sequence of this oligomer was the consensus sequence shown based on amino acids 14-25, ie the sequence determined in the original protein.

Fifteen of these 48 clones were close to the size expected from an mRNA long enough to encode the complete sequence. These were divided into two groups (2000bp and 3000bp) based on size.

The 3000 bp clone was named PN-18 and the 2000 bp clone was named PN-33.

These clones have similar coding sequences.

The clones were named PN-5, PN-8 and PN-11.

It has the same coding sequence as PN-18. The PN-9 clone has a slightly different coding sequence contained in PN-33. Due to the restriction map, these clones.

These maps are shown in Figure 2.

r'N-18 was restricted with 5au3AI and M13 (
7) Cloned into Bamll1 site. correct cd
To confirm the presence of NA, the resulting M13 subclone was screened with a 14-mer mixture. Also,
The 55 bp fragment was sequenced and found to contain the correct sequence encoding amino acids 17-34 of the original protein.

13 of the 15 clones above are 14ff
A 75obp EcoR hybridized to the single probe and likely encodes the 5' portion of this gene.
Contains the I-BglI fragment. This segment was sequenced. This determined sequence is the upper 55 bp of 5au3
It contains the codons of the old fragment and the N-terminal sequence determined above. Furthermore, it extended to the rear of the ATG. Contains codons for a 19 amino acid signal sequence.

The complete coding sequence for PN-1α is PN-18
was included in. The deduced amino acid sequence is shown in FIG. The first 19 encoded amino acids are the predicted signal sequence, and the first 34 amino acids of the predicted mature protein, starting with serine at position 20, perfectly match the N-terminus of the native protein. PN-1
8 has been deposited with the American Type Culture Collection and has the accession number ATCC 40238.

Identification of PN-18 using the PN-1 product was demonstrated by Northern blot, as shown in FIG.

A 55 bp 5au3AI fragment obtained from PN4B was labeled and used to sequence mRNA from human foreskin fibroblasts and several other cell lines incapable of producing PN-1. This probe is PN-
It hybridizes only to approximately 2.8 kb mRNA from cells that produce 1.

Also a PN-33 clone containing DNA encoding PN-Iβ. The coding region has been completely sequenced and the results, along with the deduced amino acid sequence, have been
It is shown in Figure 4. For PN-1α, the first 19 encoded amino acids are the putative signal sequence and the first 34 encoded amino acids of the mature protein starting with serine at position 20.
The amino acids correspond exactly to the N-terminus of the two native proteins. The sequence encoding PN-Iβ is the 31st part of the mature protein.
Inclusion of an additional codon corresponding to glycine after the codon at position 0 and substitution of threonine for the arginine residue at that position.

The sequence is almost identical to that of PN-Iα except for .

Therefore, mature PN-1α contains 378 amino acids; mature PN-Iβ contains 379 amino acids.

PN-18 and PN- shown in FIGS. 3 and 4
The complete sequence portion of the 33 cDNA is identical except for the codons corresponding to the amino acid sequence changes above. From this, the PN-I gene was extracted from the human genome library.

In order to recover part of the gene, PN-18 was converted into cDNA.
By using it as an A probe, it was demonstrated that there are differences in splicing mRNA. Sequencing of the region with the amino acid difference (which exists between two adjacent exons and continues into the intron separating the exons) reveals a 3 bp difference at this splice site. It was confirmed that there is. These results are shown in FIG. PN-1α
The A at the beginning of the codon at position 310 for arginine in PN-1β is spliced 3 bp further downstream into the primary exon than the corresponding A in the codon at position 310 of PN-1β.

The aforementioned Arg of PN-1α and Thr of PN-rβ
-Probes designed across this coding region containing Gly detected the relative amounts of mRNA in preparations of two foreskin fibroblasts and the cD in the corresponding cDNA library.
was used to calculate the relative amount of NA. Based on the results of these Nordan plot methods and Sachin plot methods,
It was shown that the two proteins were formed in approximately equal amounts, respectively. Therefore, it is unclear which type is "normal" or dominant.

The genomic DNA encoding this PN-I protein is.

Further studies were carried out as follows: Southern hybridization analytes to human DN isolated from SK hepatoma cells were restriction analyzed with Bgl II, l1indI [[, or EcoRI. Duplex sample sets were separated on agarose gels and electroplotted onto membrane filters. The resulting plots were hybridized to probes labeled with 22p: either the 2 kb PN insert (PN-33) or the 650 bp Bgl I fragment from the 5° end.

Based on the hybridization pattern obtained with the complete PN-1 probe, this PN gene.

It was estimated to be at least 20kb. As expected, this 5' probe hybridized to a subset of specific fragments of PN-I, and only one band was observed for each enzymatic degradation. These results are consistent with a single PN-I gene.

A cDN^ library prepared in λgtlo from mRNA extracted from mouse fibroblasts was prepared using a method similar to that described in Example 2, and a PN-18-derived 55
A 5 bp au3AI fragment is used for screening.

- The phages that hybridize to this probe are then removed and the desired mouse PN-I cDNA
(cloning).

The coding sequence derived from the EcoRI cassette of PN-33 or PN-18 was filed in an unnumbered application filed February 4, 1987 (Docket No. INVO).
The amplifiable host expression vector psT described in OIIP)
II-MDI, respectively. This vector is
It was entrusted to the same trustee, the contents of which are presented here and reproduced in part. This amplifiable IIPR sequence is under the control of a natural promoter and follows the termination sequence of the hepatitis surface antigen gene. In the completed vector, two I'N-I sequences are under the control of the SV40 early promoter and also follow the termination sequence of the hepatitis surface antigen.

DNA encoding PN-Iα and PN-1β,
Instead of the tPA expression cassette, the host vector pS
Inserted into TII-MDIF using the following three-way linkage. In this concatenation, 5” of this code sequence
The termini and 3” termini and appropriate portions of the expression system were inserted as 9 separate fragments. These vectors, psN
Substantially equivalent ligations were performed for the construction of cXH-dhfr and pSNβ1+-dhfr. However, this linkage was achieved with appropriate initiators.

psNall and pNex a-HBV3'R
For the construction of 1.

Following the hepatitis termination sequence, the vector containing the C-terminal coding portion of the gene was digested with BglII and EcoRI.

The 3° end of this expression system was isolated. The vector pSV-Nex α containing the 5' end of this expression system was digested with BglII and 5alI, and the portion of the vector containing the nexin 5' end was isolated. These fragments were ligated in three directions.

Linked to this 〇〇FR selectable marker, EcoR
psTII~MDH was excised by I/Sal1 digestion and transformed into E. coli for selection and amplification.

to the plasmid DNA representing the desired construction, pSNαH-d
hfr was isolated from successful transformants.

Exactly the same method but pNexβ-HBV3'RI and p
pSNβH-dhfr was constructed using SV-Nexβ.

Common to these constructs is a vector containing the SV40 early promoter operably linked to the nexin 5′ end common to both α and β forms.

This intermediate vector, pSV-NexBall,
PN-33, pUc18 and psVorillBV
3'. To excise the insert containing PN-1, PN-33 was cut with EcoIII and B
After cutting in the reverse direction at a131, it was digested with 5acl to obtain a tailored nexin insert containing this 5'' end through the ^TG start codon.
This fragment was ligated to the pUC18 hector fragment digested with cII/SacI to create pUCNex-Bal.
I got a T. pUCNex-BalI was cut with old ndnI and 5all, and nexin 5 common to α and β types was extracted.
``The terminal fragment was excised.

This phoxin 5′-terminal fragment is the desired pSVNex-
Give Bal r(,ll1ndI[I/Sal
pSVori-+1BV3' vector fragment (see below) cut with I.

Decompose PN-33 with EcoRI and 1lpal,
A 1650 bp fragment was isolated and pUc-Nex3'
By ligating the fragment into pUc18 digested with EcoRI and SmaI to obtain an additional vector containing the 3' sequence, psVNex3'1lBV
was built. ll1ndI [and BamHT to obtain the desired pSVNex3' HBV.
pSVori−) digested with nd m /BamHI
pUc-Nex along with the hepatitis termination sequence by ligating the phoxin 3' end into IV3' (see below).
3” was supplied.

These two additional intermediate vectors, pSVNexα
and pSVNexβ, PN-33 or PN-1
8 (whichever suitable material) and pSVNex-Bat I or pSVNex3'
were obtained using the corresponding 5' and 3' ends from HBV. In each case, pSVNex-Bal I
was digested with Bglll and Sal I, pSVNe
x3'IIBV was degraded with old ndn [and 5all and PN-Iα or l'N-3 derived from PN-18
Bgl IF of PN-Iβ derived from 3/old nd m5
A 40 bp internal fragment was ligated by a three-way ligation method,
pSVNexα and pSVNexβ were obtained, respectively.

This Bam1l r /Sac 11-degraded pUc-HB
During V3', pSVNex tx or pSVNex
By inserting the Bgl U /Sac U insert of β (whichever is appropriate), the extreme 3
These were modified to place a 'terminal HcoRI site. The resulting vector, pNexαII B V
3°RI and pNexβIIBV3°R1 were then used in the construction described above. The resulting vector (psNI
(commonly referred to as I-dhfr) is shown in FIG.

This vector can be transfected into CO5-7 cells for temporal expression or DIIFR
PN-Iα or PN-Iβ were transfected into deletion C110 cells and then amplified with methotrexate.
cultivated for the production of. The PN-I is secreted into the medium because the signal sequence is retained in the construct and is compatible with the host cell.

The culture medium of the transformed cells was prepared by Eaton, D.
,L.

Ra, Lea "Gekiyu" 1Ld Fallen, (1983) Dan 7: 17
Using the thrombin binding assay described in 5-185,
Analyze PN-1 production. Briefly, serum-free medium pre-incubated for 72 hours with cell cultures of the aggregates was centrifuged to remove cell debris. 0.1βg
37/ml labeled thrombin C2'I-Th)
This medium was incubated for 45 minutes at °C. 12J
-Th-PN complex under conditions that do not decompose, ``'I
-Th-PN complexes were analyzed by 5OS-acrylamide gel electrophoresis using a 7% gel and quantified with a gamma scintillation counter assuming that PN and Th are present in equimolar amounts in the Th-PN complex. . The formed complex was confirmed to contain PN-I by immunoprecipitation using PN-1 rabbit antiserum.

The results of this analysis indicate that successfully transfected C
HO cells or CO57 cells show production of PN-1α or PN-1β.

The construction pSVor 1HBV3° for ``expulsion use'' contains the early and late promoters of SV40 upstream of the origin of replication and the 3' terminator sequence derived from the hepatitis 8 surface antigen gene. There is an insertion site for a foreign gene between this termination sequence and this promoter. psVorit! BV3° is constructed from pML, SV40 and IIBV. p
ML was digested with EcoRI and blunt-ended with Klenow2.
Then, it is decomposed with old ndII [. A vector fragment containing the E. coli origin of replication and the ampicillin resistance gene was isolated and transformed into SV40 (which is linked to l1indI
(obtained by degradation of the SV40 ONA by the SV40 ONA and the former ncll) and this isolated 540 origin containing the early and late promoters.
The fragments were concatenated. The resulting vector (this is pSVor
(named i).

Then IIBV containing the 3' termination sequence of the surface antigen gene
The DNA is digested with BamHI to introduce the 585 bp fragment isolated from Bam1l I /Bgl U digestion. Check the exact direction with restriction analysis. Old ndlI[
and Bamll I digestion generates a 350 bp fragment from this correct vector. The resulting ligated vector, psVorillBV3', is therefore:
It contains a 5v40 promoter sequence and a replication origin sequence upstream of the HBV terminator.

The coding sequence is successfully inserted between the promoter and terminator.

A ptpA-BAt, t7, containing a tailored upstream portion of the tp^ gene in a bacterial replication vector was also prepared.

This tPA cDN^ was isolated from Penn1ca, D, et al.
Total cDNA obtained for tPA as described in Ature (1983) 301:214-221.
Bacterial vector pMoN-10 containing an insert of the sequence
Supplied from 68. Of course, any bacterial replication vector containing this coding sequence could be used as well. The restriction sites designed below are from Nature
included in the disclosed sequence of the tPA cDNA shown in the reference. In order to cut out the cDNA encoding this tPA, PMON-10 was first injected with Bamtl I
2 and then Ba1-31 to scrape off each end of this gene. Analysis of the length and sequence of the linear fragment indicates that the 5' end of this fragment is 17 bp beyond the ATG start codon.
Continue digestion with BAL-31 until it is shown that the What is the exact length of the cut?

Within the expression cassette, it is not critical as long as it is within a short enough distance to place the ATG within operable distance from the promoter. Indeed, in this fragment, separation of the 5° end fragment from the ATG of approximately 10 bp is demonstrated. This selected linear fragment was then treated with 5 acl (
This cut inside the coding sequence of the tPA gene). The resulting blunt end/5acI fragment was then isolated. this is.

Contains a properly tailored 5'' end of this gene.

To give this intermediate plasmid ptPA-BAL17.

pUc1 degraded with Sac T/Hinc II
Connected to 3.

pUc-DHFR, the DHPR-encoding sequence (this.

(which has no associated control sequences) pDHFR-1 with Fnu4111
1 (Simonsen, C.C., et al.
Proc Natl Acad 5ciUSA (19
83) Bile: 2495-2499) was digested, made blunt-ended with Klenow, and then digested with Bgll.

By isolating a 660 bp fragment as described here and ligating this fragment into pUc13, pUC-DI
IFR was constructed. This pUc13 is 1IincI
I and Bam1l I. Therefore, ρU
C-DHPR represents a direct cloning vector for DHI'R. This D 11 F R is the ptPA-B described for the 5' portion of the tPA gene above.
Similar to AL17 vector.

Finally, a separate cloning vector for the termination sequence derived from the surface antigen gene of pneumonia B, pUC-11BV.
3' to BamHI and Bgl II, as above.
It was constructed by digesting the 11BV DNA with Bam1lf, isolating a 585 bp fragment, and ligating this fragment into pUc13 digested with Bam1lf.

psv-tPA1 containing the entire tPAD-domain sequence under the control of the SV40 promoter and IIBV termination sequence.
7.

p degraded with 11indIII and Bam1l I
It was prepared as a three-way ligation of vector fragments derived from sVorillBV3'. This therefore provides the promoter and terminator along with the vector sequences; the 3' end of tPA is inserted into the Sac
obtained by I/BglIf digestion; the tailored 5' portion of this tPA coding sequence was ptPA-BAL
It was obtained as a former nd III /Sacr decomposition product of 17.

transforming the resulting ligation mixture into E. coli and selecting the transformants for ampicillin resistance;
and plasmid D containing the desired psv-tl'A17.
NA was isolated.

One vector for DHPR expression (this is pSV-
DIIFI? ) was also obtained by the three-way ligation method. psVorillBV3' old nd III
The vector fragment obtained from the /Bam1l I digestion was again used to supply the control sequences. And this D
The 5″ and 3″ parts of the II PR code sequence are
1lindlT [and 5ack (partially), respectively.

and pUC using Bgl II and TaqI (partially)
Obtained by decomposition of -DHr'R. This ligation mixture is
E. coli was transformed, ampicillin-resistant transformants were selected, and plasmid DNA designated pSV-DIIPR was isolated.

A single plasmid containing a weak expression system for this DIIPR coding sequence was also prepared. This plasmid p M D It
of.

Ikb fragment from pDR34 digested with HcoRI/Taq I (partially), vector fragment derived from pML digested with EcoRI/Sal I, and gene isolated from pSV-DIIFR digested with 5acl (partially)/Sal I using the 3' end of.

Obtained using a three-way ligation method. (This pDl?34 vector is Ga55er c, s, l et al
, Proc Natl Acad Se4 Ni (1982) Neck: 6522-6526 (supra).

This vector contains the mouse DHFR gene linked to its own promoter). Obtained vector +, p
MDl (pSV-DI) except that this DHFR gene is under the control of the mouse DHr'R promoter.
Similar to IPR. The weak expression cassette present on pMDH and the strong expression cassette present on pSV-tPA17 can be appropriately mixed to form HFI? The two constitute one embodiment of the expression system of the invention when used to transform deletion cells.

Finally, psTIl-MDH containing the expression cassettes of tPA and DHFR on a single vector was expressed as pSV-t
PA17 was constructed as a three-way ligation of appropriately isolated fragments of pMDI (and pUC-HBV3°).
pMDII was digested with EcoRI and Sma
1 and pUC-1+BV3' was cut with Sac II and EcoRI.

(Summary of the Invention) Two slightly different proteases, nexin type (P
The DNA segment encoding N-Iα and PN-1β) is a practical PN-1 protein for diagnostic and therapeutic applications.
cloned and expressed to provide 9. PN-1 is a useful serine protease inhibitor in controlling conditions mediated by proteolytic activity.

Figures 1a and 1d show the sequences of two probe mixtures used in the following examples to identify PN-I clones.

FIG. 2 shows restriction maps of cDNA clones designated I'N-9 (representative of the class to which PN-33 belongs) and PN-18. Each of them is a complete PN-
Contains the coding sequence for one protein.

FIG. 3 shows the nucleotide sequence of the coding region of PN-18 and the deduced amino acid sequence of PN-Iα.

FIG. 4 shows the nucleotide sequence of the coding region of PN-33 and the deduced amino acid sequence of PN-1β.

Figure 5 shows a Northern plot of mRNA extracts from several cell lines using the 5au3AI fragment of PN-18 as a probe.

FIG. 6 shows the splice junction region of the PN-1 gene, explaining the production of the PN~■α type and the PN-1β type.

Figure 7 shows two expression vectors: 1) SNII-dhfr.

The drawings of the present invention will be further supplementarily explained below.

Figure 1a: The codons defined for amino acid residues 20 to 24 of protease nexin are shown below this amino acid sequence. The 14ff1 mixed oligomer designed as the reverse complement of this coding sequence consists of a mixture of 24 permutations.

Figure 1b: Amino acid residues 14 to 25 of protease nexin are
Corresponds to the most well-defined coding sequence defining a 36 base oligomer. A consensus 36-mer with four mixed sites at positions 3, 6, 30 and 33 was designed by using more preferred codons to designate the remaining undefined bases as indicated.

Figure 2: Restriction map of cDNA inserts PN18 and PN9. P.N.
18 is a typical 3kd insert observed, while P
N9 is representative of the 2kd size class. Eco at the 5′ and 3′ ends of this cDNA clone.
The Rr site is the Eco site used in this cloning process.
Derived from RI linker.

Figure 5: RN^ samples were denatured, fractionated on a methyl-water-based agarose gel, and electrophoretically transferred to a SceneScreen membrane filter. This PN-18 probe is
It was prepared by brimer extension of 113 clones in the presence of '-dCTP. The RNA filter was soaked in formamide hybridization buffer (50% formamide, 5X SSC, 2X Denhardt, 20mM sodium phosphate buffer (ptl 7.0), 0.2% SO
72×IQ in S and 100 μg/related yeast RNA)
' Hybridized with 32p-labeled probe of cpm at 42°C for 36 hours. This filter is 0.
Washed at 42°C in zxssc with 2% SOS,
It was exposed to X-ray film.

Lane 1) 1 μg human fibroblast non-poly A l1lN
A.

Lane 2) 6 μg human fibroblast poly^+ RNA 1
-23° Lane 3) 8 μg human fibroblast polyA”
RNA 1-6゜lane-I) 8 μg human 293 cell poly^+ RNA.

Lane 5) 8 μg bone marrow melanoma poly^”RN^.

Lane 6) 5 μg SK hepatitis non-poly A RNA.

that's all

Claims (1)

[Scope of Claims] 1. A recombinant DNA encoding mammalian protease nexin I (PN-I), which does not contain the original DNA sequence associated with the DNA encoding PN-I. . 2. Encodes enzymatically active PN-I and at 30°C
PN-1 under moderately stringent conditions corresponding to 6 x SSC at
The D according to claim 1, which is degenerate with the DNA that hybridizes to the DNA sequence of 8 or PN-33.
N.A. 3, having the nucleotide sequence of Figure 3 or Figure 4, or the mature PN-I α or PN-I of these Figures.
D of claim 1 which is degenerate with B or with naturally occurring allelic variants thereof.
N.A. 4. The DNA according to claim 1, which is operably linked to an expression control sequence. 5. The DNA according to claim 1, which is transfected into a recombinant host cell. 6. A set transfected with a recombinant DNA encoding mammalian protease nexin I (PN-I), which does not contain the original DNA sequence associated with the DNA encoding said PN-I. A method for preparing PN-I comprising culturing a modified host cell and recovering PN-I from the culture. 7. A set transfected with a recombinant DNA encoding the mammalian protease nexin I (PN-I), which does not contain the original DNA sequence associated with the DNA encoding the PN-I. Protease nexin I prepared by a method for preparing PN-I comprising culturing a modified host cell and recovering PN-I from the culture. 8. Usually free of bound impurities, has a molecular weight of 43 kd when glycosylated, and has an N-terminal sequence: [gene sequence], and about 2.3% by weight. Protease Nexin I contains amino sugars, about 1.1% neutral sugars, and about 3.0% sialic acid. 9. A method for the purification of PN-I originally comprising subjecting a medium containing PN-I to affinity chromatography using heparin bound to a solid support, followed by gel filtration chromatography. 10, (a) Mammalian protease nexin I (PN-
A recombinant DNA encoding PN-I), the recombinant DNA encoding PN-I
culturing a recombinant host cell transfected with a recombinant DNA sequence free of the original DNA sequence associated with the DNA encoding PN-I;
or (b) subjecting the medium containing PN-I to affinity chromatography using heparin bound to a solid support, followed by gel filtration chromatography. The original method for purifying PN-I, comprising: An antibody that is immunoreactive with PN-I prepared according to the method and formed in response to immunization of a mammal.