The present invention relates to novel recombinantly derived glandular (tissue) kallikrein polypeptides and to methods for producing such polypeptides. The invention further relates to pharmaceutical compositions containing such polypeptides and to the use of such polypeptides and compositions as vasodilators and in the treatment of hypertension and male infertility.

Background of the Invention

Kallikreins are members of a closely related subfamily of serine proteases. Kallikreins are characterized by their ability to release vas.oactive peptides, kinins, from kininogen, although the physiological significance of proteolytic actions of these enzymes seems to be unrelated to the release of kinins at least in some instances, and certain kallikreins are thought to be involved in the specific processing for the generation of biologically active peptides as well as factors from their precursors. [Fukushima et al.. Biochemistry 2_4, 8037-8043 (1985)].

The cDNA sequences for mouse and rat kallikrein were isolated from a sub axillary gland and a pancreatic cDNA bank. [Nakanishi et al.. Biotechnology 3_, 1089-1098 (1985)]. cDNA clones for human kallikrein were isolated from a human pancreatic cDNA bank [Fukushima et al., supra] and from a human kidney cDNA bank, [Baker et al., DNA 4_, No. 6, 445-450 (1985)]. It was reported that the active enzyme form consists of 238 amino acids and is preceded by

a signal peptide and profragment of 24 amino acids. It was also noted that the key amino acid residues required for serine proteinase activity _g (His-41, Asp-96, Ser-190) and for the kallikrein type cleavage specificity (Asp-184) are retained in the human kallikrein as they are in mouse and rat kallikreins.

_1Q Summary of the Invention

In accordance with the present invention, a novel class of glandular kallikrein polypeptides is provided. These biologically active kallikrein , _g polypeptides have the amino acid sequence extending from the N-terminus of the formula (I):

20

25

30

35

.+1 10 20

He Val Gly Gly Trp Glu Cys Glu Gin His Ser Gin Pro Trp Gin Ala Ala Leu Tyr His

30 40

Phe Ser Thr Phe Gin Cys Gly Gly lie Leu Val His Arg Gin Trp Val Leu Thr Ala Ala

50 60

His Cys He Ser Asp Asn Tyr Gin Leu Trp Leu Gly Arg His Asn Leu Phe Asp Asp Glu

70 80

Asn Thr Ala Gin Phe Val His Val Ser Glu Ser Phe Pro His Pro Gly Phe Asn Met Ser

Leu Leu Glu Asn His Thr Arg Gin Ala Arg Leu Thr Glu Pro Ala Asp Thr He Thr Asp Ala Val Lys Val Val Glu Leu Pro Thr

130 140

Gin Glu Pro Glu Val Gly Ser Thr Cys Leu Ala Ser Gly Trp Gly Ser He Glu Pro Glu

150 160

Asn Phe Ser Phe Pro Asp Asp Leu Gin Cys Val Asp Leu Lys He Leu Pro Asn Asp Glu

170 180

Cys Lys Lys Ala His Val Gin Lys Val Thr Asp Phe Met Leu Cys Val Gly His Leu Glu

iyu 200

Gly Gly Lys Asp Thr Cys Val Gly Asp Ser Gly Gly Pro Leu tfkt Cys Asp Gly Val Leu

210 220

Gin Gly Val Thr Ser Trp Gly Tyr Val Pro Cys Gly Thr Pro Asn Lys Pro Ser Val Ala

230 238

Val Arg Val Leu Ser Tyr Val Lys Trp He Glu Asp Thr He Ala Glu Asn Ser

wherein n is 0 or 1.

The kallikrein polypeptides are characterized as the product of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast. Bacillus and mammalian cells in culture) of exogenous DNA obtained by genomic, cDNA or by gene synthesis.

10 The DNA of the present invention includes DNA useful in securing expression in an appropriate host cell of a polypeptide product having the primary structural conformation of a kallikrein polypeptide having an amino acid sequence represented by formula

₁₅ (I) above and one or more of the biological properties of naturally occurring kallikrein. The DNA of the invention are specifically seen to comprise DNA encoding the sequence of formula (I) or their complementary strands. Specifically

__ comprehended are manufactured DNA encoding kallikrein wherein such DNA may incorporate codons facilitating translation of messenger RNA in microbial hosts. Such manufactured DNA may readily be constructed according to the methods of Alton et

₂₅ al., PCT application WO 83/04053.

Also comprehended by the invention are pharmaceutical compositions comprising therapeutically effective amounts of the kallikrein .._ polypeptide products of the invention together with suitable diluents, excipients and/or carriers useful in vasodilation and male infertility applications, etc.

_3g The present invention also encompasses the various cloned genes, replicable cloning vehicles, expression vehicles and transformed cultures, all harboring the genetic information necessary to

affect the production of recombinant derived kallikrein polypeptides of the present invention. Brief Description of the Drawings

5

Figure 1 illustrates the construction of pDSHKl.

Figure 2 is a photograph of a SDS-polyacrylamide gel comparing recombinant human mature kallikrein and

₁₀ naturally-occurring urinary kallikrein wherein the molecular weight markers are designated on the right-hand side and columns 1 and 2 designate urinary kallikrein and recombinant mature kallikrein under nonreducing conditions respectively, and _ιg columns 3 and 4 designate urinary kallikrein and recombinant mature kallikrein under reducing conditions.

Figure 3 illustrates the construction of pDGHK-LlA.

20 Detailed Description of the Invention

According to the present invention, DNA sequences encoding the polypeptide sequence of human species

_2g glandular kallikrein of the present invention have been isolated and characterized. Further, the human DNA may be utilized in the eucaryotic and procaryotic expression providing isolatable quantities of polypeptides having biological and

-, _n immunological properties of naturally-occurring kallikrein as well as m. vivo and _in vitro biological activities, in particular therapeutic activity, of naturally-occurring kallikrein.

_3g The DNA of human species origin was isolated from a human genomic DNA library. The isolation of clones containing kallikrein encoding DNA was accomplished

through DNA/DNA plaque hybridization employing a pool of mixedoligon cleotide probes.

The human kallikrein gene of the present invention encodes a 262-amino acid kallikrein polypeptide: a presumptive 17-amino acid signal peptide, a 7-amino 0 acid proenzyme fragment and a 238-amino acid mature protein.

Procaryotic or eucaryotic host expression (e.g., by bacterial., yeast and mammalian cells in culture) of 5 exogenous DNA of the present invention obtained by genomic or cDNA cloning or by gene synthesis yields the recombinant human kallikrein polypeptides described herein. The kallikrein polypeptide products of microbial expression in vertebrate

20 (e.g., mammalian and avian) cells may be further characterized by freedom from association with human proteins or other contaminants which may be associated with kallikrein in its natural mammalian cellular environment or in extracellular fluids such -. _Ï‚ as plasma or urine. The products of typical yeast (e.g., Saccharomyces cerevisiae) or procaryote (e.g., E. coli) host cells are free of association with ^* any mammalian proteins. Depending upon the host employed, polypeptides of the invention may be

30 glycosylated with mammalian or other eucaryotic carbohydrates or may be nonglycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue (at position -1).

35

Illustrative of the present invention are cloned DNA sequences of monkey and human species origins and

polypeptides suitably deduced therefrom which represent, respectively, the primary structural conformation of kallikrein of monkey and human species origins having the amino acid sequences represented by Table VIII.

The microbially expressed kallikrein polypeptides of the present invention may be isolated and purified

10 by conventional means including, e.g., chromatographic separations or immunological separations involving monoclonal and/or polyclonal antibody preparations, or using inhibitors or substrates of serine proteases for affinity _ιg chromatography.. Polypeptide products of the invention may be "labeled" by covalent association with a detectable marker substance (e.g., radiolabels, e.g., ι ^~ - ^~ -= ^> or nonisotopic labels, e.g., biotin) to provide reagents useful in detection and

_ ₀ quantification of kallikrein in solid tissue and fluid samples such as blood or urine. DNA products of the invention may also be labeled with detectable markers (for example, radiolabels such as i ^* i ⁴ -- 7 ^* - ^~ - ^~ '_ ^" or

P ^* -- ² and nonisotopic labels such as biotin) and

_2g employed in DNA hybridization processes to locate the kallikrein gene position and/or the position of any related gene family in the human, monkey and other mammalian species chromosomal map. The labeled DNA may also be used for identifying the

30 kallikrein gene disorders at the DNA level and used as gene markers for identifying neighboring genes and their disorders.

The kallikrein polypeptide products provided by the _g present invention are products having a primary structural conformation of a naturally-occurring

kallikrein to allow possession of one or more of the biological properties thereof and having an average carbohydrate composition which may differ from that of naturally-occurring kallikrein.

Methods for administration of kallikrein polypeptide products of the invention include oral administration and parenteral (e.g., IV, IM, SC, or

10 IP) administration and the compositions of the present invention thus administered would ordinarily include therapeutically effective amounts of product in combination with acceptable diluents, excipients or carriers. Therapeutically effective dosages are

, _g expected to vary substantially depending upon the condition treated and may be in the range of 0.1 to 100 μg/kg body weight of the acifive material. The kallikrein polypeptides and compositions of the present invention may also be lyophilized or made

_ _n into tablets. Standard diluents such as human serum albumin are contemplated for pharmaceutical compositions of the invention, as are standard carriers such as saline.

_2g The kallikrein products of the present invention may be useful, alone or in combination with other factors or drugs having utility in vasodilation and male infertility applications.

* * *

30

The following examples are presented by way of illustration of the invention and are specifically directed to procedures carried out prior to identification of kallikrein encoding monkey cDNA

_3g clones and human genomic clones, to procedure resulting in such identification, and to the

sequencing, development of expression systems and immunological verification of kallikrein expression in such systems.

Numerous aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following detailed description which provides illustrations of the practice of the invention in its presently preferred embodiments. As used herein in the following Examples, unless otherwise specified, the term recombinant kallikrein refers to recombinant mature kallikrein represented by the amino acid sequence of formula (I) wherein n is 0.

Example 1

Protein Sequence of Human Urinary Kallikrein

5

A. Amino Acid Sequencing of Human Urinary

Kallikrein and Peptide Fragments

Human urinary kallikrein was purified from pooled urine of about 30 normal male Caucasian

-_ individuals-according to the procedures described by J. Chao et al. [J. Clinical Endocrinology & Metabolism 51: 840-848 (1980)].

The pure human urinary kallikrein thus obtained was subject to N-terminus sequence analysis and the

, _g first 40 amino acids were identified. The purified urinary kallikrein protein was derivatized upon oxidation with performic acid and by reduced alkylation with dithiothreitol and iodoacetate. The protein derivatives were then digested using

20 trypsin, Staphylococcus aureus SV-8 protease, and endolysine-C peptidase. Twenty-nine discrete peptide fragments were isolated from the digestions.

The peptide fragments thus derived from reduced and _2g alkylated human urinary kallikrein were arbitrarily assigned numbers according to the protease used (i.e., T designates peptide fragments derived from trypsin digestion; S designates peptide fragments derived from SV-8 protease; and LC designates _ ₀ peptide fragments. _. derived from endolysine C peptidase) . The fragments were analyzed by microsequence analysis using a gas phase sequencer (Applied Biosystems), and the amino acid sequence of the human urinary kallikrein was determined and is _3g represented in Table I. In addition the peptide fragments obtained from the above digests are also

represented in Table I. In Table I, single letter designations are employed to represent the deduced translated polypeptide sequence of urinary kallikrein, an asterisk (*) designates unassigned amino acids, "NT" designates N-terminal sequencing of intact protein, "#" designates determined Asn- glycosylation site and "+" designates unassigned Asn-glycosylation site.

According to the isolation and sequence analysis of two overlapping peptides, S-18 and LC-17, represented in Table I, the amino acid at position 162 of human urinary kallikrein protein sequence was identified as lysine.

Table I Amino Acid Sequence of Urinary Kallikrein

1 10 20 30 I V G G W E C E Q H S ^Q P W ^Q A A L Y H F S T F ^Q C G G I L , (NT)

•*— S-38 *•

_< s— 41 ^* **-

^• LC-6

40 50 60

V H R ^Q W V L T A A H C I S D N Y ^Q L W L G R H N L F D D E « « « » _»

.< τ-50 ^* •-• ■<

_< LC-6 ^' -

70 80 90

N T A ^Q F V H V S E S F P H P G F N M S L L E N H T R Q A D OX-T-43 + + **-

„ S-37 •. LC-6 + +

â–  ^a * + -LC-64-CBa~

100 110 10

E D Y S H D L M L L R L T E P A D T I T D A V K V V E L P T

^ 0X-T-33a >• -<— .-

^• — S-7 — J ^* - ■ S-61 »■ -t S-2 — ^*** *• >. «< LC-64-CBb ^* **-

130 140 150

Q E P E V G S T C L A S G W G S I E P E N F S F P D D L Q C T-62. T-58 # S-52 »-

-LC-54a, LC-51 #

160 170 180

V D L K I L P N D E C K K A H V Q K V T D F M L C V G H L E . ^ OX-T-41, T-52a >. -<

190 200 210

G G K D T C V G D S G G P L M C D G V L Q G V T S W G Y V P

-c T-53 _f . .< T-57 LC-50 LC-54b—

220 230

C G T P N K P S V A V R V L S Y V K W I E D T I A E N S _ _< __T-29 *** ■ T-40 ^* - ^* •

-*-- S-45 ^*** -•

.». ^ LC-42 ■+- •< LC-33- ^**• » ^* -_.

This result is consistent with the amino acid sequence deduced from human genomic kallikrein DNA sequence as set forth in Table V. This result, however, is different from the reported sequence derived from human pancreatic cDNA and kidney cDNA, wherein glutamic acid was present at position 162 of the reported mature. kallikrein, [Baker et al., DNA 4_, 445-450 (1985) and Fukushima et al.. Biochemistry

10 24, 8037-8043 (1985)].

B. Glycosylation Sites

It was determined that human urinary kallikrein contains approximately 30% carbohydrate content _ιg based on the molecular weight estimated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The amino acid sequence of human urinary kallikrein indicates that there are three potential Asn-linked glycosylation sites. Sequence

₂ _ analysis of peptide fragments obtained in Example IA., indicates that there are three Asn-linked glycosylation sites (Table II). However, Asn 141 was found only partially glycosylated (60%). This is based on the sequencing results of two peptides _g containing identical sequence (T-58 & LC-51 vs. T-62 & LC-54A) . Asn 141 when linked to carbohydrate, as in.fragments T-62 and LC-54a, could not be identified by sequence analysis of the glycopeptide.

0

5

Table II Isolation and Characterization of Glycopeptides

10

, _g Example 2

Design and Construction of Oligonucleotide Probe Mixtures

The amino acid sequence set out in Table I was reviewed

20 in the context of the degeneracy of the genetic codons for the purpose of ascertaining whether mixed probe procedures could be applied to DNA/DNA hybridization procedures on cDNA and/or genomic DNA libraries.

25 N-terminus amino acid residues 1-10 and 12-29 and Phe-Asp-Asp-Glu-Asn-Thr-Ala-Gln-Phe-Val fragment from a tryptic peptide fragment OX-T-43 (see Table I) were chosen for synthesis of deoxyoligonucleotide probe mixtures and the probe mixtures represented in Table III _ _n were designed:

35

Table I I I

Qligonucleotide Probes

Etohe Sequence

1 2 3 4 5 6 7 8 9 10 He Val Gly Gly Trp Glu Cys Glu Gin Hi s HK-la 5 ' ATT GTG GGC GGA TGG GAA TGC GAA CAA CA 3 '

G G T G G

HK-lb 5' ATT GTG GGC GGC TGG GAA TGC GAA CAA CA 3'

T G T G G

12 20 29

GLN PRO TRP GLN ALA ALA LEU TYR HIS PHE SER THR PHE GLN CYS GLY GLY ILE HK-6a 3' GTT GGI ACC GTT CGT CGG GAC ATG GTG AAG AGI TGI AAG GTC ACA CCI CCI TA 5' C C A A A

HK-6b 3' GTT GGI ACC GTT CGA CGG GAC ATG GTG AAG AGI TGI AAG GTC ACA CCI CCI TA 5' C C A A A

HK-7a 3' GTT GGI ACC GTT CGA CGG GAC ATG GTG AAG TCG TGI AAG GTC ACA CCI CCI TA 5' _a C C A A A

HK-7b 3' GTT GGI ACC GTT CGT CGG GAC ATG GTG AAG TCG TGI AAG GTC ACA CCI CCI TA 5' C C A A A

(I = Inosine)

PHE ASP ASP GLU ASN THR ALA GLN PHE VAL

KF-2b 5' TTC GAC GAC GAA AAC ACT GCC CAG TTT GT 3'

T T T G T

The probe mixtures HK-la and lb are mixtures of 64 probes 29 nucleotides in length and HK-6a, 6b, 7a and 7b are mixtures of 32 probes, 53 nucleotides in length and probe KF-2b is a mixture of 32 probes, 29 nucleotides in length.

The oligonucleotide probes were labeled at the 5' eenndd wwiitthh γγ-- ³³²² PP--AATTPP,, 77550000--88000000 Ci/mmole (ICN) using _{n 4} polynucleo ide kinase (NEN)

Example 3

Hybridization 5 Probes HK-la, lb, 6a, 6b, 7a and 7b were used to hybridize human DNA Southern blots or monkey kidney poly(A) mRNA blot to determine specific hybridization and for defining the hybridization conditions. Probe mixtures HK-lb at 45°C and HK-7b 0 at 46°C yielded specific hybridization in a hybridization buffer comprising 0.025 pmol/ml of each of the probe sequences in 0.9 NaCl/5 mM EDTA/50 mM sodium phosphate, pH 6.5/0.5% sarkosyl/100 ug of yeast tRNA per ml. As a result, _g these two sets of probe mixtures were employed in the monkey kidney cDNA library screening described in Example 4C.

Example 4 0

A. Monkey cDNA Library Construction

A monkey kidney cDNA library was constructed from poly(A) mRNA isolated from anemic cynomolgus monkey kidneys as described in PCT Patent _g Application No. WO 85/02610 and Lin et al., [Gene 44: 201-209, (1986)]. Messenger RNA was isolated from anemic monkey kidneys by the guanidinium

thiocyanate procedure of Chirgwin et al., [Biochemistry ]^: 5294-5299 (1979)] and poly(A) ⁺ mRNA was purified by two runs of oligo(dT)-cellulose column chromatography as described by Maniatis et al., ["Molecular Cloning, A Laboratory Manual", p. 197-198 Cold Springs Harbor Laboratory, Cold Springs Harbor, NY, (1982)]. The cDNA library was constructed according to a modification of the general procedures of Okayama et al. [Mol. and Cell Biol. 2 , 161-170 (1982)]. The procedures are summarized as follows: (1) pUC8 was used as the sole vector, cut with PstI and then tailed with oligo dT of 60-80 bases in length; (2) Hindi digestion was used to remove the oligo dT tail from one of the vector; (3) first strand synthesis and oligo dG tailing was carried out according to the Okayama procedure; (4) BamHl digestion was employed to remove the oligo dG tail from one end of the vector; and (5) replacement of the RNA strand by DNA was in the presence of two linkers

(GATCTAAAGACCGTCCCCCCCCC and ACGGTCTTTA) in a three¬ fold molar excess over the oligo dG tailed vector.

B. Bacterial Transformation

Transformation of DNA into E^_ coli strain DH1 was performed and transformants were selected on LM- Ap agar containing 1% (w/v) Bacto tryptone/0.5% (w/v) yeast extract - 10 mM NaCl-10 mM MgS0 ₄ , 1.5% (w/v) Bacto agar containing 50 yg ampicillin per ml. [Hanahan, J. Mol. Biol. 1^6: 557-580 (1983)]. Transformants were obtained at a level of 1.5 x 10 ⁵ per μg of poly(A) ⁺ RNA.

C. Colony Hybridization Procedures for Screening

Monkey cDNA Library

The colony hybridization procedures employed for screening the monkey cDNA library were essentially the same as described in PCT application No. WO 85/02610 and Lin et al. [Gene 4_4: 201-209, (1986)].

Transformed E^ coli were spread out at a density of

10 9000 colonies per 10 x 10 cm plate on nutrient plates containing 50 μg of ampicillin per ml. Gene Screen filters (New England Nuclear Catalog No. NEF-972) were pre-wet on a BHI-CAM plate (Bacto brain heart infusion 37 g/L, casamino acids 2 g/L _ιg and agar 15 g/L, containing 500 μg of chloramphenicol/ml) and were used to lift the colonies off the plate. The colonies were grown in the above medium for 12 hours or longer to amplify the plasmid copy numbers. The amplified colonies

₂ _ (colony side up) were treated by serially placing the filters over 2 pieces of Whatman 3 MM paper saturated with each of the following solutions:

(1) 50 mM glucose - 25 mM Tris-HCl (pH 8.0) - 10 mM EDTA (pH 8.0) for five minutes;

_2g (2) 0.5 M NaOH for ten minutes; and

(3) 1.0 M Tris-HCl (pH 7.5) for three minutes

The filters were air-dried .in a vacuum oven at 80°C for two hours and then treated with a solution

_, _n containing 50 μg of proteinase K per ml. in Buffer K, which contains 0.1 M Tris-HCl (pH 8.0), 0.1 M NaCl, 10 mM EDTA (pH 8.2) and 0.2% sarkosyl. Specifically, 5 ml of the proteinase K solution was added to each filter and the digestion was allowed

_3g to proceed at 55°C for 30 minutes, after which the solution was removed.

The filters were treated with 4 ml of a prehybridization buffer [5 x SSPE ( o.1M NaCl/5mM EDTA/ 50mM Na Phosphate,pH 6.5) - 0.5% Sarkosyl - 100 μg/ml single stranded E^ coli DNA - 5 x BFP (1 x BFP = 0.02% wt./vol. Of BSA, Ficoll (M.W. 400,000) and Polyvinylpyrrolidone) ] . The prehybridization treatment was carried out at 55°C, generally for 4 hours or longer, after which time the prehybridization buffer was removed.

The hybridization process was conducted as follows: To each filter was added 3 ml of hybridization buffer (5 x SSPE - 0.5% sarkosyl - 100 μg of yeast tRNA per ml) containing 0.075 picomoles of each of the 64 probe sequences of Table III, (the total mixture being designated a HK-lb) and the filters were maintained at 45°C for 20 hours.

Following hybridization, the filters were washed three times for ten minutes on a shaker with 6 x SSC (1 x SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7) - 0.5% sarkosyl at room temperature and washed two to three times with 6 x SSC - 1% sarkosyl at the hybridization temperature (45°C), then autoradio- graphed. The filters were incubated at 100°C in 1 x SSC, pH 7.0/0.1% sarkosyl for 2 min. to remove the hybridized probes. The filters were again prehybridized as described above and then hybridized with the HK-7b mixed probes at 46°C and washed as described above.

Four positive clones that were hybridized to both HK-7b and HK-lb probe mixtures were obtained among the 200,000 colonies screened and were further confirmed by hybridization to another set of probe mixture KF-2b at 48°C.

One of the positive clones designated as MKK80a was further analyzed and sequenced by the dideoxy method of Sanger et al., [Proc. Natl. Sci. Acad., USA, 74: 5463-5467 (1977)]. The nucleotide sequence of MKK80a clone insert is depicted in Table IV (wherein the arrow "+" designates the beginning of the monkey kallikrien sequence), which is 95% homologous to that of the coding region of the human genomic kallikrein clone λHK65a as shown in Table V. The amino acid sequence deduced from the nucleotide sequence of the monkey clone MKK80a exhibited 93% homology to that of human kallikrein as illustrated in Table VIII.

Table IV MKK80a Clone Nucleotide Sequence tø 20 30 .40 50 60 f t f ♦ i I I

Gfl ATT CCC GGG GfiT CTT A ⁴ * GAC CGT CCC CCC CCC CCC AGC TCC TCC ACC TGC CGG CCC CTG 70 80 30 100 llβ 120 f t i f f I

GAC ACC TCT GTC RTC ATG TGG TTC CIG GTT CTG TGC CTC GCC CTG TCC CTG GGG GGG ACT Mel Trp Phe Leu Val Leu Cys Leu Ala Leu Ser Leu Gly Gly Thr

130 140 150 160 170 1B0 t f t i t f

GGT CGT GCG CCC CCG ATT CAG TCC CGG ATT GTG GGA GGC TGG GAG TGT TCC CAG CCC TGG 61y Arg Ala Pro Pro lie Gin Ser Arg He Val Gly Gly Trp Glu Cys Ser Sin Pro Trp

190 200 210 220 230 24ø f t f i » I

CAG GCG 6CT CTG TAC CAT TTC AGC ACT TTC CAG TGT GGG GGC ATC CTG GTG CAT CCC CAG Gin Ala Ala Leu Tyr His Phe Ser Thr Phe Gin Cys Gly Gly lie Leu Val His Pro Gin

250 260 270 2B0 230 300

I I f i t t

TGG GTG CTC ACA GOT GCC CAT TGC ATC AGC GAC AAT TAC CflG CTC TGG CTG GGT CGC CAC Trp Val Leu Thr Ala Ala His Cys He Ser Asp Asn Tyr Gin Leu Trp Leu Gly Arg His

310 320 330 34 350 360 t t ' t t t I

AAC TTG TTT GAT GAC GAA GAC ACA GCC CAG TTT GTT CAT GTC AGT GAG AGC TTC CCfl CAC

Asn Leu Phe Asp Asp Glu Asp Thr Ala Gin Phe Val His Val Ser Glu Ser Phe Pro His

370 380 339 40 410 42 ^* 3 f t t f f t

CCT GGC TTC AAC ATG AGC CTC CTG AAG AAC CAC ACC CGC CAA GCfl GAT CAT TAC AGC CAC Pro Gly Hie Asn Het Ser Leu Leu Lys Asn His Thr Arg Gin Ala Asp Asp Tyr Ser His

430 440 450 460 470 480 t t t t f t

Etc CTC ATG CTG CTC CGC CTG ACG CAG CCI GCC GAG ATC ACA GAC GCT GTG CAG ETC GTG Asp Leu Het Leu Leu Arg Leu Thr Gin Pro Ala Glu He Thr Asp Ala Val Gln Val Val

430 500 510 520 530 540 i t t f t t

GAG TTG CCC ACC CAG BAA CCC EAA GTC GGG AGC ACC TGT TTG GCC TCC GGC TGG GGC AGC Glu Leu Pro Thr Gin Glu Pro Glu Val Gly Ser Thr Cys Leu Ala Ser Gly Trp Gly Ser

550 560 570 580 530 600 t t I f f f

ATC GAA CCA GAG AAT TTC TCA TTT CCA GAT GAT CTC CAG TGT CTA GAC CTC GAA ATC CTG He Glu Pro Glu Asn Phe Ser Phe Pro Asp Asp Leu Gin Cys Val Asp Leu Glu He Leu

610 620 630 640 650 660 f t * t f t

CCC AAT GAT GAG TGC GCC AAA GCC CAT ACC CAG AAG GTG ACA GOG TTC ATG CTG TGT GCC Pro Asn Asp Glu Cys Ala Lys Ala His Thr Gin Lys Val Thr Glu Phe Met Leu Cys Ala

670 680 630 700 710 720 f t t f f I

GGA CAC CTG GAA GGT GGC AAA GAC ACC TGT GTG GGT GAT TCA GGG GGC CCG CTG ACG TGT Gly His Leu Glu Gly Gly Lys Asp Thr Cys Val Gly Asp Ser Gly Gly Pro Leu Thr Cys

730 740 750 760 770 780 f t t t t f

GAT GGT GTG CTC CAA GGT GTC ACA TCA TGG GGC TAC ATC CCT TGT GGC AGC CCC AAT AAG Asp Gly Val Leu Gin Gly Val Thr Ser Trp Gly Tyr He Pro Cys Gly Ser Pro Asn Lys

790 800 810 820 830 840 f t f f f f

CCT GCT GTC TTC GTC AAA GTG CTG TCA TAT GTG AAG TGG ATC GAG GAC ACC ATA GCG GAG Pro Ala Val Phe Val Lys Val Leu Ser Tyr Val Lys Trp He Glu Asp Thr He Ala Glu

850 860 870 880 830 900 f t t t i f

AAC TCC TGA ATG CCC AGC CCC GTC CCC TAC CCC CAG TAA AAT CGA ATG TGC ATC AAA AAA Asn Ser —

910 920 t t

Example 5

Phage Plaque Hydridization Procedures for Isolating

_ Kallikrein Gene From the Human Genomic Library

A Ch4A phage-borne human fetal liver genomic library prepared according to the procedures described by Lawn et al., [Cell 13: 533-543 (1979)] was obtained and used in a plaque hybridization assay. The phage _ιπ plaque hybridization procedures employed were as described in PCT No. WO 85/02610 and Lin et al., [Proc. Natl. Sci. Acad., USA 821: 7580-7584 (1985)]. Phage plaques were amplified according to the procedures of Woo, [Methods Enzymol. , 389-395

₁₅ (1979)], except that Gene Screen Plus filters and NZYAM plates [NaCl, 5 g; MgCl ₂ .6H ₂ 0, 2 g; NZ-A ine A, 10 g; yeast extract, 5 g; casamino acids, 2 g; maltose, 2 g; and agar, 15 g (per liter)] were utilized. Phage particles were disrupted by alkali

₂₀ treatment and the DNA's were fixed onto filters

(50,000 phage plaques per 8.4 x 8.4 cm filter). The air-dried filters were baked at 80°C for 1 hour and then subjected to proteinase K digestion as described in Example 4. Prehybridization with a 1 M

_ ₅ NaCl/1% sarkosyl solution was carried out at 55°C for 4 hours or longer.

The monkey kallikrein MMK80a clone DNA was nick- translated with ³² P-labeled-αdCTP, and the cDNA

30 insert (-1000 bp) was cut out by double-digestions with EcoRI plus Hindlll and used as a probe to screen human fetal liver genomic library.

The hybridization buffer contained 2 xlO ^* - ^* cpm/ml of ₃₅ the nick-translated monkey kallikrein cDNA in 0.9 M NaCl/5 mM EDTA/50 mM sodium phosphate, pH 6.5/0.5% sarkosyl/100 μg of yeast tRNA per ml. Hybridization

was carried out at 55°C for 20 hours. At the completion of hybridization, the filters were washed three times with 6 x SSC, pH 7.0/0.5% sarkosyl at ■- room temperature and two times at the hybridization temperature, 10 min. per wash.

Two strongly positive clones, designated as λHK65a and λHK76a, were obtained among a total of 1.87 x _Q 10° phage plaques screened.

Both human genomic kallikrein lambda clones were subcloned into pÏ‹C118 or pUC119 and the double- stranded DNA's were sequenced according to the ₅ procedure of Chen et al., [DNA 4, 165-170 (1985)] using the dideoxy method of Sanger et al., [Proc. Natl. Acad. Sci. USA 74_: 5463-5467 (1977)], and the nucleotide sequence of kallikrein gene containing region for clone λHK65a is represented in Table V. 0

5

0

5

Table V

Clone \HK65a Genomic Sequence

ΛGΛTCTCTGGGΛC GGΛGGΛCAGGACGπGAGCACCCATΛΛCTGGGΛGCΛAGΛAGTGGGAGGCTα^ MO

GAΛACÏ€CΛCΛπCCTGGATCTGGΛGGCACTAGGAΛTGT∞TGΛTGTGTÎ ›CCC 280

GGCnATATTTCAGGGGGAACTGGCAGCTGAGATGCTAGTATCπGGCTC∞AGTGG TGCAGGGAATCAGAGCCCTAACTGCTGGTGTTTGAGGGCACCAGGTCTGGG∞ 420

©3GGCTTrGTACCAGG∞TCACTGAπCCTCCGTCπCCTGTTrCTGCπCCCXT CAGTCCCCCCTTGCrTCACT(3GCTGCTCCα 560

TGCGTC(_AGCGTGATCCAGGGCCTGCAGAACΛGGTGCTGGTTCTCCCTCCCCGT^ 700

-24 -20

MetTrpPheleuVa lleuCysLeuA l aleuSerLeuGI

CAGGGCAG(_GGTGGGGCTCTGAGGGGATAAGGGCrTTrAAAAGCCTCCCCAGGGAG GÏ€cCCCAGÏ€CCTCCACCTGCTGGCCCCTGGACACCTCTGTCACCATGTGGÏ€CCTGGÏ €CTGTGCCT∞ 840 - 10 yG l yThrG

GGGGACTGGTGAGACAGTGGGGGGATGTGGGAGGG∞AAIXGGCCCTGAnCTCπG CTGGCCTCACATCCCCCCCCGATAAGACCπCCCCTCCAACACCCCACCCTCATCCCTCT GGCCCACAGTCTATCCTAG 980

GGGTGCCCCG∞TCπATCTATCπGCGCGGCCCCACCCTAAACnCCACCCCCGCT CCTCCATGπGTCATAGTGCTCACTCCCACACCAGAπCCTGCTCCTCC^AGGAAGCCTC H 20

CAΛCGCCCTGTCCCACCGCmΛCt^ΛΛGGGGCAGΛπCTGGGCCΛTCTCTGT CT 1260 "

GCCACCTΛTπGGCTCCTGAGTCTACAAATCCCACCACACTCCAGTGACπTT7CC ATCCAGCTGTCCGCTCTCAACTGTGTCCTCCAGACCCTGACATCTGGTCTCCCCAGTCCT TCCAGGGTCCCCCAAAT^ 1400

GAAAACCCTGCTTCCCAAACTGGCATGTGGGATCCCCCATCTGGTATGGGGGGGGGT CTGCAAATGTCCCT CCCACCCAGCACCCCCAGCTCTCCCAGGAAAAACACCACACCCTCCCACCACATGCTCCT TTCCÏ€GG 1540

GGAAGπCCΛTCCCGCCCCAGGCCCCAGCCXπCTCTGCCTGAAπGπCCTACAC AGCT^ 1680

TGTGAÏ€CTACAACCTCACATCAAGTGGGTCCTGCAnGCAÏ€CI ACAGCTGCTTGGCGCCTGTTCTCACCACTTCTCCAATCCTCÏ€CCTAGGAÏ€GCCTCCAA GAGGACCCCATATCCTTCCAGTACCCTCAGACCA 1820

TGAACCCCATCCCCATGAGAAA∞AAGATCCCATCCπCACCCTGTCCGAGAAGGA CTGCCACTCACGCCTCCCTCCTCCTGAGCCCCGTCCCAGTCCTGACCGCAACTGTGTTCT CAGGGCCCC^ 1960

TGCCAπ(XTGGπCΛCΛGGAAGGACCCCΛAΛACCTCT(^ΛAGCCCACCTCT AGCCTTAACCATAGtt^ 2100

ATGTAGGGACCCCAAGTCACCCCCAAACTCπCAGTCGAGATGCTCnCTπACCTCT CAGACCTGGTTCCCTAAAGCTGCATCCTTTCCTGAACCTCAAAAGCCCAACCCAGGTCCT GTTTCCCTAGCCTAGGAGCCCC 2240 πAGCCCTCCAGAπcnCCTCAGTGAGCCTCTGATCCTGTCCCACCCπGGCGCCπGAT CCCTGGTTTCCAGAATCCCTTTCCACTCCCCTAAC^CACCACCTCCCATCCCCCAGCCCC AGACCCC 2380

GCCCAGATGCAGTGTCTGCTCCAGACACTGTCTCCTGTCTCCTACCCTGATCCCTGG AGGCTGCTCTACTGTCAGAGCACAAAATCTCCCCACCAGGTCCAGCCACCACTCCAAAAG CCCAGGGAACAGTCCAGAGTCCC 2520

ACCCCÏ€CCCAACACCCCrrGCCCATGTCCCCAACCTCCAGCCCTGGTCCTCTACCC GCAATGTCCCÏ€CAGACCCACAGCCCAGCTCCCTCTCCTAGCCTTGTCCCTGGCCTCTCC TGCCAACCCTGCCCTTCCTGACC 2660

- 1 1 10 20 30 l yA l aA l aProProl l eGl nSerArg l l βVβ lGl yG l yTrpG l uCysGl uGl oHl sSerGl nProTrpG l nA l aA l oLeuTyrHi sP eSerThrPheGl nCysGl yGl y l l eLeuVa lHi sA

CAGCACCGCCTCTGCAGGTGCTGCGCCCCCGAπCAGTCCCGGAπGTGGGAGGCTG GGAGTGTGAGCAGCAπCCCAGCCCTGGCAGGCGGCTCTGTACCATπCAGCACTTTCCA GTGTGGG∞CA 2800 rgGlnTrpValLeuThrAlaA *l°aHisCysl leSerAs

GCCAGTGGGTGCTCACΛGCTGCTCATTGCATWGC'GAGTGΛGTAGGGGCCTGGGG TCTGGGAGGAGGGCATCTATGC^^ 2940

Table V (Continued)

GA(_£CΛGAGΛGGAAG<_CTCTGGCGCAGGTα_ TGGCA^ 3080

TCTCTCTCCTCTGTCTCOTGTCTCTGCATCT^ 3220

(XTCATCCCTΛCTAAACACACACCCAGATGGACCTAAGGGAGGCCCω^ 3360

CTCTGGΛGAGΛCACΛGGGMGGGCT∞TTTCAGCGGGAGCTGGGTGGGG^ 3500

ΛGGCCACCTGCCCCTGGGGAGCCKWO^CAGCCCCAGCTGCA^ 3640

ACACTCGGTCΛCTCCTGCTTCCTCTCTGCCTCTG CTAGnCT^ 3780

CGCTGTcc GτcττττAτccπcτcAτcτGccτcrrrrπGcτcACCcτG7τrcτ cACTGCccτccccτccGccτ^ 3920

CMACTCTTTCATCÏ€C AÏ€CGCTGTCTGTATTrCTCWTAACTATATCTTTCnCCTCTGTCTCCGCCCACCCACATT ^ ₄ 060

50 60 70 pΛsnTyrG I nLeuTrpLeuG I yArgHi sAsnLeuP eAspAspG I uAsnThr A I aG I nPhβVa I Hi sVa I SβrG I uSβrP eProH i sProG I α_cmcccGτcπcτcATcccτccAπcccATC^ 4200

oG I uVa IG I y SerT rCysLeuA I aSerG I TrpG I Sβr 1 1 e I uProG I uAsnP

CGAAGTGGGGAGCACCTGTTTGGCTTO_GGCTGGGGCAGCATCGAACCAGAGAATTG TATGTGGGGGCAGACTGTGTAGCCCAAGGCGGGGATGGGG 4480

150 160 170 heSerP eProAspAspLeuGl nCysVa lAspLeuLys l l e euProAsnAspGl uCysLysLysA l aHi srfa lGl nLysVa lT rAspPhβMβtLeuCysValGl TCGGGCTGCAGCCC I I I I 1 CTCCCGGGnαSTAGTCTCATTTCCAGATGATCTCCAGTGTGTGGACCTCAAAATCCTGC CTAATGATGAGTGCAAAAAAGCCCACGTCCAGAAGGTGACAGACTTCATGCTGTGTGTCG G 4620

180 yHi sLeuG l uGl yG l yLysAspT rCysVa l ACACCTGGAAGGTGGCAAAGACACCTGTGTGGTGAGGCAGCCCTGCCCCCAGGGTCTGGA AGGGCTGAGGGAGGGGACTCAGCCTCTGAACTGGCnCTGAGAGCTAACC^GGCATCTGCT TCACTGCTTCCCAGCTAGC 4760

TGTAGCCACTCGCCCCATCAGTGCCCCAGCTCCCCCTCCTTCCC _GCCCATCCAGGGACAACTGCATCTCACCCCCCACACCAGAGTTCACCGTTCCTCGTGGT AATGTGTTGTTACCGTra 4800

GAGAGGTGGCCTCTGCGATGTGCCCGCAGGGGCAGCGTCCTGCAGATGGTCCTGCCC CTCCTCCCCCTAACCTGTCTGCAGGCACTGTCCACCTGGACCCTGCCCCATGTGCAGGAG CTGGACCCTGAGGTCCCTTCCCC 5040

CπGGCCAGGACTGGAGACCCTGTCCCCTCTGTGGGAATCCCTGCCCACCTTCCTCT G∞^ 5180

190 200 210 220

G I y AspSerG I G I yProLeuHetCy SASDG I y a I teuG I nG I Va I ThrSerTr G I yTyrVa I ProCysG I yT rPro sn LysProSer a I A I aVa I ArgVa I LβuSerTy r Va I L

T(XCACCÏ€GTGCTCCCAGGGTGAT CAGGGGGCCCGCTGATGTGTGATGGTGTGCTCCAAGGTGTCACATCATGGGGCTACGTCC CTTGTGGCACCCCCAATAAGCCÏ€CTGTCGCCGTCAGAGTGCTGTCTTA 5320

230 238 ysTrp l l eGl uAspTbr l IβA l aG I uAsnSerEnd

AGTGGATCGAGGACACCATAGCGGAGAACTCCTGAACGCCCAGCCCTGTCCCCTACC CCCAGTAAAATCAAATGTGCATCCAATGTGTGTCACGTTCTGCCATCACCTATCTTTCCA GATGTGGTGCACCTGGACCCACT 5460

CGGCTGAGGCTGGGGCCACCCCAGCTGTGTCAATCTCATGCCTGGAAGTCTGAGGTC ACCAGCAGGTGGGGAAAGGA^ 5600

GGACGCCAGAACAGTTCACTCACACTAGGATGGACCCTGCCCTGGTGGGGAGGTGGG GGGCAATGGAAGGTCTGAGGCTGAGAGGT 5686

Example 6

Table VI represents the nucleotide sequence of a manufactured DNA encoding the recombinant mature kallikrein polypeptide of the present invention. The manufactured DNA was constructed according to the methods described by Alton et al., PCT application WO 83/04053. This manufactured (synthetic) gene has codons preferred for E_^_ coli expression. EcoRI and Ndel sites were added 5 ¹ to the initiation codon ATG, and PstI and BamHl sites 3 ¹ to the termination codon TAA. The resulting sequence is depicted in Table VI-A. The entire synthetic gene was cloned into pUC119 cut with EcoRI and BamHl and the resulting plasmid then sequenced. The gene was removed by digestion with Ndel and BamHl, then inserted into the E. coli expression vector pCFM1156 as described in commonly owned U.S. Ser. No. 004,379 hereby incorporated by reference. The resulting expression plasmid was used to transfect E. coli host FM5 as described in

Burnette et al., BIO/TECHNOLOGY, Vol. 6, 699 (1988). The transformed E^ coli was grown in brain/heart infusion (BHI) medium containing 20 μg/ml kanamycin at 28°C until OD ₆₀₀ = 0.1, then shifted to 42°C for 4-6 hr for maximal expression. The level of kallikrein expression was approximately 25% of the total cellular protein as estimated from SDS - Polyacrylamide gel electrophoresis analysis of whole cell lysates.

The E^ coli expressed kallikrein was extracted from inclusion bodies isolated from a bacterial cell paste by solubilizing in 8 M urea, pH 3.5 for 2 hr. The lysate was clarified by centrifugation at

5000 x g for 30 minutes. The clear lysate was diluted 10-fold and adjusted to pH 9-11 with sodium hydroxide. The solution was left stirring overnight at 4°C, then 2-mercaptoethanol added to a final concentration of 0, 50, or 100 mM. At the completion of oxidation, pH of the solution was adjusted to 8 with acetic acid and then reclarified as before.

The efficiency of refolding into an immunologically detectable molecule was determined by RIA as described in Example 7 to be approximately 0.42% of the proteins present in inclusion bodies.

Table VI

30 NCOl 60

ATG ATT GTA GGC GGT TGG GAA TGT GAA CAA CAT AGC CAG tCA TGG CAG GCT GCG CTG TAT M«t He Val Gly Gly. Trp Glu Cys Glu Gin Mis Ser Gin Pro Trp Gin Ala Ala Leu Tyr

90 120

CAC TTT TCT ACC TTT CAA TGC GGC GGT ATC CTG GTG CAC CGT CAG TGG GTT CTG ACC GC5 His Phe Ser Thr Phe Gin Cys. Gly Gly lit Leu Val Hit Arg Gin Trp Val Leu Thr Ala

150 180

GCA CAC TGC ATC AGC GAT AAT TAT CAA CTG TGG CTC GGC CGC CAC AAC CTG ^* TTC GAT GAC Ala His Cys He Ser Asp Asn Tyr Gin Leu Trp Leu Gly Arg His Asn Leu F'he Asp Asp

210 2A0

GAA AAC ACT GCA CAG TTC GTT CAC GTG AGC GAA TCC TTT CCG CAC CCG GGC TTC 'AAC ATS Glu Asn Thr Ala Gin Phe Val His Val Ser Glu Ser Phe Pro His Pro Gly Phe. Asn Met

XhoT 270 300

TCT CTG L'I LI tiAfc ^* AAT CAC ACC CGT CAG GCG GAT GAA GAC TAT AGC CAT GAC CTG ATG CT£J Ser Leu Leu Glu Asn His Thr Arg Gin Ala Asp Glu Asp Tyr Ser His Asp Leu flel Leu

330 360

CTG CGT CTG ACC GAA CCG GCA GAT ACC ATC ACC GAT GCG GTT AΛA GTG GTT GAA CTG CCG Leu Arg Leu Thr Glu Pro Ala At Thr He Thr Asp Ala Val Lys Val Val Glu Leu Pro

390 420

ACT CAG GAA CCG GAA GTG GGC TCC ACC TGT CTG GCG TCT GGT TGG GGC AGC ATC GΛA CCG Thr Gin Glu Pro Glu Val Gly Ser Thr Cys Leu Ala Ser Gly Trp Gly Ser He Glu Pro

450 480

GAA AAC TTC AGC TTC CCG GAT GAC CTG CAA TGC GTG GAC CTG AAA ATT CTG CCG AAC GAC Glu Asn Phe Ser Phe Pro Asp A=.p Leu Gin Cys V*l Asp Leu Lys lie Leu Pro Asn Asp

BstEII 54-0

GAΛ TGC GAA AAA GCG CAC GTG CAA AAG GTT ACC GAT TTC ATG CTG TGC GTG SS CAT CT& Glu Cys Glu Lys Ala His Val Gin Lys Val Th ASP Phe Met Ltu Cys Val Gly His Leu

570 ^" 600

GAG GGT GGT AAA GAT ACG TGT GTG GGT GAT TCT GGC GGC CCG CTG ATG TGC GAC GGT GTT Glu Gly Gly Lys Asp Thr Cys Val Gly Asp Ser Gly Gly Fro Leu Met Cys ASP Gly Val

630 660

CTT CAG GGC GTT ACC AGC TGG GGT TAC GTT CCG TGT GGT ACC CCG AAC AAA CCG TCT GT& Leu Gin Gly Val Thr Ser Trp Gly Tyr Val Pro Cys Gly Thr Pro Asn Lys Pro Ser Val

690 720

GCG GTT CGT GTG CTG AGC TAC GTT AAA TGG ATC GAA GAT ACC ATT GCG GAG AAC AGC TAΛ Ala Val Ars Val Leu Ser Tyr Val Lys Trp He Glu Asp Thr 111 Ale Glu Asn Ser End

Table VI-A

EcoRI Ndel

ΛATTCCΛT

GGTΛ

30 Ncol 60

ATδ ATT GTΛ GGC GGT TGG GAA TGT CAA CAA CAT AGC CAG CCA TGG CΛG GCT GCG CTG TΛT

TAC TAA CAT CCG CCA ACC CTT ACA CTT GTT GTA TCG GTC GGT ACC GTC CGA CGC GΛC ATA

90 120

CAC TTT TCT ACC TTT CAA TGC GGC GGT ATC CTG GTG CAC CGT CAG TGG GTT CTG ΛCC GCG GTG AAA AGA TGG AAA GTT ACG CCG CCA TAG GAC CAC GTG GCA GTC ACC CAA GΛC TGC CGC

150 180

GCΛ CAC TGC ATC ΛGC GAT ΛΛT TΛT CΛΛ CTG TGG CTC GGC CGC CΛC ΛΛC CTG TTC CAT CΛC CGT GTG ACG TAG TCG CTA TTA ΛTA GTT GΛC ΛCC GΛG CCG GCG GTG TTG GΛC AAG CTA CTG

210 240

GAA ΛΛC ACT GCA CAG TTC GTT CAC GTG AGC GAA TCC TTT CCG CAC CCG GGC TTC ΛΛC ΛTC CTT TTG TGA CGT GTC AAG CAA GTG CAC TCG CTT AGG AAA GGC GTG CGC CCG AΛC TTC TΛC

Xhol 270 300

TCT CTG CTC GAG AAT CAC ACC CGT CAG GCG GAT GAΛ GAC TAT AGC CAT GAC CTG ATG CTG AGA GAC GAG CTC TTA GTG TGG GCA GTC CGC CTA CTT CTG ATA TCG GTA CTG CAC TAC GΛC

330 - 360

CTG CGT CTG ΛCC GAA CCG GCA GAT ACC ΛTC ACC GAT CCG GTT AΛA GTG GTT GΛA CTG CCG GAC GCA GAC TGG CTT GGC CGT CTA TGG TAG TGG CTA CGC CΛA TTT CAC CΛA CTT GAC GGC

390 420

ACT CAG GAA CCG GAA GTG GGC TCC ACC TGT CTG GCG TCT GGT TGG GGC AGC ATC GΛΛ CCG TGA GTC CTT GGC CTT CAC CCG AGG TGG ACA GAC CGC AGA CCA ACC CCG TCG TΛG CTT GGC

450 480

GΛA AAC TTC AGC TTC CCG GAT GAC CTG CAA TGC GTG GAC CTG ΛAA AAT CTG CCG ΛAC GΛC CTT TTG ΛAG TCG AAG GGC CTA CTG GAC GTT ACG CAC CTG GΛC TTT TΛA CAC CCC TTG CTG

BstEII 540

CAA TGC GAA ΛAA GCG CAC GTG CAA ΛAG GTT ΛCC GΛT TTC ATG CTG TGC GTG CCC CAT CTG CTT ACG CTT TTT CGC GTG CAC GTT TTC CAA TGG CTA AAG TAC GAC ACG CAC CCC GTA GAC

510

570 600

GAG GGT GGT AAA GAT ACG TGT GTG GGT GAT TCT GGC- GGC CCG CTG ATG TGC GAC GGT GTT CTC CCA CCA TTT CTA TGC ACA CAC CCA CTA AGΛ CCG CCG GGC GΛC TAC ACG CTG CCΛ CΛA

630 660

CTT CAG GGC GTT ΛCC AGC TGG GGT TAC GTT CCG TGT GGT ACC CCG AΛC ΛΛΛ CCG TCT GTG GAA GTC CCG CΛA TGG TCG ΛCC CCΛ ΛTG CAΛ GGC ΛCΛ CCΛ TGG GGC TTG TTT GCC ΛGΛ CΛC

690 720

GCG GTT CGT GTG CTG ΛGC TΛC GTT ΛΛA TGG ΛTC GΛA GΛT ACC ΛTT GCG GΛG ΛΛC ΛGC TΛΛ CGC CAA GCA CAC GAC TCG ATG CAA TTT ACC TAG CTT CTΛ TGG TAA CGC CTC TTG TCG ATT

Pst I CTGCAG GACGTCCTAG BamHl

Example 7

( Radioimmunoassay for Kallikrein The radioimmunoassay procedure employed for quantitative detection of kallikrein was a modification of the procedure described by Shimamoto et al., [J. Clin. Endocrinol & Met. 51: 840-848 (1980)J:

The assay buffer employed was phosphate-buffered saline {PBS; 0.14 M NaCl in 0.01 M Na ₂ HOP ₄ -NaH ₂ P0 ₄ , pH 7.0) containing 0.1% BSA. The assay utilizes the following reagents and samples: 200 μl aliquots of samples or purified human urinary kallikrein (24 pg - 6250 pg) standards containing appropriate dilutions in the assay buffer; 100 μl of rabbit antiserum raised against purified human urinary kallikrein. at 1:250,000 dilution; 100 μl of ¹²⁵ i- human urinary kallikrein (Sp. Act. ~1.5 x 10 ⁸ cpm per μg kallikrein) 50,000 cpm. All samples and standards were assayed in duplicate. Assay tubes were incubated at 37°C for 2 hours then at 4°C overnight. Antibody bound kallikrein was separated from free kallikrein by adding a formalin fixed staphylococcus aureus (Cowan Strain) cell suspension, IgGsorb (Enzyme Center, Inc) . To each tube, fifty μl of 10% IgGsorb was added and let ^' the reaction mixture stand at room temperature for 30 min. The tubes were centrifuged and the resulting pellet was washed twice with a wash buffer comprising 0.05 M Tris-HCl, (pH 8.9), 2% BSA, 0.1% SDS and 0.1% Triton X-100 and counted in a gamma counter. The assay detects kallikrein in a range from about 24 pg to about 6250 pg. The kallikrein content of an unknown sample was determined by

comparison to a standard containing a known quantity of pure human urinary kallikrein.

Example 8

Human Grandular Kallikrein Gene Sequences

10 Nucleotide sequence analyses of the two independent positive human genomic kallikrein clones designated as λHK65a and λHK76a were carried out and results obtained for the kallikrein gene containing region of clone λHK65a are set out in Table V. Both clones , _g have identical kallikrein protein coding sequences and identical ihtron sequences that were completed and the restriction endonuclease map of the human kallikrein gene from clone λHK65a is shown in Table VII.

20

The protein coding region of the gene is divided by four intervening sequences or introns. Since the transcription initiation site of the mRNA for kallikrein has not been determined due to lack of

_2g human tissue mRNA, the boundary on the 5' side of exon I is undefined. The exons were identified by comparison of the nucleotide sequence to the amino acid sequence of human urinary kallikrein shown in Table I and by comparison with the cDNA sequence ₀ published [Baker et al., supra, and Fukushima et al., supra]. The exon-intron boundaries of the kallikrein gene conform to consensus splice rules, [Mount, Nucleic Acids Res. 1 : 459-472 (1982)]. In Table IV, the initial sequence appears to comprise ₅ the 5' end untranslated region (802 bp) of the gene which may contain enhancer/promoter like functions.,

Table VII

I II III IV nt803 nl 2678 nl4t06 nt4SI4 nl 5199

< ♦ \ i »

848 2837 4395 4650 5351 aa-24 — -9 aa-9— 45 aa 5— 142 aa 142-»I87 aaiaa — 23a

IVSI IVSII IVSIII IVSIV

(1829 nl) (1268 nl) (lie πi) (548 nl)

that leads up to a translated DNA region, the first exon, coding for the first 15 amino acids (-24 through -10, and part of residue -9). Then follows an intervening sequence (IVS) of 1829 base pairs. The second exon immediately followed codes for amino acid residues glycine -9 through aspartic acid 45. The second IVS that follows comprises 1268 base pairs. The third exon codes for amino acid residues aspartic acid 45 through phenylalanine 142, the third IVS of 118 base pairs. The fourth exon encodes amino acid residues phenylalanine -142 through valine 187, the fourth IVS of 548 base pairs. The last exon codes for amino acid residues Glycine 188 through serine 238 and a stop codon (TGA).

There is a 46 bp untranslated region at the 3' end of the last exon. The nucleotide sequence 15 bp upstream from the site contain a sequence AGTAAA resembling the consensus polyadenylation signal sequence AATAAA and the related sequences normally found at this location. [Nevins, Annu. Rev. Biochem. 5_^, 441-466 (1983)]. The kallikrein gene encodes a protein of 262 amino acids. Based on the NH^-terminal amino acid sequence of purified human urinary kallikrein, the last 238 residues correspond to the mature active protein with a calculated M _r of 26403 in an unglycosylated form. The sequence of the first 17 amino acids, predominantly hydrophobic residues, is consistent with this region encoding a signal peptide, [Watson, Nucleic Acids Res. _T2: 5145-5164 (1984)] and the following 7 amino acid residues being the activation peptide or propeptide,

[Takahashi et al., J. Biochem 99, 989-992 (1986)].

The amino acid sequence starting at position -7 through -1 corresponds to the propeptide

sequence in the recombinant prokallikrein and that starting at +1 corresponds to the sequence of the amino terminus of expressed recombinant mature kallikrein product of the present invention in CHO cells. As indicated in Table V, the mature protein has three potential sites for Asn-linked glycosylation, amino acid residues 78, 84 and 141 in the third exon of the gene, according to the rule of Asn-Xaa-Ser/Thr, [Marshall, Biochem. Soc. Symp. 40, 17-26 (1974)], and has 10 cysteine residues.

Example 9

Table VIII below illustrates the extent of polypeptide sequence homology between human and monkey kallikrein of t _* he present invention. In the upper continuous line of the Table, three letter designations for amino acids are employed to represent the deduced translated polypeptide sequences of human kallikrein of the present invention commencing with residue -24 and the amino acid residues appearing in the lower continuous line shows the differences in the deduced polypeptide sequence of monkey kallikrein also commencing at assigned residue number -24. Dashes are employed to highlight missing amino acid residues in the monkey kallikrein sequence that are present in human kallikrein of the present invention.

Table VIII

Comparison of Amino Acid Sequences of Human and Monkey Kallikrein

-24

Met Trp Phe Leu

-20 -10 -1

Val Leu Cys Leu Ala Leu Ser Leu Gly Gly Thr Gly Ala Ala Pro Pro lie Gin Ser Arg

Arg

+1 10 20 lie Val Gly Gly Trp Glu Cys Glu Gin His Ser Gin Pro Trp Gin Ala Ala Leu Tyr His

30 40 Phe Ser Thr Phe Gin Cys Gly Gly He Leu Val His Arg Gin Trp Val Leu Thr Ala Ala

Pro

50 60 His Cys lie Ser Asp Asn Tyr Gin Leu Trp Leu Gly Arg His Asn Leu Phe Asp Asp Glu

70 80 Asn Thr Ala Gin Phe Val His Val Ser Glu Ser Phe Pro His Pro Gly Phe Asn Met Ser Asp

90 100

Leu Leu Glu Asn His Thr Arg Gin Ala Asp Glu Asp Tyr Ser His Asp Leu Met Leu Leu Lys -

110 120

Arg Leu Thr Glu Pro Ala Asp Thr He Thr Asp Ala Val Lys Val Val Glu Leu Pro Thr Gin Glu — Gin

130 140

Gin Glu Pro Glu Val Gly Ser Thr Cys Leu Ala Ser Gly Trp Gly Ser He Glu Pro Glu

150 160

Asn Phe Ser Phe Pro Asp Asp Leu Gin Cys Val Asp Leu Lys He Leu Pro Asn Asp Glu

Glu _m .

170 180

Cys Lys Lys Ala His Val Gin Lys Val Thr Asp Phe Met Leu Cys Val Gly His Leu Glu Ala Thr Glu Ala

190 200

Gly Gly Lys Asp Thr Cys Val Gly Asp Ser Gly Gly Pro Leu Met Cys Asp Gly Val Leu

Thr

210 220

Gin Gly Val Thr Ser Trp Gly Tyr Val Pro Cys Gly Thr Pro Asn Lys Pro Ser Val Ala

He Ser Ala Phe

230 238

Val Arg Val Leu Ser Tyr Val Lys Trp He Glu Asp Thr He Ala Glu Asn Ser

Example 10

Construction of the Plasmid pDSHKl for the Expression of Human Kallikrein Gene in Mammalian Cells

For the expression of the human kallikrein gene, a 7 kb Bgl II-XbaI fragment from lambda clone λHK65a, that contains the entire kallikrein gene, was first isolated and then inserted into BamHl/Xbal doubly- digested plasmid vector pÏ‹C118. The resulting pUC118-based kallikrein clone was subjected to the Henikoff deletion procedures [Henikoff, Gene 28; 351-359 (1984)] to generate clones containing the _g human kallikrein gene with its 3'-flanking sequences deleted to various extent. In particular, the plasmid was opened by digesting with restriction enzymes SphI and Sail. The combination of ExoIII nuclease and SI nuclease was utilized to digest the _ insert from its 3' end (the Xbal site) for various time intervals. Thereafter, the plasmid DNA was treated with DNA polymerase large fragment (Klenow enzyme) to fill the ends for subsequent blunt-end ligation with T ₄ DNA ligase. The recircularized _g plasmid DNA was used to transform the DH5 E^ coli host, (Bethesda Research Laboratories, Cat. No. 8263SA) . The resulting transformants were analyzed by sizing the inserts and by DNA sequencing. One of the deletion clones, pHK102, contains the entire ₀ human kallikrein gene insert with 801 bp upstream from the protein initiation codon and 232 bp downstream from the termination codon. The pHK102 contains a Dral site 67 bp upstream from the protein initiation codon of the kallikrein gene and a _g Hindlll site which was carried over from the pÏ‹C118 237 bp downstream from the termination codon. The pHK102 DNA was digested with Dral and Hindlll and

the approximate 4.85 Kb Dral-Hindlll fragment was isolated and briefly digested with Bal31-Slow nuclease. The resulting DNA fragment was. ligated to _g the BamHl cleaved expression vector pDSVL (pDSVL contains a murine dihydrofolate reductase gene and SV40 late promoter as described in PCT application No. WO 85/02610), that have been end-filled with DNA polymerase large fragment (Klenow enzyme). The

₁₀ kallikrein expression plasmid thus obtained was designated as pDSHKl, which contains the entire human kallikrein gene including 64 bp 5' to the initiation codon and 232 bp 3* from the termination codon. The detailed construct of pDSHKl is depicted

, _g in Figure 1. The pDSHKl plasmid contains a murine DHFR minigene as a EcoRI-PstI fragment from pMgl; SV40 origin of replication and early/late promoters in the Hindlll-BamHl fragment; SV40 nt 2538-2770 in the BamHl-BclI fragment; pBR322 nt 2448-4362 in the

₂ _ Hindlll-EcoRI fragment; and the 4.85 kb Dral-Hindlll fragment of the human kallikrein gene. The SV40 late promoter is used to drive the expression of the human kallikrein gene. This pDSHKl plasmid was used to transfect African green monkey kidney cells COS-1

₂₅ for transient expression or transfect DHFR ^" CHO cells for stable expression.

Example 11

0 A. ^• Transient Expression of Human Kallikrein Gene in African Green Monkey Kidney Cells, COS-1 Cells A calcium phosphate microprecipitate of covalent circular DNA, pDSHKl, at 1.5 yg DNA plus 10 μg mouse liver DNA as carrier per 4 x 10 ⁵ cells was used to _g transfect COS-1 cells. The cells were grown in high glucose DMEM medium supplemented with 10% fetal bovine serum plus penicillium, streptomycin and

glutamine. A three-day conditioned medium was determined to contain 5.1 ng immunoreactive kallikrein per ml using the radioimmunoassay procedure of Example 7.

B. Expression of Human Kallikrein Gene in Chinese Hamster Ovary (CHO) Cells with Plasmid pDSHKl

(1) Transfection of CHO Cells With the Circular

Plasmid DNA of pDSHKl

Chinese hamster ovary DHFR ^" cells (CHO DHFR ^" cells) [ϋrlaub et al., (1980) Proc. Nat. Acad. Sci. (U.S.A.), 77, 4461] lack the enzyme dihydrofolate reductase (DHFR) due to mutations in the structural genes and therefore require the presence of glycine, hypoxanthine, and thymidine in the culture media for growth. The cells were grown as a monolayer in medium C comprising Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum, 1% nonessential amino acids, 1% hypoxanthine and thymidine, and penicillin, streptomycin, and glutamine. One day prior to transfection, cells were plated at the approximate density of 3 x 10° cells per 10 cm culture dish. A calcium phosphate microprecipitate procedure by a modification of the methods of Graham et al., [Virology 5_^: 456-467 (1973)], and Wigler 0 et al., [Cell lit 223-232 (1977)] was employed to introduce pDSHKl DNA with a final concentration of 6 to 7 μg of plasmid DNA per 10 cells. Cells that had been transformed with and were expressing the DHFR gene (and thereby _g the kallikrein gene) survived and proliferated in the selective medium comprising DMEM supplemented with 10% dialyzed fetal bovine

serum, 1% nonessential. amino acids, glutamine, penicillin, and streptomycin, but no hypoxanthine and thymidine. The cells that grew in the selective medium were considered as stable transformants. The culture conditioned medium of the stable transformants was collected and assayed for the immunoreactive human kallikrein by the radioimmunoassay procedure, 0 described in Example 7. The results indicated that the immunologically reactive recombinant human kallikrein products were secreted at the level of 6 ng/ml from a 3-day conditioned medium. 5

(2) Transfection of CHO cells with linear DNA fragment of pDSHKl ^{••}

Plasmid pDSHKl (9.85 kb) was digested by restriction enzymes EcoRI and Hindlll. The 0 resulting digest was applied to a 0.7% agarose gel electrophoresis to separate the kallikrein insert containing fragment from the prokaryotic DNA fragment which originated from pBR plasmid. The 7.9 Kb DNA fragment, that contains _g the DHFR gene, the SV40 regulatory sequences, and the kallikrein gene, was isolated from the agarose gel. The transfection of CHO cells with this linear DNA fragment was carried out in 60 mm culture dishes at the cell density of 1 x 10 ₀ per dish with 10 μg of 7.9 Kb DNA fragment added per dish as previously described in Example 11B(1) for the closed circular DNA. Seventeen hours after adding the DNA to the cells, the medium was aspirated and replaced with a fresh _g medium. Three days later, the cells were trypsinized from two of 60 mm dishes and transferred to two 75-cm culture flasks. On

the following day, the medium was replaced with the selective medium. From this point on, the cells were maintained and subcultured in the selective medium for approximately four weeks and were designated as stable transformants. The stable transformants have a secretion level of 0.27 μg/ml of conditioned medium obtained from a 14-day culture in serum-free condition. Ham's F-12/DME 1-:1 mixture supplemented with glutamine.

C. Amplification of the Cloned Human Kallikrein Gene (HK) in CHO DHFR ^" Cells Containing pDSHKl The quantity of recombinant kallikrein produced by the stable transformants may be increased by gene amplification with methotrexate treatment to yield new cell strains having higher productivity. To amplify the cloned kallikrein gene, the stable transformants were subjected to a series of increasing concentration of methotrexate treatment. Initially, the cells were shifted from the selective medium to a methotrexate medium. The methotrexate medium is composed of DMEM supplemented 5 with 5% fetal calf serum, 1% nonessential amino acids,, glutamine, penicillin and streptomycin, plus methotrexate at desired concentration.

Cell strain CHO-DSHK1-C1 (stable transformants that 0 originate from circular DNA transfected cells) was subjected to treatments with increasing methotrexate concentrations (0 nM, 20 nM, 60 nM, 300 nM, and 1 μM) to select out the higher producing cell strains. After about four weeks of culturing in a _g given ethotrexate-containing media, the near confluent cells were then fed with serum-free media without methotrexate. Representative 7-day medium samples from serum-free culture (Ham's F-12/DMEM 1:1

mixture) from each amplification step were assayed using the radioimmunoassay procedure of Example 7 and determined to contain 10, 42, 110, 693 and 708 ng human kallikrein/ml, respectively. On the other hand, the cell strain CHO-DSHK1-L1 (stable transformants that originate from linear DNA trans¬ fected CHO cells) was also subjected to amplification with increasing concentrations of methotrexate (0 nM, 30nM, 60 nM, 300 nM, 1 μM and 5 μM) . The kallikrein content in the conditioned media was determined using the radioimmunoassay (RIA) procedure described in Example 7. The results based on 7-day samples are shown in Table IX.

Table IX

Expression of Kallikrein by CHO-DSHK1-L1 Cells Different Stages of Amplification with Methotrexate

Kallikrein

Methotrexate Culture Days of Concentration Concentration Media Conditioned Media (μg/ml)

SF ^J 14 0.27

60 nM SF 7 1.44 SF+PC-1 ^: 7 1.92

300 nM SF+PC-1 7 2.34 SF+PC-1+Gln ³ 7 3.57 •SF+PC-1 ⁴ . 7 4.06

1 μM SF+PC-1 7 1.93 10 2.80 12 4.87

5% serum 7 1.74

5 μM 5% serum 0.82

SF = serum-free medium, i.e. Ham's F12/DMEM 1:1 mixture.

PC-1 = a supplement for serum-free culture, obtained from Ventrex (Portland, Maine) .

Glutamine concentration is 8 mM instead of the regular 2 mM.

The cells used in this experiment had been previously maintained for 7.days in PC-1 supplemented serum-free medium, and subsequently 3 days in methotrexate medium.

The highest expression level was 4.06 μg human kallikrein/ml for a 7-day conditioned medium, ₍ observed with 300 nM methotrexate treated cells that have been transfected with linearized kallikrein

DNA. ^"

D. Expression of Human Kallikrein Gene in CHO cells with Plasmid pDGHK-LlA

(1) Construction of pDGHK-LlA Expression Vector pDGHK-LlA as depicted in Figure 3, consists of the following: (i) pBR322 nt 2448-4362 as a Hindlll-EcoRI fragment; (ii) a DHFR minigene as an EcoRI-PstI fragment from pMgl with a deletion in the 3' untranslated region made by removing a Bglll-Bglll fragment of 556 bp, and then end filled with klenow enzyme; (iii) A bidirectional SV40 termination sequence of 237 bp originally as a BamHI-BclI fragment (SV40 nt 2770-2553), adding synthetic linker-1 to Bell (destroying the Bell site and generating a PstI site), and adding synthetic linker-2 to BamHl end of the fragment to create a Sail site; (iv) The kallikrein gene (nt 801- nt 5370 in Table V) with synthetic linker-3 added to Drain site at nt 801 and with a Sail site was created by insertion of GA using site-directed mutagenesis between nt 15 and nt-16 past termination codon TGA, generating the sequence TGAACGCCCAGCCCTGTA(GA)C; (v) A rat glucose- regulated protein (GRP78) gene promoter which contains Smal-Bsshll fragments (GRP78 nt-722 to nt-37) [Shin C. Chang et al_. , Proc. Natl. Acad. Sci. 84:680-684 (19R7)]; and (vi) synthetic linker-4 to reinstate the sequence including the mRNA cap site and also to create a BamHl cloning site. The GRP promoter-kallikrein gene fusion was made by ligating the BamHl site of linker-4 to the

Bglll site of linker-4 to the Bglll site of linker-3. Linker-5 was added to the Smal end of the GRP promoter sequence and it was joined to the pBR322 derived sequence at Hindlll.

Linker 1:

GCAATGGCAACAACGTTGCCCG

ACGTCGTTACCGÏ€GÏ€GCAACGGGCCTAG

Pstl (Bel l)

Linker 2: Sail BamHl TCGACTG GACCTAG

Linker 3:

Bglll (Dralll) GATCTCAAACAGACAACAT AGTTTGTCTGTT

Linker 4: CGCGCTCGATACTGGCTGTGACTACACTGACTTGGACACTTGGCCTTTTGCGGGTTTGAG

GAGCTATGACCGACACTGATGTGACTGAACCTGTGAAOGGGAAAACGCCCAAACTCC TAG Bsshll BamHl

Linker 5;

Hind III EcoRI . Smal

AGCTTGAGTCCTGAATTCGAGCTCGGTACCC

ACTCAGGACTTAAGCTCGAGCCATGGG

(2) Transfection of CHO Cells with Plasmid pDGHK-LlA:

^• CHO D~ cells were grown in high glucose, high glutamine DMEM (Gibco Laboratories, Cat. #320-1965) supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acids, 13.6 μg/ml hypoxanthine, 7.6 μg/ml thymidine, and 2 mM glutamine. The 60 mm plates were seeded at a density of approximately 3.3 x 10 ⁵ cells/dish containing 5 ml of the medium and allowed to grow for about 20 hours at 37°C, 5% C0 ₂ . The media from each plate was aspirated, fresh media added and cells allowed to grow an additional five hours at 37°C, 5% C0 ₂ . The

media was again aspirated and the cells washed once with PBS, then once with transfection buffer before cells were transfected with the linearized plasmid DNA, pDGHK-LlA. The transfection procedure was essentially the same as described by Bond and Wold (Bond, V.C. and Wold, B., Mol. & Cell. Biol. 7:2286-2293, 1987). The transfection buffer consisted of 5

10 mM NaCl, ^' 120 mM KCl, 1.5 mM a ₂ HP0 ₄ and 25 mM Tris, pH 7.5; then adding CaCl ₂ to 1.4 mM and MgCl to 0.5 mM. After mixing, the buffer was sterilized by filtering through a 0.22 μm Costar Microstar filter. Plasmid pDGHK-LIA, linearized _ιg with restriction enzyme Pvul was extracted once with phenol-chloroform, 1:1, saturated with 0.05 M Tris, 0.02 M EDTA, 0.01 M 2-mercaptoethanol, pH 8.0, then once with chloroform only. The extracted plasmid digest was precipitated with

20 1/2 volume 7.5 M ammonium acetate and 2 1/2 volumes absolute ethanol at -20°C overnight. Precipitated plasmid DNA was redissolved in the sterile transfection buffer to give a final concentration of approximately 2.5 μg/ml and 5

25 μg/ml. Poly-L-ornithine (Sigma, Cat. #P4638), 200 mg/ml in 0.01 M Tris pH 7.5 sterilized through a 0.22 μm Costar Microstar filter, was added to 17 μg/ml and a plasmid DNA concentration of 2.5 μg/ml transfection mixture,

30 and was added to 25 μg/ml and a plasmid DNA concentration of 5 μg/ml plasmid DNA transfection mixture . Transfection mixtures (0.5 ml/60 mm dish) were carefully layered onto cells containing dishes and incubated for 1 hour

- ₅ at room temperature in a laminar flow hood. At the end of the transfection treatment, 5 ml of culture media were added to each dish, which was then returned to 37°C, 5% C0 ₂ . The following

day, i.e., approximately 16 hours later, the culture dishes were aspirated and fresh media added, returning the dishes to 37°C, 5% C0 ₂ for 48 hours. Cells were trypsinized and replated to four 100-mm dishes (10 ml medium each) for each 60 mm dish. One day later, the media was aspirated and the cells were washed twice with PBS. Then the .selective media (it contains high glucose, high glutamine DMEM supplemented with 10% dialyzed FBS, 2 mM glutamine, 0.1 mM non¬ essential amino acids) was added. Selective media was changed approximately every 3 days until 15 days post transfection, when individual colonies were visible. Colonies were tabulated, trypsinized and transferred to 25-cm ² culture flasks, at . a density of approximately 50 pooled, colonies/flask. From 1 μg of transfected DNA, about 40-50 colonies were obtained.

(3) Amplification of the Cloned HK Gene in CHO DHFR-Cells Containing pDGHK-LlA Amplification procedure for CHO DHFR~cells containing linearized pDGHK-LlA was essentially the same as that described in Example 11C with the following modifications.

Cell strain CHO-DGHK-L1A-8 (Stable transformants that originate from pDGHK-LlA transfected cells) was subjected to two different schemes of treatment with methotrexate (MTX) . One scheme of MTX concentration was:

0-20nM- ^* -60nM-200nM-600nM MTX and the other was:

0-30nM+100nM-300nM->900nM. The kallikrein content as secreted by the cells into the media was determined using RIA as described in Example 7. 60nM MTX and lOOnM MTX

amplified cells secreted the highest level of kallikrein, 1350ng/ml/day and 1725 ng/ml/day respectively. The results are shown in Table X below.

TABLE X c

The Expression Level of Kallikrein by CHO-DGHKL1A-8 Cells at Different Stages of Amplification with Methotrexate

Daily Yield MTX (nM) of rHK (ng/ml)

0 630

30 561

60 1350

100 1725

200 .191

300 61

600 167

2

Data was obtained from 175-cm culture flasks containing 40 ml medium and approximately 1.5 x 10^ cells per flask. The medium used was DMEM/F12 1:1 containing PC-1 (lOml/500 ml medium), 6mM Glutamine and 0.1 mM non-essential amino acids.

Example 12

Isolation of Recombinant Human Kallikrein Cell free culture media containing recombinant human kallikrein produced by CHO cells was pooled and con¬ centrated using a diafiltrator having a 10,000 dalton molecular weight cutoff membrane filter. The concentrated sample was buffer exchanged with 10

10 volumes of 10 mM Tris-HCl buffer (pH 7.8). The crude recombinant kallikrein was added to a QA- sepharose column packed and equilibrated with 10 mM Tris-HCl buffer (pH 7.8). The column was washed with the equilibration buffer and the recombinant

, _g kallikrein was eluted with a linear gradient of from 0 to 0.5 M NaCl in the same buffer. Both active kallikrein and enzymatically inactive prokallikrein were eluted at fractions containing 0.3 to 0.4 M NaCl. The recombinant kallikrein and prokallikrein

__ fractions were pooled and dialyzed against 10 mM

Tris-HCl (pH 7.8) buffer to remove excessive sodium chloride. The dialysis proceeded for 2-3 days at 4°C, at which time prokallikrein was completely activated by kallikrein to generate mature

₂₅ kallikrein. The dialyzed activated kallikrein pool was added to a benzamidine-sepharose affinity column which was pre-equilibrated with 10 mM Tris-HCl (pH 7.8). The column washed with the equilibration buffer and kallikrein was eluted with the same _n buffer containing 2 M guanidine HC1 The kallikrein fractions were pooled then precipitated with 70% saturated ammonium sulfate in 10 mM Tris-HCl (pH 7.8). The ammonium sulfate precipitated kallikrein fractions were dissolved in 10 mM Tris-HCl buffer _g (pH 7.8) and the dissolved sample was applied to a sephacryl S-300 gel filtration column which is equilibrated with the same buffer. The kallikrein fractions were eluted at molecular weights ranging

from 30,000 to 45,000. The pooled kallikrein fractions contained highly purified active recombinant human mature kallikrein. A C ₄ HPLC column (Vydac C ₄ column 25 cm x 4.6 mm) chromatography was used to check the purity of the final product. The buffer gradient conditions used were as follows:

A = 0.1% TFA (Trifluoroacetic acid)

B = 90% CH ₃ CN/0.1%TFA (CH3CN = Acetonitrile)

Flow = 1 ml/min

The results are shown below in Table XA:

Isolation of Recombinant Human Tissue Kallikrein (TCHK009) Derived from CHO Cells

70% active form

Frx B 33 17 13407 788.65 11.3 7% profor 93% active

HOK concentration of 1 mg/ml has an OD = 1.6 at 280 nm.

Specific activity = 1100 U/mg assayed by AcPheArgOet coupling enzyme procedure.

Kallikrein activity was assayed by protease activity using AcPheArgOet as a substitute (Fiedler et al. , Methods in Enzymology 8j): 493-532, 1980).

*** Frx=Fraction

The resulting purified kallikrein was enzymatically active as determined using the following synthetic peptide: Ac-Phe-Arg-OEt-Nitroanilide as a substrate for an esterase assay according to the procedures described by Geiger et al., [Adv. Biosci. 11_, 127 (1979) ] .

From different batches of culture media both kallikrein (the mature, active form) and prokallikrein (the inactive form) have been purified using similar purification procedures. The purified proenzyme was not enzymatically active. Active kallikrein was isolated from prokallikrein after autoactivation or after removal of the activation peptide in prokallikrein by incubation with trypsin.

Example 13

Sequence Analysis of Recombinant Kallikrein and Prokallikrein

Sequence determination of the purified recombinant kallikrein revealed that the purified protein is homogeneous and contains a single amino terminus.

The partial N-terminal sequence is determined as:

1 2 3 4 5 ^" 6 7 8 9 10 11 12 13 14 NH

-Ile-Val-Gly-Gly-XXX-Glu-XXX-Glu-Gln-His-Ser-Gln-Pro -XXX-

15 16 Gln-Ala- wherein XXX denotes residues that cannot be positively assigned. The result indicated that the N-terminal sequence of recombinant kallikrein is identical to the previously determined N-terminal sequence of human urinary kallikrein.

N-terminal sequence analysis of recombinant prokallikrein revealed that the proform contains a heptapeptide leader followed by the amino acid sequence of mature kallikrein, i.e.,

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ala-Pro-Pro-Ile-Gln-Ser-Arg-Ile-Val-Gly-Gly-XXX-Glu- XXX-

15 16 17 18 19 20 GLU-Gln-His-Ser-Gln-Pro ,

where XXX denotes residues that cannot be positively assigned. This proform N-terminal sequence is also identical to the N-terminal sequence reported by Takahashi et al., supra.

Example 14

Kinongenase Assay for Kallikrein The procedure used for measuring the kinin- generating activity of the recombinant kallikrein polypeptide of the present invention was essentially the same as the described by Shimamoto et al., Jap. Circ. J. 4_3: 147-152), (1979):

Purified human urinary kallikrein standards or recombinant kallikrein samples diluted (20 μl or 40 μl) in 0.1 M sodium phosphate (pH 8.5)/30 mM Na _? EDTA/3 mM phenanthroline containing.3 μg of purified bovine low molecular weight kininogen in a total volume of 0.5 ml were incubated at 37°C for 30 min. The reaction was terminated by boiling for 10 min. and 50 μl aliquots in duplicate were used to measure the amount of kinin-released by a kinin RIA

with a rabbit antiserum against kinin. The kininogenase activity of recombinant kallikrein is expressed as the amount of generated kinin in μg/30 min/mg kallikrein.

Example 15

In Vivo Assay for Blood Pressure-Lowering Effect of Kallikrein

A. Rats

Male species of spontaneous hypertensive rats, about 250 grams body weight, were used in the study. Rats were anesthetized with sodium pentobarbital (50mg/Kg body weight) intraperitoneally. The common carotid artery was cannulated and then connected to a Statham pressure transducer. The right jugular vein was cannulated for administering kallikrein and changes in blood pressure were measured with a polygraph. Upon administration of the recombinant mature kallikrein of the present invention, lowering blood pressure levels in the hypertensive rats was ₀ observed.

B. Pigs

In order to carry out conscious animal experiments _g for the drug test the following protocol was used.

Normal farm pigs weighing approximately 50kg, were

sedated with 25 mg ketamine HC1, per Kg, IM, and anesthesia was induced with 20 mg sodium thya ylol per Kg, IV and was maintained with 1-2% halothane. A left thoracotomy was performed at the fourth or fifth rib space. Both an aortic and left atrial catheter were implanted and exteriorized through the back.

The animals were allowed to recover one week before undergoing an experiment. During the recovery period each animal was trained for the drug testing procedure. All animals were monitored daily to insure good health.

The animals were loaded into the experimental cart and the two catheters of each animal were flushed. The aortic line was used to continuously monitor pressure. During each experiment the animal received 10,000 U heparin/hour to prevent clot formation. Once a stable blood pressure was reached i.e., mean arterial pressure was maintained ±5mmHg for five minutes, the test began. The drug was then administered through the left atrial catheter with continual pressure monitoring. The time interval between drug tests was dependent on the animal and the dose previously given. The two preparations of pure recombinant kallikrein (rHK) designated as K- 011 and K-012 were used in the tests. For K-011, assuming 1 mg rHK = 1.5 2gg, 0.25 ml solution used in the test is equivalent to the dosage of 2.5 μg rHK/Kg body weight, and for preparation K-012, 0.25 ml solution is equivalent to 1.25 μg rHK/Kg body weight.

The results are shown below in Tables XI - XIV. Even at a concentration as low as 1.25 μg/kg body weight can cause significant reduction in mean atrial pressure in the conscious pig (see Table XIV)

TABLE XI

DRUG K-011

PIG 500

dose=0.25 ml dose=0.50 ml

Time HR BP MAP Time HR BP MAP

Time HR BP MAP Time HR BP MAP

cont. = control

HR = heart rate

BP = blood pressure

MAP = mean atrial pressure

TABLE XII

DRUG K-011

PIG 14

dose=0.25 ml dose=0.50 ml

Time HR BP MAP Time HR BP MAP

Time HR BP MAP Time HR BP MAP

cont. = control

HR = heart rate

BP = blood pressure

MAP = mean atrial pressure

TABLE XIII

DRUG K-012

PIG 14

dose=0.25 ml dose=0.50 ml

Time HR BP MAP Time HR BP MAP

108 73 77 81 86 86 86 86

Time HR BP MAP

cont. = control

HR = heart rate

BP = blood pressure

MAP = mean atrial pressure

TABLE XIV

DRUG K-012

PIG 43

dose=2.0 dose=4.0

0 Time HR BP MAP Time HR BP MAP

5

Q cont. = control

HR = heart rate

BP = blood pressure 5 MAP = mean atrial pressure

The longest response obtained was six minutes.

0

5

Example 16

Kininogenase Activity and Esterase Activity of Purified Recombinant Human Kallikrein Recombinant human kallikrein acted on the substrate kininogen to release kinin, i.e., kininogenase activity, an activity associated with naturally- occurring kallikrein. As represented in Table XV, the activity of the purified recombinant human mature kallikrein of the present invention was similar to the activity obtained from purified human urinary kallikrein.

Table XV Kininogenase Esterase Specific Activity (i) Unit (2)

Recombinant kallikrein 29.0 45.1 Human urinary kallikrein 28.9 57.5 Buffer alone 0 0

(1) Kininogenase specific activity is expressed as μg kinin generated/30 min/mg kallikrein and is determined in accordance with the procedures of Example 12.

(2) Esterase unit is measured using: 3H- T s-Arg-OMe as substrate and expressed as E.U./mg kallikrein and is determined in accordance with the procedures of Beaven et al., [Clin. Chi . Acta. 32, 67 (1971)].

The recombinant human kallikrein exhibited a dose response curve parallel to that of the purified human urinary kallikrein as measured by radioimmunoassay procedure of Example 7. This result indicates that the kallikrein produced by the recombinant cells have the same immunological property as naturally-occurring kallikrein produced by native cells.

The purified recombinant human mature kallikrein was analyzed in a 8-25% gradient sodium dodecyl sulfate- polyacrylamide gel and both recombinant human mature kallikrein, and naturally-occurring urinary kallikrein, have similar molecular weight a

nd both showed heterogeneity in size (Figure 2). Size heterogeneity is common for glycoproteins due to the variation in the sugar chain length.

Example 17

Effect of Recombinant Human Kallikrein on Human

Sperm Motility

10

Sperm were obtained from fertile normal donors. Purified human kallikrein (0.68 A _j 3g) was added at the final dilution of 1:10 and 1:20 to l-2xl0 ⁶ sperms in 100 μl final volume. , _g Sperm motility was measured using the procedure described by Mathur et al.. Fertility and Sterility, Vol. 46, No. 3, 484(1986) and Mathur et al. , American Journal of Reproductive Immunology and Microbiology, 12:87-89(1986) hereby incorporated by

20 reference. The results from a 48hr observation were as follows:

% Motile

Control Media 29.13

25 r-HuK (1:10 dilution) 42.76 r-HuK (1:20 dilution) 41.26

The recombinant kallikrein significantly enhanced the sperm motility. This biological activity of recombinant kallikrein is important as a therapeutic 0 agent for treating male infertility.

* * *

Numerous modifications and variations in the practice of the invention are expected to occur to _g those skilled in the art upon consideration of the foregoing illustrative examples. Consequently, the

invention should be considered as limited only to the extent reflected by the appended claims.