Minactivin compositions and antibodies to minactivin

A novel human protein, minactivin, can be produced by recombinant DNA technology, Biologically active native minactivin, peptides derived from minactivin, and their amino acid sequences can also be purified.

TECHNICAL FIELD 
The present invention relates to the production of a novel human protein, 
minactivin, by recombinant DNA technology, the characterization of the DNA 
sequence of the gene, and the expression and purification of large 
quantities of biologically active minactivin from a recombinant host. It 
also relates to the purification of biologically active native minactivin, 
as well as peptides derived from minactivin and their amino acid 
sequences. 
BACKGROUND ART 
Minactivin (PAI-2) is a naturally occurring inactivator of urokinase-type 
plasminogen activators. This type of plasminogen activator is found in 
abnormally high levels in many major human carcinomas, most notably lung, 
colon, breast and prostate. Plasminogen activators are serine proteases 
which are thought to mediate the proteolytic cascade involved in cellular 
translocation, migration and invasion. As such, they appear to be 
associated with tissue destruction and remodelling, and have been 
implicated in tumor growth and metastasis. They may also have a role in 
inflammatory reactions. 
Plasminogen activators are generally found to be of two types: 1) 
urokinase--type and 2) tissue--type. Tissue-type plasminogen activator is 
mainly found in the blood and blood vessel walls and where it is 
responsible for activating the fibrinolytic defence system against 
thrombosis. Urokinase-type plasminogen activators do not appear to play a 
role in normal thrombolytic processes but have been implicated in those 
pathological events associated with invasion and tissue destruction, in 
particular, tumor metastasis and inflammatory reactions. 
Several inhibitors specific for plasminogen activators have been described 
with include one isolated from placenta (Holmberg, L. Biochim. Biophys. 
Acta 544, 128-137 (1978) and another (PAI-1) which is produced in cultured 
vascular endothelial cells (Van Mourik, J. A. Lawrence, D. A., Loskutoff, 
D. J., J. Biol. Chem., 259, 14914-14921 (1984)). Minactivin was found to 
be produced by blood monocytes and U937 cells and appears to be 
immunologically related to the placental inhibitor. The relationship 
between these various inhibitors is presently unknown. 
As is the case with most other potent biologically active proteins, 
minactivin is produced in very small amounts in vivo, and as such, is 
difficult to purify and characterise by conventional biochemical 
approaches. Therefore,as large quantities of purified minactivin are 
required for further evaluation of its properties and biological efficacy 
in clinical applications, it is desirable to produce the protein using 
recombinant DNA techniques; that is, by cloning the minactivin gene into 
an alternate host, such as bacteria or animal cells. In order to clone 
minactivin it is desirable to purify to homogeneity the small amounts that 
can be so purified of naturally occurring minactivin in order to produce 
antibodies, amino acid sequences, peptide fragments and synthetic 
oligonucleotides derived from said purified minactivin. These reagents are 
of use in cloning strategies. 
ABBREVIATIONS 
HPLC--High pressure liquid chromatography 
M.sub.r --relative molecular mass 
MW--molecular weight 
PMA--4-phorbol-12-myristate-13-acetate 
SDS-PAGE--sodium dodecyl sulfate polyacrylamide gel electrophoresis 
TFA--trifluoroacetic acid 
HPA--human plasminogen activator 
bp--base pairs 
kb--kilobase pairs 
PU--Pluog 
DESCRIPTION OF INVENTION 
In a first embodiment, the invention provides a DNA sequence comprising a 
first DNA sequence which acts as a coding sequence for amino acid 
sequences of all, part, analogues, homologues, derivatives or combinations 
thereof of minactivin, a DNA sequence which hybridizes to said first DNA 
sequence, a DNA sequence related by mutation, including single or multiple 
base substitutions, deletions, insertions and inversion, to said first DNA 
sequence or hybridizing sequence or a DNA sequence which on expression 
codes for all, part, analogues, homogues, derivatives or combinations 
thereof a polypeptide which is minactivin or which displays similar 
immunological or biological activity in minactivin. 
A preferred DNA sequence and fragments and derivatives thereof, according 
to the invention codes for a polypeptide displaying an immunological or 
biological activity of minactivin. 
Such DNA sequences can be prepared for example from mammalian cells by 
extracting total DNA therefrom and isolating the sequences by standard 
techniques for preparation of recombinant molecules. 
Also with the scope of the invention is a process for selecting a DNA 
sequence coding for a polypeptide displaying an immunological or 
biological activity of minactivin from a group of DNA sequences, which 
process comprises the step of: determining which of said DNA sequences 
hybridises to a DNA sequence known to code for a polypeptide displaying 
said activity. 
The selected sequence may be, for example for natural sources, synthetic 
DNA sequences, DNA sequences from recombinant DNA molecules and DNA 
sequences which are a combination thereof. 
A preferred embodiment of the invention provides a process for the 
manufacture of a cDNA sequence which acts as a coding sequence for amino 
acid sequences of minactivin, which process comprises the steps of: 
stimulating cells to produce minactivin; obtaining RNA from said 
stimulated cells; isolating mRNA therefrom; and producing said cDNA from 
said mRNA. Preferably the cells are U937 cells. 
The more preferred process for molecular cloning the cDNA for minactivin 
and expression of the protein in a recombinant host includes the following 
methods: 
1. induction of a cell line for stimulated minactivin production and 
expression. 
2. isolation of mRNA from the appropriate cell line. 
3. in vitro translation of the mRNA and immunoprecipitation of the 
minactivin translation product by complex formation with urokinase. 
4. fractionation of mRNA from (2) and identification of the fraction 
containing minactivin translation activity. 
5. construction of cDNA libraries from them RNA from (2) and (4). 
6. cloning of the cDNA libraries from (5) into suitable hots, for example, 
E. coli or bacteriophage lambda. 
7. identification of clones containing the minactivin gene by: 
a) hybrid-select translation employing (3); 
b) hybridization to a chemically synthesized DNA sequence probe, especially 
a probe comprising a synthetic olignoucleotide probe according to the 
invention; 
c) differential hybridization using labelled cDNA synthesized from induced 
and noninduced mRNA; 
d) immunological screening of cDNA expression libraries using antibodies 
directed against minactivin or other immunologically related molecules; 
e) screening of cDNA expression libraries for biological activity using 
labelled urokinase or urokinase and antibodies to urokinase. 
8. extension of the cloned gene by generating dDNA libraries using 
oligonucleotides primers obtained from partial minactivin gene sequences, 
especially oligonucleotide sequences disclosed within the scope of the 
invention. 
9. determination of the nucleotide sequence of the minactivin gene, 
10. expression of the minactivin gene in E. coli and refolding to obtain 
biologically active product. 
11. expression of biologically active recombinant minactivin by cloning 
into alternate hosts, for example, eukaryotic cells. 
12. purification of recombination and clinical assessment of tis biological 
properties. 
In a second embodiment, the invention provides a recombinant DNA molecule 
with includes a first DNA sequence comprising a first DNA sequence which 
acts as a coding sequence for amino acid sequences of all, part, 
analogues, homologues, derivatives or combinations thereof of minactivin, 
a DNA sequence which hybridizes to said first DNA sequence, a DNA sequence 
related by mutation, including single or multiple base substitutions, 
deletions, insertions and inversions, to said first DNA sequence or 
hybridizing sequence or a DNA sequence which on expression codes for all, 
part, analogues, homologues, derivatives or combinations thereof of a 
polypeptide which is minactivin or which displays similar immunological or 
biological activity to minactivin. 
Preferred recombinant DNA molecules of the invention include an expression 
control sequence operatively linked to a first DNA sequence comprising a 
first DNA sequence which acts as a coding sequence for amino acid 
sequences of all, part, analogues, homologues, derivatives or combinations 
thereof of minactivin, a DNA sequence which hybridizes to said first DNA 
sequence, a DNA sequence related by mutation, including single or multiple 
base substitutions, deletions, insertions and inversions, to said first 
DNA sequence or hybridizing sequence or a DNA sequence which on expression 
codes for all, part, analogues, homologues, derivatives or combinations 
thereof a polypeptide which is minactivin or which displays similar 
immunological or biological activity to minactivin. 
A preferred recombinant DNA molecule of the invention is a plasmid which 
acts as a coding sequence for amino acid sequences of minactivin. 
A preferred plasmid of the invention has a first DNA sequence coding for a 
means of controlling expression of the DNA sequence of the invention 
linked to the DNA sequence of the invention. 
The invention also provides a fused gene comprising a portable promoter, a 
translation start site, and a gene coding for human minactivin. 
Also within the scope of the invention is a process for the manufacture of 
a recombinant DNA molecule, which process comprises the step of: 
introducing into a cloning vehicle, a first DNA sequence comprising a 
first DNA sequence which acts as a coding sequence for amino acid 
sequences of all, part, analogues, homologues, derivatives or combinations 
thereof of minactivin, a DNA sequence which hybridizes to said first DNA 
sequence, a DNA sequence related by mutation, including single or multiple 
base substitutions, deletions, insertions and inversions, to said first 
DNA sequence or hybridizing sequence or a DNA sequence which on expression 
codes for all, part, analogues, homologues, derivatives or combinations 
thereof a polypeptide which is minactivin or which displays similar 
immunological or biological activity to minactivin. 
Preferably the process also includes the step of introducing an expression 
control sequence in the cloning vehicle. 
The invention further provides a process for the manufacture of a plasmid 
which acts as a coding sequence for amino acid sequences of all, part, 
analogues, homologues, derivatives or combinations thereof of minactivin, 
which process comprises combining a plasmid with a DNA sequence which acts 
as a coding sequence for said amino acid sequences, and preferably with an 
expression control sequence. The DNA sequence is preferably a cDNA 
sequence. 
In a third embodiment, the invention provides a host transformed with at 
least one recombinant DNA molecule which includes a first DNA sequence 
comprising a first DNA sequence which acts as a coding sequence for amino 
acid sequences of all, part, analogues, homologues, derivatives or 
combinations thereof minactivin, a DNA sequence which hybridizes to said 
first DNA sequence, a DNA sequence related by mutation, including single 
or multiple base substitutions, deletions, insertions and inversions, to 
said first DNA sequence or hybridizing sequence or a DNA sequence which on 
expression codes for all, part analogues, homologues, derivatives or 
combinations thereof of a polypeptide which is minactivin or which 
displays similar immunological or biological activity to minactivin. 
Suitable hosts include bacteria, yeasts, other fungi, mice or other animal 
hosts, plant hosts, insects hosts and other eukaryotic hosts e.g. 
mammalian, including human tissue cells. Suitable bacteria include E. 
coli, Pseudomonas species, and Bacillus spaces. 
Especially preferred is a microorganism with the genetic information for 
the biosynthesis of minactivin. 
Also included within the invention is a process for transforming a host, 
which process comprises the step of: introducing into a host a recombinant 
DNA molecule which includes a first DNA sequence comprising a first DNA 
sequence which acts as a coding sequence for amino acid sequences of all, 
part, analogues, homologues, derivatives or combinations thereof of 
minactivin, a DNA sequence which hybridizes to said first DNA sequence, a 
DNA sequence related by mutation, including single or multiple base 
substitutions, deletions, insertions and inversions, to said first DNA 
sequence or hybridizing sequence or a DNA sequence which on expression 
codes for all, part, analogues, homologues, derivatives or combinations 
thereof a polypeptide which is minactivin or which displays similar 
immunological or biological activity to minactivin. 
The invention also provides a process for the manufacture of a 
microorganism with the genetic information for the biosynthesis of all, 
part, analogues, homologues, derivatives or combinations thereof of 
minactivin, which process comprises transforming a microorganism with a 
plasmid or other vector which acts as a coding sequence for amino acid 
sequences of all, part analogues homologues, derivatives or combinations 
thereof minactivin. 
In a fourth embodiment, the invention provides a process for the 
preparation of peptides derived from purified minactivin which process 
comprises purifying minactivin to homogeneity then obtaining amino acid 
sequences unique to minactivin. 
A preferred embodiment of this process comprises: 
a) Culturing a cell line capable of expressing minactivin; 
b) harvesting the supernatant; 
c) concentrating the supernatant; 
d) dialysing the supernatant, then centrifuging said culture supernatant to 
remove residual cell debris and protein which may have precipitated during 
dialysis; 
e) fractionating the culture supernatant chromatographically and 
electrophoretically; 
f) concentrating the fraction containing minactivin activity; 
g) analysing the fraction containing minactivin activity to demonstrate 
purity; 
h) obtaining amino acid sequences unique to minactivin. 
In a preferred form the process comprises: 
a) culturing a minactivin producing culture or cell line; 
b) harvesting the culture supernatant and concentrating said culture 
supernatant; 
c) dialysing the culture supernatant, then centrifuging said culture 
supernatant to remove residual cell debris and protein which may have 
precipitated during dialysis; 
d) fractionating the culture supernatant by ion exchange chromatography; 
e) pooling and concentrating the eluates of highest minactivin specific 
activity; 
f) fractionating the pooled, concentrated eluates by gel filtration 
chromatography; 
g) concentrating the eluate then isoelectrofocussing said eluate; 
h) probing fractions isolated from the isoelectrofocussing gel with 
antibodies reactive with minactivin, to locate the minactivin band; 
i) concentrating the fraction containing minactivin activity; 
j) further fractionating the fraction containing minactivin activity by 
partition chromatography than analysing the purified fraction containing 
minactivin activity by gel electrophoresis; 
k) digesting the purified minactivin and separating the resulting peptides 
by partition chromatography. 
In a more preferred form the culture is of the human macrophage cell line 
U937. Preferred culture conditions include culturing in the absence of 
serum and/or in the presence of a sufficient amount of a substance or 
substances which will inhibit urokinase production or induce constitutive 
production of minactivin. A suitable substance for this purpose is 
dexamethasone which is preferably used at a concentration of 1 .mu.M. The 
culture may also be grown in the presence of PMA. A preferred 
concentration range of PMA in the culture is 1-300 ng/ml, more preferably 
10-30 ng/ml. 
A preferred volume of harvested culture supernatant is 4-5 liters. The 
initial concentration step is preferably a 10-fold concentration step. A 
suitable apparatus for this concentration is an Amicon DC2 Hollow Fibre 
Dialysis/Concentration unit equipped with a 30,000MW cut of cartridge. 
The dialysis according to step c) is preferably with a dialysate such as 50 
mM glycine, ph7.8 More preferably the 50 mM glycine pH7.8 dialysate should 
be used at at least equal volume to the volume of the sample being 
dialysed against said dialysate. 
The ion exchange chromatography according to step d) is preferably 
performed on a phenyl-sepharose column, the elution being preferably a 
step pH elution. More preferably, for the pH step elution, the ionic 
strength of the supernatant should be adjusted to 2M, especially this may 
be by the addition of solid NaCl, then the pH should be adjusted to 5.5 
preferably with citric acid. A preferred equilibrant for the 
phenyl-sepharose column is a solution of 50 mM Na citrate pH5.5, 2M NaCl 
and 1 mM EDTA. The column may be eluted initially with equilibration 
buffer, then with 50 mM Na citrate pH5.5 containing 0.5M NaCl and 1 mM 
EDTA and finally with 50 mM glycine pH9.0. 
The concentration of the sample according to step g) is preferably 
performed on an Amicon YM10 membrane, with a final concentrate volume of 3 
ml. The isoelectrofocussing step is preferably performed on a preparative 
flatbed gel of Ultrodex containing Ampholines in the pH range 4.5 to 6.0. 
More preferably the gel is electrofocussed at 10.degree. C. for 23 hours. 
On an LKB Multiphor isolelectrofocussing apparatus. A preferred elutant 
for proteins from the electrofucssing gel is 1M glycine containing 1 mM 
EDTA pH9.0, more preferably in a 10 ml volume. Suitable antibodies 
according to step h) include goat anti-placental inhibitor antibodies. 
The concentration according to step 1) may be performed on an Amicon YM10 
membrane. 
The partition chromatography according to step j) is preferably HPLC, more 
preferably performed on a Vydac C-4 reverse phase column using a Waters 
high pressure liquid chromatograph. The elution gradient is preferably 
acetonitrile in 0.1% TFA. Gel electrophoresis according to step j) is 
preferably SDS-PAGE. 
Digestion of the purified minactivin, according to step k) is preferably 
with endoproteinase LysC. Suitable digestion conditions include 3-5 .mu.g 
minactivin with 0.1 .mu.g endoproteinase LysC in 20 mM Tris-Cl ph8.5., 5M 
urea, at a volume of 50 .mu.l and 22.degree. C. for 8 hours. A suitable 
form of partition chromatograph is reverse phase HPLC, particularly 
employing a Synchropak RP-P(C-8) column with a gradient of acetonitrile in 
0.1% TFA. 
In a fifth embodiment the invention provides minactivin in substantially 
pure form. Preferably said minactivin is purified to homogeneity. 
In a sixth embodiment the invention provides purified minactivin when 
prepared by a process according to the invention. 
In a seventh embodiment the invention provides peptides derived from 
purified minactivin and peptides displaying similar immunological or 
biological activity to said peptides. 
Preferred peptides according to the invention include peptides of the 
following sequences and which are also set forth in SEQ. ID. Nos. 1, 2, 3, 
4 and 5, respectively; 
AQILELPY-GDV-MFLLLP-3 . . 
GRANFSGMSE-NDLF. . . 
MAE-EVEVYIPQFKLEE-Y. . . 
LNIGYIEDLK 
IPNLLPEG-V 
The invention also provides peptides according to the invention when 
prepared by a process according to the invention. 
In an eighth embodiment, the invention provides a microbiologically 
prepared peptide, all or part of which contains the amino acid sequence of 
all, part, analogues, homologues, derivatives or combinations thereof 
minactivin. 
A peptide and fragments and derivatives thereof which display an 
immunological or biological activity of minactivin are also within the 
scope of the present invention. 
The preferred peptide or fragments or derivatives thereof are coded for by 
a DNA sequence which hybridises to a DNA sequence which acts as a coding 
sequence for amino acid sequences of minactivin and displays the 
biological or immunological activity of minactivin, which activity is 
destroyed by antisera to minactivin. 
The invention also provides a process for the manufacture of all, part, 
analogues, homologues, derivatives or combinations thereof of 
unglycosylated minactivin, which process comprises the steps of: obtaining 
the genetic information for the biosynthesis of minactivin using mRNA from 
cells of monocytic lineage; incorporating the resulting gene into a 
microorganism; selecting and culturing said microorganism to produce said 
minactivin; and collecting said minactivin. 
The invention further provides a process for the manufacture of a peptide 
displaying an immunological or biological activity of minactivin, which 
process comprises the steps of: culturing a host which has been 
transformed with recombinant DNA molecule which includes a first DNA 
sequence comprising a first DNA sequence which acts as a coding sequence 
for amino acid sequences of all, part, analogues, homologues, derivatives 
or combinations thereof of minactivin, a DNA sequence which hybridizes to 
said first DNA sequence, a DNA sequence related by mutation, including 
single or multiple base substitutions, deletions, insertions and 
inversions, to said first DNA sequence or hybridizing sequence or a DNA 
sequence which on expression codes for all, part, analogues, homologues, 
derivatives or combinations thereof a polypeptide which is minactivin or 
which displays similar immunological or biological activity to minactivin. 
The invention also provides a reagent for locating and defining the 
boundaries of tumours in histological specimens or in vivo which reagent 
comprises suitable labelled minactivin, especially recombinant DNA derived 
minactivin, or fragments of minactivin and the associated method of 
locating and defining the boundaries of tumours is histological specimens 
or in vivo whichcomprises applying or administering suitably labelled 
minactivin or fragments thereof and subsequently imaging to determine the 
site of concentration of the label. 
The invention further provides a method of inhibiting tumour invasion and 
treating tumours comprising administering to a patient requiring such 
treatment a therapeutically effective amount of minactivin, suitably 
labelled minactivin, fragments of minactivin or labelled fragments of 
minactivin; a method of treatment of chronic inflammation such as 
rheumatoid arthritis comprising administering to a patient requiring such 
treatment a therapeutically effective amount of minactivin or fragments of 
minactivin; and a method of monitoring chronic inflammation comprising the 
detection of minactivin in samples of body fluids and tissues using 
antibodies prepared against minactivin or fragments of minactivin. 
Also included within the invention are antibody preparations prepared 
against minactivin including recombinant minactivin, purified natural 
minactivin and fragments thereof. The invention also provides therapeutic, 
diagnostic or phrophylactic compositions which comprise minactivin, 
especially recombinant DNA derived minactivin, fragments of minactivin or 
antibodies to minactivin or fragments of minactivin and a pharmaceutically 
acceptable non-toxic carrier or diluent therefor. 
The invention further provides synthetic olignoculeotide probes, the 
sequence of said probes comprising a first nucleotide sequence which on 
expression codes for the amino acid sequence of a peptide according to the 
invention, a nucleotide sequence sufficiently related to said first 
nucleotide sequence to hybridize to said first nucleotide sequence or a 
DNA sequence related by mutation including single or multiple base 
insertions, inversions deletions or substitutions to said first nucleotide 
sequence. 
Included within the scope of the invention is a process for the production 
of said synthetic oligonucleotide probes which process comprises 
determining the amino acid sequence of peptide fragments derived from 
purified minactivin and synthesizing corresponding oligonucleotides. In a 
preferred form said synthesis is performed on an Applied Biosystems 380A 
DNA synthesizer. 
The invention provides formulations comprising synthetic olignoucleotide 
probes according to the invention. 
Preferably said formulations are diagnostic reagents. 
The invention also provides a method for the detection of human carcinomas 
and inflammatory conditions and susceptibility thereto which method 
comprises using a formulation comprising said synthetic oligonucleotide 
probe in an assay designed for the detection of DNA coding for minactivin. 
Deficiency in ability of tissues to produce minactivin may be related to 
susceptibility to carcinomas and inflammatory conditions. Detected 
deficiencies may be treated by administration of purified minactivin to 
the patient, and may also serve as a marker for tissues affected by 
carcinomas and inflammation.

BEST MODE OF CARRYING OUT THE INVENTION 
Induction of U937 cell line for enhanced minactivin synthesis 
Minactivin has been found to be produced by induced human monocytes, 
certain macrophages, and transformed cells of monocytic lineage (refer to 
international patent application WO86/01212). The transformed cell line 
U937 (ATCC CRL 1593) was found to produce minactivin constitutively in the 
presence of dexamethasone. The level of minactivin secreted by these cells 
under serum free conditions was found to be only about 0.06% of the total 
protein secreted by these cells. It was found that this level could be 
enhanced by approximately an order of magnitude to 0.4% with the addition 
of 4-phorbol-12-myristate-13-acetate (PMA). The effect of PMA on 
minactivin secretion with time followed biphasic course with an initial 
lag per of 6 hours, followed by a linear increase in minactivin activity 
up to 60 hours (FIG. 1). No differences were observed by increasing the 
PMA concentration from 10 ng/ml to 30 ng/ml. Furthermore, it was 
determined that the phorbol esters were tightly associated with the cells, 
as radioloabelled PMA could be detected only in small amounts (less than 
10%) in the culture supernatants ever after 17 hours. 
The following examples illustrate preferred embodiments of the invention. 
They should not be construed as limiting on the scope of the invention. 
Unless otherwise stated, all parts and percentages are by weight. 
EXAMPLE 1 
Minactivin activity was measured by a modification of the method of Coleman 
and Green N.Y. Acad. Sci. 370, 617 (1981), as described by Stephens et al 
Eur. J. Biochem. 136, 517-522 (1983), in which the inhibitory activity of 
minactivin was determined by quantifying the loss of urokinase activity in 
the colorimetric assay using a urokinase reference standard (Calbiochem). 
The minactivin samples were preincubated with 4 mPU urokinase for 90 
minutes at 23.degree. C. before the addition of plasminogen. One unit of 
minactivin activity was defined as that amount which inhibited 1 Plough 
wait of urokinase. Human urokinase was purchased from Calbiochem Behring 
Corp. La Jolla, Ca. Plasminogen was purified from fresh human plasma by 
lysine-sepharose (Pharmacia) affinity chromatography (Unkeless, J. C. et 
al. (1974), J. Biol. Chem. 249, 4295-4305). 
The protein concentration was determined according to the method of 
Bradford, M. M., Anat. Biochem. 72, 248-254 (1976) using bovine serum 
albumin as the standard. Specific activity is defined as the minactivin 
activity as measured by colorimetric assay divided by the protein 
concentration. 
Proteins were separated by SDS-polyacrylamide gel electrophoresis using 11% 
Laemmli gels (Laemmli, U. K., nature 227, 680-685, 1970) or on 
SDS-urea-gradient polyacrylamide gels using a modified Laemmli buffer 
system as described by Mattick, J. S. et al Eur. J. Biochem. 114, 643-651 
(1981). Western (Transfer) blotting was performed by electrophoretic 
transfer to nitrocellulose as described previously (Towbin, H. et al., 
Pro. Natl. Acad. Sci. USA, 76, 4350-4354 and Johnson, D. A. et al, Gene 
Anal. Tech. 1, 3-8 1984). 
Cell Culture 
The human macrophage cell line, U937, was cultured in RPMI 1640 containing 
10% foetal calf serum and 1 micromolar dexamethasone, either in T175 
culture flasks or in a 10 liter Braun fermenter. The cells were maintained 
at densities of 1- 3.times.10.sup.6 cells/ml. Although minactivin was 
secreted by the cells during this growth phase, the cells were transferred 
to serum-free medium to obtain supernatants for minactivin purification. 
The cells were pelleted by low speed centrifugation, washed by 
resuspension in phosphate buffered saline and recentrifugation and then 
resuspended in serum free RPMI 1640 containing 1 micromolar dexamethasone, 
and cultured for a period of three days. The level of minactivin secreted 
by these cells under serum free conditions could be enhanced by 
approximately an order of magnitude to 0.4% with the addition of PMA. 
The cells were then harvested and the supernatants used in the purification 
scheme which follows. 
EXAMPLE 2 
Purification of Homogeneous Minactivin 
a) Concentration of Serum Free Minactivin Supernatants 
Typically, 4 to 5 liter of culture supernatant was concentrated10-fold 
using an Amicon DC2 Hollow Fiber dialysis/Concentration unit equipped with 
a 30,000 MW cut-off cartridge. The concentrate was then dialysed using the 
DC-2 Hollow fibre unit by repeated concentration and dilution using at 
least an equal volume of 50 mM glycine pH 7.8 for 3 to 6 hours at room 
temperature, to remove all traces of dye. 
b) Centrifugation of Minactivin Concentrate 
The dialysed concentrate was centrifuged in a JA10 rotor at 8000 rpm for 30 
min at 4.degree. C. to pellet residual cell debris and protein that may 
have precipitated during dialysis. The clarified supernatant is then 
aliquoted and frozen at -20.degree. C. until required for subsequent 
purification. 
c) Phenyl-Sepharose Chromatography using a Step pH Elution 
Minactivin was further purified from ten-times concentrated culture 
supernatant obtained from cells cultured in the absence of PMA by step pH 
elution using phenyl-sepharose as follows. 
The ionic strength of the supernatant (200 ml; 12000 units; specific 
activity 102 units/mg) was adjusted to 2M by the addition of solid NaCl 
and the pH adjusted to 5.5 with citric acid. This solution was applied to 
a phenyl-sepharose column (4.4 cm.times.5.0 cm) equilibrated in 50 mM Na 
citrate, pH5.5, 2M NaCl and 1 mM EDTA and eluted with the same buffer 
until the baseline absorbance at 280 nm (A280) returned to baseline. The 
column was then eluted with 50 mM sodium citrate, pH5.5 containing 0.5M 
NaCl and 1 mM EDTA and again the A280 monitored until the absorbance 
returned to baseline. The minactivin was then eluted from the column with 
50 mM glycine, ph9.0. FIG. 14 shows the elution profile. 
The recovery of minactivin by this method was 9553 units which represents 
80% of the units applied to the column. The material of highest specific 
activity was pooled (6700 units: specific activity 1343 units/mg) and 
concentrated to 3 ml on an Amicon YM10 membrane. 
d) Sephacryl S-200 Gel Permeation Chromatography 
The pooled, concentrated minactivin was applied to a 2.2 cm.times.78 cm 
column of Sephacryl S-200 equilibrated with 0.1M sodium borate, ph9.0. 
Fractions of 5.0 ml were collected at a flow rate of 0.46 ml/min. FIG. 8 
shows that minactivin was eluted at the tailing edge of the major protein 
peak. The fractions containing minactivin activity were pooled (4480 
units; specific activity 1355 units/mg) and concentrated to 3 ml using a 
YM10 membrane. Calibration of this column with known M.sub.r standards 
indicated that minactivin had an M.sub.r of 45-48 kD. 
e) Isoelectric Focussing 
The concentrated minactivin solution was applied to a preparative flat bed 
gel of Ultrodex containing Ampholines in the pH range 4.5-6.0 and 
electrofocussed for 23 hrs at 10.degree. C. on an LKB Multiphor 
isoelectric focussing apparatus. Following completion of the run, 30 zones 
across the length of the gel were scraped out and the protein eluted from 
each with 10 ml of 1M glycine containing 1 mM EDTA, pH9.0. Aliquots of 
each fraction were assayed for minactivin activity and electrophoresed on 
15% SDS-polyacrylamide gels to locate protein. FIG. 15 illustrates that a 
significant amount of protein has been removed from the fractions 
containing the minactivin activity. Under these conditions minactivin 
focusses between pH5 and pH5.2 and within this region of the gel 15% of 
the total activity applied to the gel was recovered. 
In fact, in the region of the isoelectric focussing gel containing 
minactivin activity, only two protein bands are visible (FIG. 15). To 
determine which of these bands is minactivin the protein on an equivalent 
polyacrylamide gel was transferred onto nitrocellulose and probed with 
antibodies made in goat to placental inhibitor. Due to similar biological 
properties it was considered likely that the two proteins would be 
immunologically related. As shown in FIG. 16 the protein band of M.sub.r 
=45-48 kD specifically cross reacts with the anti-placental inhibitor 
antibodies suggesting that this protein band is minactivin. Furthermore, 
this observation is consistent with the M.sub.r of 45-48 kD determined for 
native minactivin on gel permeation chromatography. 
f) High Pressure Liquid Chromatography 
The fractions from the isoelectric focussing above which contained 
minactivin activity were concentrated 10-fold on an Amicon YM10 
ultrafiltration membrane and further fractionated on a Vydac C-4 reverse 
phase column using a Waters high pressure liquid chromatograph. The 
proteins were eluted from the reverse phase column using a gradient of 
acetonitrile in 0.1% TFA as shown in FIG. 17. Each of the absorbance peaks 
was examined by SDS-PAGE and peak 5 was found to contain pure minactivin 
(FIG. 18). 
EXAMPLE 2a 
(a) Gel Filtration 
Call free supernatants were processed through steps (a) and (b) as 
described in Purification Example 2 of WO86/01212, and then through 
Phenyl-Sepharose using a step pH elution as described in Purification 
Example 1 of WO86/01212. The fractions containing minactivin activity were 
pooled, concentrated by precipitation with 85% saturated ammonium sulphate 
and applied to a 2.2 cm.times.80 cm column of Sephacryl S-200 equilibrated 
in 0.1M sodium borate, pH9.0. Fractions of 3.5 ml were collected at a flow 
rate of 0.46 ml/min. FIG. 8 shows that minactivin was eluted as the 
tailing edge of the major protein peak and had a peak specific activity of 
2206 Units/mg representing an overall increase in specific activity of 31 
fold. Under these conditions the minactivin behaves as a molecule with a 
Stokes radius similar to ovalbumin, suggesting a molecular size of 
45-49.times.10.sup.3 daltons. 
(b) Phenyl-Boronate Agarose Chromatography 
Cell free supernatants were processed through steps (a) and (b) as 
described in Purification Example 2 of WO86/01212. one ml of the 
supernatant was made to 10 mM in MgCl.sub.2 and the pH then adjusted to 
pH8.5 with sodium hydroxide. This solution was applied to a column of 
phenyl-boronate agarose -30 (PBA 30) (0.8 cm.times.2.5 cm) equilibrated in 
50 mM glycine, pH8.5 containing 10 mM MgCl.sub.2 at 4.degree. C. The 
column was then washed with 9 ml of the above buffer and then serially as 
follows: 
a) 10 ml of 50 mM glycine, pH8.5 containing 10 mM EDTA 
b) 10 ml of 50 mM glycine, pH8.5 containing 100 mM sorbitol 
c) 10 ml of 100 mM Tris-HCl, pH8.5 
d) 10 ml of 50 mM sodium acetate, pH 5.0. 
Fractions of 5 ml were collected and dialysed against 50 mM glycine, pH7.8 
overnight at 4.degree. C. prior to minactivin activity and protein 
determinations. The results shown in FIG. 9 illustrate that two distinct 
peaks of activity elute from the column under different conditions. The 
first peak, eluted with EDTA, contains 35% of the total activity loaded 
onto the column with an increase in specific activity of 14 fold. The 
second peak represents 32% of the initial activity with a 4.4 fold 
increase in specific activity. 
(c) Chromofocussing 
Cell free supernatants were processed through steps (a) and (b) as 
described in Purification Example 2 of WO86/01212. Four ml of this 
supernatant was dialysed against 25 mM imidazole-HCl buffer, pH7.4 
overnight at 4.degree. C. and then applied to a PBE 94 chromofocussing 
column (1 cm.times.27 cm) equilibrated in the above buffer. A linear pH 
gradient was then established by applying 200 ml of polybuffer pH 4.0 and 
4 ml fractions were collected into 4 ml aliquots of 1M Tric.HCl, pH7.5. 
Every 10 fractions were pooled, concentrated and washed on a centricon 30 
and assayed for minactivin activity and protein concentration. FIG. 10 
shows that the majority of the activity eluted near pH5. The overall 
recovery of activity was 87% and there was a 2 fold increase in specific 
activity. 
(d) Isoelectric Focussing 
Cell free supernatants were processed through steps (a) and (b) as 
described in Purification Example 2 of WO86/01212, and then through phenyl 
Sepharose using a step pH elution as described in Purification Example 1 
of WO86/01212. The fractions containing minactivin activity were pooled, 
concentrates by precipitation with 85% saturated ammonium sulphate and 
dialysed overnight against 50 mM glycine pH9.0. This solution was applied 
to a preparative flat bed gel of Ultrodex containing Ampholines in the pH 
range 4.5-6.0 and electrofocussed for 23 hrs at 10.degree. C. on a LKB 
Multiphor isoelectric focussing apparatus. Following completion of the 
run, 30 zones across the length of the gel were scraped out and the 
protein eluted from each with 10 ml of 1M glycine containing 1 mM EDTA, 
pH9.0. Aliquots of each fraction were assayed for minactivin activity and 
electrophoresed on 15% SDS-polyacrylamide gels to locate protein. FIG. 11 
illustrates that a significant amount of protein has been removed from the 
fractions containing the minactivin activity. Under these conditions 
minactivin focusses between pH5 and pH5.2 and within this region of the 
gel 39% of the total activity applied to the gel was recovered. 
(e) Immunoaffinity Chromatography 
Cell free supernatants were processed as through Purification Example 1. A 
4.6 ml aliquot of this minactivin preparation (2300 Units, 2.25 mg, 
specific activity 1020U/mg) was made 0.05M in sodium phosphate, 0.5 in 
NaCl, 0.01% in TritonX-100, 0.1% in sodium azide, 1 mM in EDTA and the pH 
adjusted to 7.5 . This solution was diluted to 15 ml with the above buffer 
and added to 15 ml of Sepharose 4B to which 10 mg of anti-placental 
inhibitor antibody had been chemically coupled using the 
1,1'-carbonyl-diimidazole method of Bethell, G. S. et al J. of Biol. Chem. 
254 (8) 2572-2574 (1979). The slurry was shaken overnight at 4.degree. C. 
and then poured into 2.5 cm.times.3.1 cm column. Unbound protein was 
drained from the column and the column washed with the above buffer until 
the absorbance at 280 nm returned to baseline. The column was then eluted 
with 3M KSCN containing 10 mM tris. HCl, pH8.0. The elution profile is 
shown in FIG. 12. The fractions eluted by the KSCN were concentrated 8.5 
fold on a Centricon 10, washed with 40 mM glycine, pH 7.8 and analysed for 
minactivin activity and by SDS-PAGE. The majority of the minactivin 
activity did not bind to the antibody column. However, a small amount of 
minactivin activity (8.5 units) is bound specifically and is eluted with 
3M KSCN. This indicates that under these conditions the antibody column 
has been overloaded with minactivin. Furthermore, minactivin loses over 
90% of its activity in the presence of KSCN over a comparable period of 
time suggesting that the low recovery of minactivin activity may be due in 
inactivation of the molecules in KSCN. The SDS-PAGE results show that the 
vast majority of the protein elutes unretarded from the column. The KSCN 
eluate however contains a major protein band of molecular weight ca 
45,000, similar to the molecular size of minactivin on gel filtration (see 
Example 2A(a) (FIG. 12a). Western analysis of this minactivin preparation 
showed a single immunologically cross reactive species migrating 
identically with the protein band observed following SDS-PAGE (FIGS. 
12b-12c). 
Under certain conditions, minactivin has been observed to have a molecular 
size of approximately 60-70,000 (as detailed in PCT191-85). This 
discrepancy may be due to altered mobility due to the degree of 
glycosylation of minactivin. 
EXAMPLE 3 
Isolation and Sequence of Peptide Fragments from Minactivin 
Minactivin was purified from PMA induced U937 cells as described in Example 
2 above. The minactivin (3-5 .mu.g) was then digested with endoproteinase 
Lys C (0.1 .mu.g) n 20 mM Tri-HCl, pH 8.5 containing 5M Urea in a final 
volume of 50 .mu.l for 8 h at 22.degree. C. The resultant peptides were 
separated by reverse phase high pressure liquid chromatography on a 
Synchropak RP-P (C-8) column using a gradient of acetonitrile in 0.1% TFA 
(FIG. 19). The peptides indicated by the asterisks were sequenced on an 
Applied Biosystems 470A gas phase sequencer and the sequences are as 
follows (and are also set forth in SEQ. ID. NOs. 1, 2, 3, 4 and 5, 
respectively). 
Peptide 13: AQILELPY-GDV-MFLLLP-E . . . 
Peptide 11; GRANFSGMSE-NDLF . . . 
Peptide 10: MAE-EVEVYIPQFKLEE-Y . . . 
Peptide 6: LNIGYIEDLK 
Peptide 9: IPNLLPEG-V 
EXAMPLE 4 
Molecular Cloning of Minactivin 
a) Isolation of mRNA 
From FIG. 1, the optimal time of transcription for PMA induced U937 cells 
could be estimated to be between 15 and 25 hours. Therefore, a four liter 
serum-free culture of U937 cells at a cell density of 1.2.times.10.sup.6 
cells/ml was incubated for 19 hours in the presence of PMA, the cells 
harvested, and quick frozen in liquid nitrogen until further use. Non-PMA 
stimulated U937 cells from three day serum-free cultures were also 
retained for mRNA isolation. Human blood monocytes prepared as described 
international patent application WO86/01212, and cultured for 3 days in 
vitro were also used as a source of mRNA. 
Total RNA from each of the above sources was extracted by a modification of 
the Guanidin-HCL method [Chirgwin, J. M. et al Biochemistry 18 5294 
(1979)]. The cell pellet was homogenized in 20 volumes (per gram weight) 
of buffer containing 4M quanidine isothiocyanate, 50 mM Tris HCl, ph7.5, 
10 mM EDTA, 0.5% Sarkosyl, 0.1M 2-mercaptoethanol in a blender at low 
speed for three minutes at 4.degree. C. The suspension was then 
centrifuged at 5,000 x g for 10 minutes at 4.degree. C. to remove debris. 
Subsequent centrifugations were carried out at 5-10,000 x g unless 
specified otherwise. Nucleic acids were precipitated from the supernatant 
by the addition of acetic acid to 25 mM and 0.75 volumes of cold ethanol, 
and incubated overnight at -20.degree. C. The suspension was centrifuged 
again for 30 minutes at -10.degree. C., and the pellet dissolved in buffer 
containing 7.5M guanidine HCl, 20 mM sodium acetate pH5.0, 1 mM 
dithiothreitol at 20% of the original volume. After centrifuging to remove 
any undissolved material, the RNA was reprecipitated with 0.55 volumes of 
cold ethanol at -20.degree. C. for 1-3 hours. The RNA was recovered by 
centrifugation, redissolved in the guanidine HCl buffer, and 
reprecipitated. The last step was repeated 3 times. Following the last 
precipitation,the pellet was dissolved in 20 ml of 20 mM EDTA, pH7.0 and 
extracted with an equal volume of chloroform: butanol (4:1). RNA was then 
precipitated from the aqueous phase by the addition of sodium acetate, 
pH5.0 to 0.3M and two volumes of cold ethanol at -20.degree. C. overnight. 
The RNA was recovered by centrifugation and treated with 100 mg/ml 
proteinase K in 20 mM HEPES, pH7.4, 0.5% sodium dodecyl sulfate for 4 
hours at 50.degree. C. to remove any residual protein. The RNA was then 
recovered by precipitation in the presence of 0.2M sodium acetate, pH5.0 
and two volumes of ethanol at -20.degree. C. Following recovery by 
centrifugation, any residual DNA was removed by precipitation of the RNA 
in the presence of 3M sodium acetate, pH6.0, overnight at 4.degree. C. The 
RNA was recovered by centrifugation at 15,000 x g at 4.degree. C. for 1 
hour and precipitated in the presence of 0.2N sodium chloride and two 
volumes of ethanol. The RNA was again recovered by centrifugation. Poly 
A.sup.+ mRNA was then isolated by two cycles of adsorption and elution 
from oligo (dT)-cellulose [Aviv, H. Leder, P. Proc. Natl. Acad. Sci. USA 
69 1408 (1972)]. 
The poly A.sup.+ mRNA was enriched 10 to 20 fold for minactivin mRNA by 
sucrose density gradient centrifugation. The sample was layered on a 15 to 
34% (w/w) sucrose gradient and centrifuged in a Beckman SW41 rotor at 
33,000 rpm for 16 hours at 4.degree. C. FIG. 2 shows a gel analysis under 
denaturing conditions of the size fractionated mRNA preparation. 
Minactivin mRNA was detected in those fractions (Fractions 16 and 17) 
centered around the 18S ribosomal RNA standard as determined by in vitro 
translation and immunoprecipitation (method described below) as shown in 
FIG. 3. 
b) Identification of the Minactivin Translation Product 
Minactivin mRNA was identified by in vitro translation in a cell free 
reticulocyte lysate followed by immunoprecipitation of the minactivin 
translation product utilizing its natural substrate, urokinase. 
Rabbit reticulocyte lysate commercially available from Amersham, was used 
primarily according to the manufacturer's instructions with the addition 
of calf liver tRNA (Boehringer Mannheim) at a concentration of 100 ng/ml. 
.sup.35 S-methanionine (Amersham) was added at a concentration of 2 mCi/ml 
to allow detection of the translation products by autoradiography. Poly 
A.sup.+ mRNA prepared as described above was translated at a concentration 
of 50 mg/ml for 90 minutes at 30.degree. C. Twenty-five microliters of the 
translation mixture was used for each immunoprecipitation. Following 
incubation and removal of a washed suspension of whole Staphylococcus 
aureus cells (Pansorbin, Calibiochem) to minimize nonspecific binding, the 
sample was incubed with 50 mPU of urokinase (Calibochem) for 90 minutes at 
room temperature. This step allows complex formation between the 
minactivin translation product and urokinase. The complex was removed from 
the solution by the addition of 1-2 microliters of anti-urokinase 
antiserum (Green Cross Corp.), or antibodies against placental inhibitor 
and incubated at room temperature for 30 minutes and overnight at 
4.degree. C., and then precipitated by the addition of 25 microliters of 
washed Pansorbin. After centrifugation the 
minactivin-urokinase-antibody-Pansorbin pellet was washed by repeated 
centrifugation and resuspension in 0.05% Nonidet-P40, 0.15M NaCl, 5 mM 
EDTA, 50 mM Tris HCl pH 8.0, 0.025% sodium azide, disrupted by boiling in 
the presence of 2% SDS, and 2-mercaptoethanol, and the products analyzed 
by gel electrophoresis followed by autoradiography. 
Immunoprecipitation of the .sup.35 S-labelled translation products with 
antibodies against urokinase yielded urokinase specific translation 
products having M.sub.r S of 69,000 and 79,000. These protein bands 
represent specific complexes of minactivin with urokinase as: 
1) they are not present in the absence of urokinase or mRNA; 
2) they do not precipitate in the absence of antibody, and; 
3) they compete with the unlabelled purified minactivin and placental 
inhibitor (Calibiochem) preparations for urokinase binding (FIG. 4). 
The immunoprecipitated product was found to represent 0.05% of the total 
protein synthesized from mRNA obtained from PMA induced U937 cells. No 
immunoprecipitation products could be detected from mRNA obtained from 
non-induced U937 cells, presumably due to the decreased levels of 
minactivin mRNA in this preparation. 
Immunoprecipitation of the urokinase--minactivin translation products using 
antibodies to placental inhibitor yielded identical results. Several 
anti-placental inhibitor antibody preparations precipitated the 
distinctive urokinase-minactivin translation product complexes at 69,000 
and 79,000 MW (FIG. 5). 
A comparison of the immunoprecipitation products obtained in the presence 
and absence of urokinase allows direct identification of the minactivin 
translation product as shown in FIG. 6. It is present as a distinct band 
at a M.sub.r of 43,000. This molecular weight appears to be slightly less 
than that observed for the native protein possibly due to glycosylation. 
In the presence of urokinase, this band disappears and the characteristic 
urokinase-minactivin translation product is detected at 69,000 M.sub.r. 
The additional protein band at 79 to 80,000 M.sub.r observed previously 
appears to represent a non-reduced form of the complex as the samples were 
analyzed under partially reduced conditions. 
Furthermore, it was found that complex formation with the minactivin 
translation product was dependent on the presence of the low molecular 
weight form of urokinase (HPA 33). Pure preparations of HPA 52 and HPA 33 
was obtained (Calibiochem) and verified to be predominantly one species or 
the other by fibrin overlay (FIG. 7). In addition, plasminogen/plasmin was 
added to HPA 33 to convert any residual traces of HPA 52 in the 
preparation to the low molecular weight form. The distinctive 
urokinase-minactivin translation product complex at 69,000 MW appeared 
only when the urokinase preparations used contained HPA 33. The 
explanation for this result is unknown. Addition of trasylol to the lysate 
mixture to inhibit possible proteolysis had no effective on this result. 
In summary, in vitro translation of mRNA from U937 cells clearly yields a 
biologically active minactivin translation product of M.sub.r 
approximately 43,000 which can be easily identified by the formation of 
its complex with urokinase giving a characteristic M.sub.r of 69,000. 
c) Construction of Complementary DNA Libraries 
cDNA libraries were constructed from total poly A+mRNA or sucrose density 
gradient fractionated mRNA using a variety of established methods [see in 
general Maniatis, T. et al Molecular Cloning (1982)]. By way of example, 
the first strand complementary DNA was generally synthesized from the mRNA 
using primer initiated reverse transcriptase. Second strand was then 
synthesized, for example, by (1) conventional hairpin-loop primed DNA 
synthesis using DNA polymerase or reverse transcriptase [Maniatis, T. et 
al. Molecular Cloning (1982)]; (2) RNase H-DNA polymerase I--mediated 
second strand synthesis [Grubler, U. Hoffman, B. J. Gene. 25 (1983) 
263-269, Laperye, B. Amabric, F. Gene 37 (1985) 215-220]; or (3) 5'-tailed 
priming method of Land, H. et al Nucleic Acid Research 9 2251-2266 (1981). 
After treatment with S1 nuclease (if required), the DNA is methylated and 
blunt ends generated using standard methods of filling-in, e.g. DNA 
polymerase, the Klenow fragment, or T4-polymerase. Subsequently, the 
cDNA's can be cloned by joining them to suitable plasmid (e.g. pBR322, UC 
or pUR systems) or bacteriophage (e.g. lambda gt 11) vectors through 
complementary homopolymeric tails or cohesive ends created with synthetic 
linker segments containing appropriate restriction sites using standard 
procedures, and then transforming a suitable host. 
EXAMPLE 5 
A preferred method of constructing the cDNA libraries is as follows. 
Methods for purifying DNA from both E. coli and bacteriophages lambda, and 
subsequent standard manipulations such as digestion with restriction 
enzymes, ligations and transformations and radiolabelling of DNA with 
.sup.32 P-ATP, as well as phenol:chloroform extraction and ethanol 
precipitation of DNA which are used in Examples 5 to 10 are as described 
by Maniatis, T. et al. Molecular Cloning (1982). cDNA was synthesized from 
6 micrograms of total poly A+mRNA using Moloney murine leukemia virus 
reverse transcriptase (BRL, 200U/microgram mRNA) in the presence of 50 mM 
Tris HCl, 75 mM KCl, 10 mM DTT, 3 mM MgCl, 1 mM each of dATP, dCTP, dGTP, 
and dTTP, 10 micrograms/ml Oligo (dT).sub.12-18 and 100 micrograms/ml BSA. 
A 200 microliter reaction volume was incubated at 37.degree. C. for 40 
minutes. Second strand was synthesized by hairpin loop primed synthesis 
using the Klenow fragment of DNA polymerase I. The reaction was heated at 
70.degree. C. for 10 minutes to separate DNA/RNA duplexes, diluted to 
twice the volume and Klenow added to 325U/ml in the presence of 10 
microCuries of dATP (1800 Ci/mmole). The reaction was allowed to incubate 
for 1 hour at 15.degree. C. Following phenol:chloroform (1:1) extraction 
and ethanol precipitation [as described by Maniatis, T. et al Molecular 
Cloning (1982)], the DNA was dissolved and the hairpin loop was removed by 
treatment with 80 units of S1 Nuclease (P/L Biochemicals) in the presence 
of 0.2M NaCl, 50 mM sodium acetate pH 4.5, 1 mM ZnSO.sub.4 and 0.5% 
glycerol and precipitated as described previously. 
The double stranded cDNA was then methylated using 20 Units of EcoRl 
Methylase (Biolabs) in the presence of 100 mM Tris-HCl pH 8.0 10 mM EDTA 
and 80 micro-molar S-adenosyl methionine. The DNA was repaired by the 
addition of 2.5U of T4 DNA Polymerase in the presence of 33 mM Tris 
acetate pH8.0, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM 
dithiothreitol, 0.1 mg/ml BBA and 0.5 mM each of dATP, dCTP, dGTP, and 
dTTP for 1 hour at 37.degree. C., followed by the addition of T4 
polynucleotide kinase (20U) and 0.1 mM ATP. Following phenol:chloroform 
(1:1) extraction and ethanol precipitation [as described by Maniatis, T. 
et al Molecular Cloning (1982)], Eco R1 linkers were added to the 
redissolved DNA (2 micrograms linkers/micogram cDNA) using T4 DNA ligase 
(IBI; 1.2U/microgram, DNA). The reaction was carried out on a concentrated 
cDNA solution (167 micrograms/ml) at 26.degree. C. for 4 hours. After 
treatment with EcoRl, the free linkers were separated from the dDNA by gel 
filtration chromatography on Biogel A 50M, as described by Huynh, T. V. et 
al DNA Cloning Vol 1 p 49-78 (1985). Fractions containing cDNA were 
analysed by agraose gel electrophoresis followed by autoradiography and 
those fractions containing cDNA of average length greater than 1,000 b.p. 
were pooled and the cDNA concentrated by lyophiulization to near dryness 
and precipitated by the addition of two volumes of ethanol. The yield of 
cDNA was 2.5 micrograms. 
cDNA libraries were prepared in both lambda gt 11 and gt 10. cDNA (100 ng) 
was ligated to EcoRl- cleaved, phosphatased lambda gt 11 (1 microgram), at 
a DNA concentration of 220 micrograms/ml at 4.degree. C. for 16 hours. The 
DNA was packaged using prepared packaging preparations from Vector Cloning 
Systems. Phages were amplified by adsorption to E. coli strain Y1088 and 
screened in Y1090. The lamba gt 11 library contained approximately 
8.times.10.sup.6 recombinants per microgram cDNA (94% of total phages). 
The proportion of recombinants that contained cDNA molecules was 
determined by screening the library with cDNA synthesized in the presence 
of alpha[.sup.32 P]-dATP. Around 90% of white plaques hybridized with this 
probe. 
For the library prepared in lambda et 10, cDNA (200 ng) was ligated to 
EcoRl cleaved, phosphatased lambda gt 10 (1 microgram), at a DNA 
concentration of 240 micrograms/ml at 25.degree. C. for 4 hours. The DNA 
was packaged as above using E. coli strain C600 hfl. 
The lambda gt 10 library contained approximately 7.5.times.10.sup.6 
recombinants per microgram cDNA. The proportion of recombinants that 
contained cDNA molecules was determined by screening the library with 
radiolabelled cDNA. Greater than 90% of plaques hybridized with this 
probe. 
EXAMPLE 6 
Identification of Clones containing the Minactivin Gene 
The clone(s) containing the gene encoding minactivin may be identified with 
the probes described in the following examples using established 
techniques [see generally Maniatis, T. et al Molecular Cloning (1982]. 
EXAMPLE 6a 
cDNA clones containing sequences complementary to minactivin mRNA may be 
identified by hybridization selection [Maniatis, T. et al Molecular 
Cloning (1982]. The cloned DNA is denatured, immobilized to a solid matrix 
such as nitrocellulose, and hybridized to preparations of total mRNA. The 
RNA/DNA duplex is heated to release the mRNA which is then translated in 
the in vitro rabbit raticulocyte lysate cell free system as described 
above. The translation product may then be identified as described in 
Example 4b. 
EXAMPLE 6b 
DNA Probes Complementary to the Minactivin Gene Sequence 
Using the amino acid sequence obtained for peptides of minactivin as 
described in Example 3, oligonucleotide sequences which would code for the 
amino acid sequence can then be predicted and oligonucleotide probes 
synthesised using conventional established technology [Beaucage, S. L. and 
Carruthers, N. H. Tetrahedron Letts 1859-1862 (1981)]. Using this sequence 
data, a number of oligonucleotide probes were synthesized using an Applied 
Biosystems 380A DNA synthesizer. The sequences of these oligonucleotides 
are set forth in SEQ. ID. NOs. 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, 
respectively, and as follows: 
##STR1## 
The specific oligonucleotide probe may be radiolabelled and then used to 
screen cDNA libraries by in situ hybridization of bacterial colonies or 
bacteriophage plaques using standard techniques [Maniatis, T. et al 
Molecular Cloning (1982] to identify clones containing all or part of the 
minactivin gene. 
EXAMPLE 6C 
Immunological Screening 
The clones may be screened using established procedures (Young, R. A. and 
Davis, R. W. Science 222 778-782) with antibodies which cross-react with 
the native minactivin protein. 
Antibodies to minactivin are prepared by standard methods, for example, 
each rabbit is immunized with 10 to 100 micrograms of purified minactivin 
in the presence of a suitable adjuvant, such as Freunds complete or 
montanide. Following a boost of an equivalent amount approximately four 
weeks later, the rabbit is bled and rabbit serum obtained which is assayed 
for antibodies to minactivin. Antiserum prepared in this way was used to 
immunoprecipitate the minactivin translation product as shown in FIG. 5 
using Ab2 (bled 1 week after boost) and Ab3 (bled 2 weeks after boost) 
antisera. These antisera could be used to screen expression libraries 
prepared in lambda gt 11 according to published methods [Huynh, T. V. et 
al DNA Cloning Vol 1 49-78 (1985)]. 
EXAMPLE 6d 
Screening for Biological Activity 
Clones containing the minactivin gene maybe tested or screened for 
minactivin activity using either radiolabelled urokinase or urokinase and 
antibodies to urokinase. This would be accomplished using standard 
techniques of immunological screening, as described in Example 6c. 
Urokinase and antibodies to urokinase may be obtained commercially. 
Radiolabelled urokinase may be prepared as described below. 
Commercially obtained urokinase (Calibiochem) was affinity purified by 
chromatography on p-aminobenzamidine sepharose according to published 
protocols [Holmberg, L., Bladh, B., Astedt, B. Biochim. Biophys. Acta 445, 
215-222 (1976), and Goldfabr, R. H., Quigley, J. P. Biochemistry 19, 
5463-5471 (1980]. Seventy-five Ploug units of the purified urokinase was 
iodinated by conjugation with N-succinimidyl 3-(4-hydroxy, 5-[I.sup.125 
]iodophenyl) proprionate according to the Bolton Hunter procedure. The 
I.sup.125 -labelled urokinase was separated from the free label by 
chromatography on Sephadex G-25M equilibrated in 0.1M sodium phosphate, pH 
7.0, 0.4M sodium chloride, 0.1% Triton X100 and 1% carrier bovine serum 
albumin. 
Analysis of the I.sup.125 -labelled urokinase preparation by 
SDS--polyacrylamide gel electrophoresis under non-reducing conditions 
showed the presence of the characteristic high (M.sub.r 55,000) and low 
(M.sub.r 33,000) molecular weight forms of urokinase (FIG. 13). Under 
reducing conditions, the high molecular weight form is dissociated into 
its characteristic 33,000 and 22,000 M.sub.r subunits. Iodination of the 
urokinase preparation resulted in a loss of 10 to 15% plasminogen 
activator activity as measured by the assay of Coleman and Green [Ann. 
N.Y. Acad. Sci. 370, 617 (1981)], but had no effect on minactivin 
inhibition of the enzyme. Dot blot assays in which various dilutions of 
minactivin were spotted on nitrocellulose paper, incubated overnight with 
radiolabelled urokinase, washed, dried,and autoradiographed, showed that 
the radiolabelled urokinase could detect minactivin bound to a solid phase 
at a sensitivity of 10 mU or approximately 0.1 ng minactivin. 
EXAMPLE 7 
Identification of the Minactivin Gene 
The preferred method of identifying the minactivin gene is as follows. 
Following synthesis, the oligonucleotide probes 7-10 described in Example 
6b and set forth in SEQ. ID. NOs. 7, 8, 9 and 10 were purified by 
polyacrylamide gel electrophoresis, and labelled with polynucleotide 
kinase (IBI, 1U/pmole DNA) and gamma-.sup.32 P-ATP and purified by ion 
exchange chromatography using standard procedures [see generally Maniatis, 
T. et al Molecular Cloning (1982)]. 
The lambda gt10 library as described in Example 5 was screened by in situ 
hybridization according to standard experimental procedures [see generally 
Maniatis, T. et al. Molecular Cloning (1982)]. Hybridization conditions 
were adjusted to allow specific binding with minimum background and were 
determined to be as follows: 
Probes 7 and 8 (SEQ. ID. NOs. 7 and 8): 3 hours at 50.degree. C. in 6xSSC, 
5.times.Denhardt's, 0.1% SDS, 20 .mu.g/ml tRNA, following prehybridization 
for 1 hour at 42.degree. C. in 6xSSC, 5xDenhardt's 0.5% SDS, 0.2 mg/ml 
sheared calf thymus DNA. 
Probes 9 and 10 (SEQ. ID. NOs. 9 and 10): 16 hours at 37.degree. C. in 10x 
Denhardt's, 5xSSC, 0.05% sodium pyrophosphate. 
.sup.32 P-labelled oligonucleotide probes of specific activity greater than 
10.sup.8 cpm/.mu.g were used at approximately 0.5 pmole/ml. The filters 
were washed in 0.5X or 2xSSC containing 0.1% SDS at increasing 
temperatures to raise the stringency for selection of positive clones. 
Plaques giving positive signals were picked, rescreened, and the phase DNA 
purified using standard procedures (see for example Maniatis et al. 
Molecular Cloning 1982). 
Two recombinant bacteriophage clones MINID and MIN611, containing sequences 
which cross-hybridized to each other were obtained, with EcoRI-linkered 
cDNA inserts of 2100 and 1060 base pairs respectively. These inserts were 
subcloned into plasmid pUC18 to create plasmids pBTA440 and pBTA441 
respectively and mapped by restriction enzyme analysis as shown in FIG. 
20. Southern blot analysis of clone MIN1D located the binding region of 
oligonucleotide probes 7 and 8 (SEQ. ID. NOs. 7 and 8) within a 320 base 
pair XbaI-NcoI restriction fragment as illustrated in FIG. 20. 
That these clones contained genes which code for minactivin was verified by 
hybrid-select translation and DNA sequence analysis. 
Hybrid Select Translation 
Purified pBTA440 was immobilized on nitrocellulose filters at a 
concentration of 20 .mu.g per 3 mm.times.3 mm filter according to the 
procedure described by Maniatis et al. (Molecular Cloning 1982). After 
washing, each filter was incubated with 50 .mu.g of total mRNA and 
hybridized for 3 hours at 50.degree. C. After thorough washing, the 
specifically hybridized mRNA was eluted at 65.degree. C. and then 
translated in vitro using a commercial rabbit reticulocyte lysate 
preparation (Amersham). 
As illustrated in FIG. 21, the hybridized mRNA was shown to specifically 
code for a translation product of M.sub.r 43,000 by gel electrophoresis, 
characteristic of the minactivin translation product described in Example 
4b. Furthermore in the presence of urokinase, this band disappeared and 
the characteristic urokinase-minactivin complex was detected at 
69,000M.sub.r. 
DNA Sequence Analysis 
Restriction fragments of pBTA440 were subcloned into the single stranded 
phase vectors M13mp9, M13mp18 and M13mp19 and the DNA sequence of the 
2,100 bp inserted was determined using the Sanger chain termination 
method. Examination of the DNA sequence indicated that the 2100 bp insert 
did not contain the entire coding sequence of the minactivin gene. 
Primer Extension 
To obtain the remainder of the DNA sequence encoding the N-terminal region 
of minactivin a second cDNA library was constructed using primer extension 
(Luse, D. S. et al, Nucleic Acid Research 9 (17) 4339-4355 (1981]. The 
library was prepared by priming 5 micrograms of poly A.sup.+ mRNA with the 
oligonucleotide set forth in SEQ. ID. No. 16 being complementary to the 
previously sequenced nucleotides 391 to 420. EcoRI-linkered cDNA inserts 
were subsequently cloned in lambda gt10 using standard techniques. 
Approximately 5.3.times.10.sup.3 of the 7.2.times.10.sup.4 clones obtained 
were screened with a second oligonucleotide as set forth in SEQ. ID. NO. 
17 (complementary to nucleotides 310-335). Of the 100 positive clones 
obtained, 15 were purified and the clone (clone 13) with the largest cDNA 
insert (430 bp) was subcloned into plasmid pUC18 to create plasmid 
pBTA442. The DNA sequence of pBTA442 was determined as described above 
(see also FIG. 20). 
The coding sequence of the minactivin gene, contained in pBTA440 and 
pBTA443, a plasmid containing the 430 bp 5' minactivin sequence in pUC18 
in opposite orientation to pBTA442, was made contiguous by recombining 
certain DNA restriction fragments to crate pBTA438 as shown in FIG. 22. E. 
coli K-12, strain JM109 containing pBTA438 has been deposited with the 
American Type culture collection, 12301 Parklawn Drive, Rockville, 
Md.20852. United States of America on 11 Feb. 1987 under accession number 
ATCC 53585. 
The complete cDNA sequence (SEQ ID NO:18) of the minactivin gene and the 
deduced amino acid sequence of the minactivin protein are given in FIG. 
23. The complete translation product consists of 415 amino acids (M 46 
543). The gene encodes the 5 peptides (as set forth in SEQ ID. NO:18) 
obtained from the amino acid sequence (SEQ ID NO:19) analysis of native 
minactivin as illustrated in FIG. 23. 
The DNA sequence analysis reveals that minactivin is a member of the serine 
protease inhibitor superfamily, (known as serpins) albeit specific for 
urokinase type plasminogen activators. 
EXAMPLE 8 
Expression of Biologically Active Minactivin 
High-level expression of the biologically active molecule is obtained, for 
example, by integration of the full-length cDNA present in pBTA438 into 
various vectors which can direct the synthesis of the protein in a variety 
of hosts such as bacteria or eukarotic cells (such as mammalian cells 
transfected or transformed with the vector). The vector preferably 
contains a nucleotide sequence capable of controlling expression of the 
nucleotide sequence coding for minactivin. This second nucleotide sequence 
may include, by way of example, a promoter sequence, polyadenylation 
sequences, or nucleotide sequences which allow the protein to be expressed 
as a hybrid molecule fused with another protein. 
EXAMPLE 9 
Bacterial Expression of Minactivin 
The general approach is the preparation of an expression vector or cloning 
vehicle replicable in E. coli, which contains a DNA sequence which codes 
for the expression of minactivin. 
Minactivin may be expressed in its native form or as a hybrid molecule 
fused to another protein. These constructions are shown n FIGS. 24 and 26. 
One series of plasmid constructs used the lambda P.sub.L expression vectors 
pLK57, and pLK58 (Botterman et al. Gene 37; 229-239, 1985) to express 
native or near-native (N-terminal amino acid modified) minactivin. 
As shown in FIG. 24, the plasmid pBTA438 was digested with EcoRI and DraI 
and a 1610 bp EcoRI-DraI restriction fragment was isolated from an agarose 
gel. This fragment was ligated with T4 ligase to vector pLK57 which had 
been digested with EcoRI and EcoRV. The derivative plasmid pBTA444 
contains the lambda P.sub.L promoter controlling the expression of native 
minactivin. 
The expression vector pBTA444 was used to transform E. Coli K-12 strain 
N4830 (Joyce et al. PNAS 80, 1830-1834, 1983) which contains the 
thermolabile CI repressor of lambda. Cells transformed with pBTA444 were 
grown overnight in MED medium (Mott et al PNAS 82, 88-92, 1985) 100 
micrograms/ml ampicillin at 28.degree. C. Cells were diluted in MEB 
medium, grown at 28.degree. C. to an OD.sub.600 of 1.0 when prewarmed 
(65.degree. C.) MEB medium was added in equal volume to equilibrate the 
temperature to 42.degree. C. 
Following 4 hours of growth at 42.degree. C. the cells were harvested and 
membrane and soluble protein fractions prepared by resuspending washed 
cells (after -70.degree. C. freezing and thawing) in 200 .mu.of 20% 
sucrose 30 mM Tris-HCl pH8.1. and mg/ml lysozyme solution followed by the 
addition of 3 mls of 3M EDTA pH7.3. The cells extract was clarified by 
brief sonification and membrane and insoluble proteins pelleted by 
centrifugation (27,000xg), 60 mins). The soluble proteins were 
precipitated by the addition of trichloroacetic acid (10% w/v) to the 
supernatant and the pellet dissolved in water. The pelleted membranes was 
also dissolved in water. Samples of these fractions for both uninduced 
(28.degree. C.) and induced (42.degree. C.) cells were analysed by 
SDS-polyacrylamide gel electroploresis and immunological detection of 
minactivin by western transfer using antiserum against human placental 
inhibitor. As shown in FIG. 25 a minactivin protein band (Mr 40-50K), 
visualized by western transfer using antibodies to human placental 
inhibitor and rabbit anti-goat IgG coupled to alkaline phosphate (Sigma) 
is present in both the induced (42.degree. C.) soluble and membrane 
fractions. 
An alternative method for producing native minactivin is also shown in FIG. 
24. The plasmid pBTA442 was digested with XhoII and a 243 bp XhoII 
restriction fragment was purified from an agarose gel. This fragment was 
ligated with T4 ligase to vector pLK58 digested with BglII. The derivative 
plasmid pBTA445 was digested with PvuII and SmaI and a 2800 bp fragment 
purified and ligated with T4 ligase to a purified 1320 bp PvuII-DraI 
restriction fragment from pBTA438. The derivative plasmid pBTA446 was 
linearized with BdlII and ligated to a synthetic double stranded 26 mer 
oligonucleotide containing a bacterial ribosome binding site and the 
initial nucleotides of the native minactivin gene, creating plasmid 
pRTA447. When pBTA447 is transformed into an appropriate host, such as 
N4830, induced and analysed as described above, minactivin is again 
produced as shown in FIG. 25. In both cases, for pBTA444 and pBTA447 
containing cells, minactivin was present in both the induced (42.degree. 
C.) soluble and membrane fractions. 
To assess the biological activity of minactivin produced in E. coli N4830, 
soluble and membrane fractions were incubated for 90 mins with high and 
low molecular weight urokinase as described in Example 4. Samples were 
then precipitated with acetone, resuspended in water, and run on a 
reducing SDS-polyacrylamide gel. Minactivin and minactivin-urokinase 
complexes were visualized by Western transfer as described above. As shown 
in FIG. 25 minactivin in the soluble fraction from induced E. coli N4830 
containing pBTA447 complexes with urokinase under standard assay 
conditions. This indicates that minactivin produced from these bacterial 
cells retains biological activity. 
Two examples of a method for producing a protein that is the fusion of all 
or part of one protein coding sequence and all or part of the minactivin 
coding sequence follows. As shown in FIG. 26, the plasmid pBTA440 was 
digested with SspI and DraI and a 1110 bp fragment was isolated from an 
agarose gel. This fragment was ligated to the vector pBTA449 digested with 
EcoRV creating pBTA450. pBTA450 was then digested with AvaI and a purified 
2800 bp fragment ligated to the plasmid pLK57 digested with AvaI to create 
plasmid pBTA586. This places part of the minactivin coding sequence under 
the control of the lambda P.sub.L promoter and fused to the coding 
sequence of the first 80 amino acids of traT gene, the first 20 of which 
constitutes a signal sequence that results in the fusion appearing in the 
outer member of E. coli. This signal sequence is cleaved off during 
transport to the outer membrane, which is the normal location of the traT 
protein. 
When plasmid pBTA586 is transformed into an appropriate host, such as N4830 
and induced with temperature shift as above, the TraT-Minactivin fusion 
protein appears in the outer membrane, as shown in FIG. 27. 
A second example of a method for producing a fusion is shown in FIG. 26. In 
plasmid pBTA440, the minactivin coding sequence is fused in frame with a 
portion of the .beta.-galactosidase gene present on plasmid pUC18. 
When plasmid pBTA440 is transformed into an appropriate host, such as 
JM101, or any E. coli strain which contains the lacI.sup.q gene, and 
induced by addition of isopropyl-thio-.beta.-D-galactopyranoside (final 
concentration 1 mM), minactivin production can be detected as described 
above (FIG. 27). 
EXAMPLE 10 
Expression of Recombinant Minactivin in Eukaryotic Cells 
A fragment of pBTA438 containing the entire coding region of minactivin was 
inserted into a series of vectors capable of stable integration and 
expression of eukaryotic genes in mammalian cells. These included 1) pKC3 
(derived from pKO-neo, Van Doren, Hanahan, D., Gluzman, Y., J. Virol. 50 
606-614 (1982) wherein the minactivin cDNA sequence is placed under the 
control of the SC40 early promoter; 2) pZipNeoSV(X)1 (Cepko, C. L. 
Roberts, B. E., Mulligan, R. C. , Cell 37 1053-1062 (1982)), a Molony 
Murine Leukemia virus-derived retroviral shuttle system in which the 
minactivin gene is placed downstream from the retroviral LTR promoter and 
selection is based on the neo gene which confers kanamycin resistance in 
prokaryotes and G418 resistance in eukaryotes; and 3) pMSG (commercially 
available from Pharmacia), wherein regulated expression of minactivin is 
achieved by utilizing a dexamethasone inducible promoter contained within 
the Mouse Mammary Tumor-Virus (MMTV) 5'-LTR. 
The construction of these three vectors is shown in FIG. 28 and the details 
are as follows. The coding region of the minactivin gene was isolated from 
pBTA438 as a 1610 bp EcoRI-DraI fragment and inserted into the following 
vectors as described below. 
The 1610 bp EcoRI-DraI fragment was ligated into pKC3 which had been 
digested with EcoRI and SmaI, and then transformed into E. coli 
C600.gamma.. The resultant plasmid was designated pBTA587. 
In the second construction, the 1610 bp EcoRI-DraI fragment was rendered 
flush-ended using the Klenow fragment of DNA polymerase I, ligated into 
the SmaI site of pMSG, and transformed into a suitable E. coli K-12 host. 
Colonies containing the minactivin gene in pMSG were detected by colony 
hybridization using the .sup.32 P-labelled oligonucleotide (29 mer) 
previously described in Example 7 (complementary to nucleotides 310-335). 
The resultant plasmid was designated pBTA588. 
In the third construction, the flush-ended EcoRI/DraI fragment described 
above was ligated into pUC7 which had been digested with HincII giving the 
construction designated pBTA589. As the HincII site in pUC7 is flanked by 
BamHI sites, this allowed the minactivin gene to be isolated following 
BamHI digestion and ligated into the BamHI site of pZIPNeo SV(X) 1. 
Following transformation into a suitable E. coli K-12 host, colonies 
containing the minactivin gene were detected by colony hybridization as 
described above. The resultant plasmid was designated pBTA590. 
Transfection of Eukaryotic Cells 
All plasmids were transfected into eukaryotic cells by the calcium 
phosphate method. Approximately 1-2.times.10.sup.5 cells were seeded into 
a T25 flask in 5 ml of Dulbecco modified Eagle medium supplemented with 
10% (v/v) foetal calf serum, 3.6 mM glutamine 200 mM, 45IU/ml penicillin 
and 45 mg/ml streptomycin (complete medium). Approximately 1 to 5 .mu.g of 
CsCl gradient purified DNA was precipitated with calcium phosphate and 
added to the cells. After 4 hours, the cells were treated to a glycerol 
shock, and cultured in complete medium for 3 days. The culture supernatant 
was then removed for measurement of transient expression. The cells were 
then trypsinized and split 1/3 into T75 flasks with complete medium 
containing the appropriate antibiotic selection (see below). The cells 
were washed every 6 to 7 days with the same medium and transfectants 
picked at 14 to 28 days and cultured individually until confluent growth 
was achieved. 
The conditions of transfection for each of pBTA587, pBTA588 and pBTA590 
were as follows: 
pBTA587. As pKC3 does not contain a selectable marker, pBTA587 was 
cotransfected with pZIPNeo SV(X)1 at a molar ratio of 7.5:1, pBTA587: 
pZIPNeo SV(X)I. Transfectants were selected with complete medium 
containing 0.4 mg/ml G418. Transfections were carried out in COS cells. 
pBTA588. As pMSG contains the E. coli xanthine-guanine 
phosphoribosyltransferase (gpt) gene expressed from the SV40 early 
promoters,stably transfected cells were selected in HAT medium containing 
hypoxanthine, aminopterin and mycophenolic acid. Transfections were 
carried out using NIH3T3 cells. 
pBTA590. Transfectants were selected using complete medium containing 0.4 
mg/ml G418. Transfections were carried out in NIH3T3 cells. 
Analysis of Expression of Recombination Minactivin in Eukaryotic Cells 
Following transfection, transient expression of recombinant minactivin is 
detected by culturing the cells in the presence of .sup.35 S-methionine 
and specific immunoprecipitation of the recombinant radiolabelled 
minactivin using antibodies to placental inhibitor essentially according 
to the method described in Example 4b. For example, forty-eight hours 
after transfection of pBTA587 into COS cells, the supernatant was removed 
and the cells cultured in the presence of 1 ml methionine-free EMEM 
(Flow), supplemented with .sup.35 S-methionine (Amersham). Following 
immunoprecipitation with 50 mg goat anti-placental inhibitor antibodies 
and 200 ml washed Pansorbin, the complexes were analysed by 
SDS-polyacrylamide gel electrophoresis (reducing conditions) and 
visualized by autoradiography as shown in FIG. 29. Recombinant minactivin 
is detected as a band of Mr 45-48,000, which is not observed in the 
corresponding control transfection containing the vector (pKC3) alone. 
When urokinase (15 Plough units, Calbiochem) is added to the supernatant 
prior to immunoprecipitation, this band disappears which is characteristic 
of biologically active minactivin. A band is observed at M.sub.r 69,000 
which is indicative of the minactivin urokinase complex, but is somewhat 
obscured by a nonspecific protein band at the same position. Some of the 
recombinant minactivin also appears to have been proteolytically nicked 
following the addition of the urokinase preparation, as evidenced by the 
M.sub.r 35-37,000 band detected. 
That the recombinant minactivin produced was biologically active was 
determined by culturing the cells in the absence of serum for 4 hours and 
quantitating the inhibition of urokinase activity by the colorimetric 
assay essentially as described in Example 1. A level of inhibition was 
detected which corresponded to approximately 1 unit/ml minactivin activity 
above background. 
Transfectants containing the minactivin gene can also be analyzed for 
minactivin activity using radiolabelled urokinase prepared as described in 
Example 6 or according to the method of Baker. Culture supernatants are 
incubated with the radiolabelled urokinase in order to allow complex 
formation between the recombinant minactivin and urokinase. The complex is 
then removed from the solution by the addition of rabbit antibodies 
prepared against urokinase (Green Cross Corp.) and precipitated by the 
addition of washed Pansorbin or anti-rabbit antibodies covalently attached 
to immunobeads (Biorad). After centrifugation, the 
minactivin-urokinase-antibody pellet is washed, disrupted by boiling with 
2% SDS and the products analysed by gel electrophoresis followed by 
autoradiography. The presence of biologically active recombinant 
minactivin produced by the transfected cells is evidenced by the shift in 
molecular weight of urokinase from Mr 55,000 (or 33,000) to a higher Mr 
(69 to 92,000) (see Example 4b) characteristic of the formation of the 
minactivin-urokinase complex. 
EXAMPLE 11 
Purification and Recover of Biologically Active Protein 
Following the establisment of conditions for the expression of minactivin 
in E. coli at high levels the cells harbouring the plasmid encloding the 
minactivin gene are harvested at late log phase. One volume of packed 
cells are suspended in two volumes of lysis buffer (0.1M sodium phosphate. 
pH7.0 containing 1 mM EDTA and 1 mM phenyl methyl sulphenyl fluoride) and 
lysed by three passages through a French Press at 15,000 psi. The 
suspension is centrifuged at 23,000 xg for 20 minutes and the pellet 
resuspended in two volumes of lysis buffer containing 5% Triton X-100. The 
suspension is again centrifuged at 23,000 xg for 20 minutes and the pellet 
suspended in three volumes of 0.1M Tris-Cl, pH8.0 containing 8M urea and 
0.1M DTT. The solution is flushed with nitrogen and incubated in a sealed 
tube at 37.degree. C. for 2 hours. Following incubation the pH of the 
solution is lowered to approximately pH3.5 by the addition of 50 ml of 
glacial acetic acid for every ml of solution. The suspension is clarified 
by centrifuging as above and the supernatant applied to a Sephadex G-75 
column (3.2 cm.times.90 cm) equlibrated in 0.1M acetic acid. The fractions 
containing the minactivin are located by SDS-PAGE. The fractions 
containing the minactivin are pooled and dialysed against 10 mM Tris-Cl, 
pH8.0 containing 8M Urea and 0.1 mM DTT at room temperature for 16 hours. 
The analysed solution is then applied to a DEAE-Sephadex column (2.2 
cm.times.25 cm) equilibrated in the above buffer and the column washed to 
elute unbound material. The minactivin is then eluted from the column 
using a linear gradient of sodium chloride from 0 to 0.5 M in the same 
buffer. The fractions containing the minactivin are indentified by 
SDS-PAGE and dialysed extensively against distilled water. The protein, 
which precipitates during this procedure, is recovered by lyophilization. 
The lyophilized protein is redissolved in 0.1% trifluoroacetiac acid and 
applied to a Vydac C-4 reverse phase column attached to a Waters high 
pressure liquid chromatograph. The pure minactivin is eluted from the 
column using a linear gradient of acetonitrile from 0 to 80% in 0.1% 
trifluoroacetic acid. The A.sub.220 peak corresponding to minactivin is 
identified by SDS-PAGE, the fractions pooled and lyoplilized. 
The lyophilized, purified minactivin is dissolved in 0.1M Tris-Cl, pH8.0 
containing 8 urea at a concentration of 10 mg/ml and diluted to 10 mgm/ml 
into 0.1M Tris Cl, pH8.0 containing 1 mM reduced glutathione and 0.1 mM 
oxidized glutathione. The refolding reaction is allowed to proceed at 
room-temperature for 24 hours and then the solution concentrated and 
diafiltered against0.1M sodium phosphate pH7.0 on an Amicon stirred cell 
using a YM10 membrane. The resultant solution containing active minactivin 
is assayed using the assay described above (Example 1). 
The recovery of biologically active minactivin secreted at high levels from 
mammalian cells employs the same procedures as described in Example 2 for 
the purification of the native minactivin from U937 cells. This involves 
initially a ten fold concentration of the cell free supernatant using an 
Amicon DC-2 hollow fibre concentrator equipped with a 30,000 dalton 
cut-off cartridge. The concentrate is then dialysed against at least an 
equal volume of 50 mM glycine, pH7.8, to remove all traces of dye. The 
dialysed concentrate is centrifuged in a JA10 rotor at 8000 rpm for 30 min 
at 4.degree. C to pellet residual cell debris and protein that may have 
precipitated during dialysis. The clarified supernatant is then aliquoted 
and frozen at -20.degree. C. until required for subsequent purification. 
Minactivin is further purified from ten-time concentrated culture 
supernatant by step pH elution using Phenyl-Sepharose as follows. 
The ionic strength of the supernatant is adjusted to 2M by the addition of 
solid NaCl and the pH adjusted to 5.5 with citric acid. This solution is 
applied to a Phenyl-Sepharose column (4.4 cm.times.5.0 cm) equilibrated in 
50 mM Na citrate, pH5.5, 2M Nacl and 1 mM EDTA and eluted with the same 
buffer until the baseline absorbance at 280 nm (A280) returned to 
baseline. The minactivin is then eluted from the column with 50 mM 
glycine, pH9.0. Fractions containing the highest specific activity 
minactivin are pooled and concentrated on an Amicon YM10 membrane. 
The pooled, concentrated minactivin is then applied to a 2.2 cm.times.78 cm 
column of Sepharcyl S-200 equilibrated with 0.1M sodium borate, pH9.0. 
Fractions of 5.0 ml are collected at a flow rate of 0.46 ml/min. The 
fractions containing minactivin activity were pooled and concentrated in 3 
ml using a YM10 membrane. Calibration of this column with known M.sub.r 
standards indicates that minactivin has a M.sub.r of 45-48 kD. 
The concentrated minactivin solution is applied to a preparative flat bed 
gel of Ultrodex containing Ampholines in the pH range 4.5-6.0 and 
electrofocussed for 23 hrs at 10.degree. C. on an LKB Multiphor 
isoelectric-focussing apparatus. Following completion of the run, 30 zones 
across the length of the gel are scraped out and the protein eluted from 
each with 10 ml of 1M glycine containing 1 mM EDYA, pH9.0. Aliquots of 
each frction are assayed for minactivin activity and electrophoresed on 
15% SDS-polyacrylamide gels to locate protein. Under these conditions 
minactivin focusses between pH5 and pH5.2 and is highly purified. This 
material is again concentrated on an Amicon YM10 membrane and stored at 
-20.degree. C. in 50 mM glycine, pH9.0, containing 1 mM EDTA and 50% 
glycerol. 
Industrial Application 
As a specific inactivator of urokinase-type plasminogen activiators, 
minactivin has a range of potential industrial applications as a clinical 
reagent for the diagnosis and possible treatment of various human 
carcinomas and inflammatory conditions. 
Similarly, oligonucleotide probes derived from the amino acid sequence of 
peptides derived from purified minactivin or antibodies to minactivin can 
be used as diagnostic tools in assays for monitoring the status of 
diseases such as inflammation and cancer metastasis particularly during 
prescribed courses of treatment. 
Studies of cell transformation in vitro by tumor viruses (Ossowski, 1 et 
al. J. Exp. Med. 137, 112, 1973) and by chemical carcinogens (Sisskin, et 
al. Int. J. Cancer, 26, 331, 1980) both show that plasminogen activator 
secretion is the most constituent early biochemical event associated with 
transformation. Furthermore, the ability of cell lines to metastasize in 
vivo has been found to correlate with their ability to express plasminogen 
activator (Wang et al, Cancer Research 40, 288, 1980). It is also well 
established that tumor cells of several of the most prevalent human 
cancers, i.e. carcinoma of the lug, breast, prostate and colon, produce 
high levels of urokinase-type plasminogen activator (Duffy, M. J., 
O'Grady, P. Eur. J. Clin. Oncol. 20 (5) 577-582, 1984). 
Our previous studies (Stephens, R. S. et al. Blood 66, 333-337, 1985) on 
malignancy in colon mucosa and conditions which predispose to malignancy, 
i.e. adenomatous polyps, polyposis coli and inflammatory conditions of the 
colon such as Crohn's disease and ulcerative colitis, have demonstrated 
that human colon cancers produce significantly greater amount of 
urokinase-type plasminogen activator than that occurring in adjacent 
noninvolved tissue. Minactivin was found to be capable of binding to and 
inhibiting this tumor associated plasminogen activator (Stephens et al. 
Blood 65, 333-337, 1985). Thus, it follows that minactivin has industrial 
application as a reagent for identifying and defining tumors both in vivo 
and in histological specimens. For imaging tumors in vivo, minactivin may 
be labelled with an appropriate isotope, such as Technetium-99 m 
(Richardson, V. J. Brit. J. Cancer 40, 35, 1979) or Iodine-131 (Begent, R. 
H. J. Lancet, Oct 2, 1982). Following administration of the minactivin 
preparation, the location and boundaries of the tumor may be determined by 
known raidoisotopic methods, such as gamma-camera imaging. Thus, 
minactivin offers a sensitive method for enabling the identification of 
small metastatic cancers particularly those arising after surgical 
intervention. In the analysis of histochemical specimens, minactivin or 
its antibody, may be labelled with an isotope such a I.sup.131, or 
conjugated to an appropriate enzyme or other chemical reagent. On contact 
with a histological specimen, such as a biopsy section, minactivin will 
bind to the tumor type plasminogen activator at its place of secretion, 
thereby identifying the tumor boundaries and potentially the metastatic 
state of the tumor. In addition to its diagnostic applications, minactivin 
is also indicated for use in the direct treatment of tumors. As a specific 
inhibitor of the enzyme implicated in the process by which tumors invade 
surrounding tissues (Dano, K. et al., Adv. in Cancer Res. 44, 139,1985), 
regulation and, in particularly, inhibition of tumor growth and metastases 
can be achieved. Furthermore, minactivin can be used as a drug delivery 
system to deliver lectins or toxins directly to growing tumors. It will be 
appreciated that this system could offer many advantages in terms of 
specificily and extremely potent tumoricidal capability. 
Other biological processes in which urokinase-type plasminogen activators 
have been implicated involve those physiological events associated with 
invasion and tissue destruction, such as chronic inflammatory conditions 
including rheumatoid arthritis. As minactivin is part of the natural host 
response to tissue degradation, it will provide a useful marker for 
monitoring the status of the disease particularly during prescribed 
courses of treatment. Labelled antibodies or DNA probes derived from 
minactivin have industrial application as diagnostic reagents for 
monitoring minactivin levels in blood plasma, in macrophages of tissue 
biopsies and in synovial fluid for corelations with diseased states. 
Similarly, minactivin itself is also indicated to have a therapeutic 
effect when administered in vivo in amellorating such conditions. 
EXAMPLE 12 
Studies of cell transformation in vitro by tumor viruses (Ossowski, 1 et 
al. J. Exp. Med. 137, 112, 1973) and by chemical carcinogens (Sisskin, et 
al. Int. J. Cancer, 26, 331, 1980) both show that plasminogen activator 
secretion is the most consistent early biochemical event associated with 
transformation. Furthermore, the ability of cell lines to metastasize in 
vivo has been found to correlate with their ability to express plasminogen 
activator (Wang et al. Cancer Research 40, 288, 1980). It is also well 
established that tumor cells of several of the most prevalent human 
cancers, i.e. carcinoma of the lug, breast, prostate and colon, produce 
high levels of urokinase-type plasminogen activator (Duffy, H. J. O'Grady, 
P. Eur. J. Clin. Oncol. 20(5) 577-582, 1984). 
Our previous studies (Stephens, R. S. et al. Blood 66, 333-337, 1985) on 
malignancy in colon mucosa and conditions which predispose to malignancy, 
i.e. adenomatous polyps, polyposis coil and inflammatory conditions of the 
colon such as Crohn's disease and ulcerative colitis, have demonstrated 
that human colon cancers produce significantly greater amount of 
urokinase-type plasminogen activator than that occurring in adjacent 
noninvolved tissue. Minactivin was found to be capable of binding to and 
inhibiting this tumor associated plasminogen activator (Stephens et al. 
Blood 66, 333-337, 1985). Thus, it follows that minactivin has industrial 
application as a reagent for identifying and defining tumors both in vivo 
and in histological specimens. For imaging tumors in vivo, minactivin may 
be labelled with an appropriate isotope, such as Technetium-99 m 
(Richardson, V. J. Brit. J. Cancer 40; 35, 1979) or lodine-131 (Begent, R. 
H. J. Lancet, Oct. 2, 1982). Following administration of the minactivin 
preparation, the location and boundaries of the tumor may be determined by 
known radioiostopic methods, such as gamma-camera imaging. Thus, 
minactivin offers a sensitive method for enabling the identification of 
small metastatic caners particularly those arising after surgical 
intervention. In the analysis of histochemical specimens, minactivin or 
its antibody, may be labelled with an isotope such a I.sup.131 or 
conjugated to an appropriate enzyme or other chemical reagent. On contact 
with a histological specimen, such as a biospy section, minactivin will 
bind to the tumor type plasminogen activator at its place of secretion, 
thereby identifying the tumor boundaries and potentially the metastatic 
state of the tumor. In addition to its diagnostic applications, minactivin 
is also indicated for use in the direct treatment of tumors. As a specific 
inhibitor of the enzyme implicated din the process by which tumors invade 
surrounding tissues (Dano, K. et al. Adv. in Cancer Res. 44, 139,1985), 
regulation and, in particularly, inhibition of tumor growth and metastases 
can be achieved. Furthermore, minactivin can be used as a drug delivery 
system to deliver lectins or toxins directly to growing tumors. It will be 
appreciated that this system could offer many advantages in terms of 
specificily and extremely potent tumoricidal capability. 
Other biological processes in which urokinase-type plasminogen activators 
have been implicated involve those physiological events associated with 
invasion and tissue destruction, such as chromic inflammatory conditions 
including rheumatoid arthritis. As minactivin is part of the natural host 
response to tissue degradation. It will provide a useful marker for 
monitoring the status of the disease particularly during prescribed 
courses of treatment. Labelled antibodies or DNA probes derived from 
minactivin have industrial application as diagnostic reagents for 
monitoring minactivin levels in blood plasma, in macrophages of tissue 
biopsies and in synovial fluid for correlations with diseased states. 
Similarly, minactivin itself is also indicated to have a therapeutic 
effect when administered in vivo in amellorating such conditions. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 19 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AlaGl nIleLeuGluLeuProTyrXaaGlyAspValXaaMetPheLeu 
151015 
LeuLeuProXaaGlu 
20 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GlyArgAlaAsnPheSerGlyMetSerGluXaaAsnAspLeuPhe 
15 1015 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetAlaGluXaaGluValGluValTyrIlePro GlnPheLysLeuGlu 
151015 
GluXaaTyr 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LeuAsnIleGlyTyrIleGluAspLeuLys 
1510 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
IleProAsnLeuLeuProGluGlyXaaVal 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TTRAAYTGNACNATRTA 17 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TANACYTCNACYTC14 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A ) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TCBARNATNTGVGC14 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA olgonucleotide 
(iv) ANTI-SENSE: YES 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 18 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 24 
(D ) OTHER INFORMATION: /mod.sub.-- base=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTGAAYTGNACNATGTANACYTCNACYTC29 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucelic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 15 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TCYTCNATRTANCCNATRTT20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /mod.sub.-- base=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AANTTNGCNCKNCC 14 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ATATGTTTCCTCGAGCTTGAACTGAGGGATGTACACCTCGACTTCGCTCTCTGCCAT57 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
TTCATCAGGCAACAGGAGGAACATGCTCACATCTCCGGCGTAAGGGAGTTCCAGGATCTT60 
CATTTT66 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
CTCCTCCAGCTTGAACTGGGGGATGTAGACCTCCAC CTC39 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CTTG AACTGRGGRATGTASACCTCCACCTC30 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
TTCCAGTAAATAATTCCCTGTGGATGCATT30 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid; 
(A) DESCRIPTION: Synthetic DNA oligonucleotide 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GCCTGCAAAATCGCATCAGGATAACTACC29 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2409 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA to genomic RNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 49..1296 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GTCAGACAGCAACTCAGAGAATAACCAGAGAACAACCAGATTGAAACAATGGAGGAT57 
MetGluAsp 
CTTTGTGTGGCAAACACACTCTTTGCCCTCAATTTATTCAAGCATCTG105 
LeuCysVal AlaAsnThrLeuPheAlaLeuAsnLeuPheLysHisLeu 
51015 
GCAAAAGCAAGCCCCACCCAGAACCTCTTCCTCTCCCCATGGAGCATC153 
AlaLysAlaSerProTh rGlnAsnLeuPheLeuSerProTrpSerIle 
20253035 
TCGTCCACCATGGCCATGGTCTACATGGGCTCCAGGGGCAGCACCGAA201 
SerSerThrMetA laMetValTyrMetGlySerArgGlySerThrGlu 
404550 
GACCAGATGGCCAAGGTGCTTCAGTTTAATGAAGTGGGAGCCAATGCA249 
AspGlnMetAla LysValLeuGlnPheAsnGluValGlyAlaAsnAla 
556065 
GTTACCCCCATGACTCCAGAGAACTTTACCAGCTGTGGGTTCATGCAG297 
ValThrProMetThr ProGluAsnPheThrSerCysGlyPheMetGln 
707580 
CAGATCCAGAAGGGTAGTTATCCTGATGCGATTTTGCAGGCACAAGCT345 
GlnIleGlnLysGlySerTy rProAspAlaIleLeuGlnAlaGlnAla 
859095 
GCAGATAAAATCCATTCATCCTTCCGCTCTCTCAGCTCTGCAATCAAT393 
AlaAspLysIleHisSerSerPheArgS erLeuSerSerAlaIleAsn 
100105110115 
GCATCCACAGGGAATTATTTACTGGAAAGTGTCAATAAGCTGTTTGGT441 
AlaSerThrGlyAsnTyrLeuLeu GluSerValAsnLysLeuPheGly 
120125130 
GAGAAGTCTGCGAGCTTCCGGGAAGAATATATTCGACTCTGTCAGAAA489 
GluLysSerAlaSerPheArgGlu GluTyrIleArgLeuCysGlnLys 
135140145 
TATTACTCCTCAGAACCCCAGGCAGTAGACTTCCTAGAATGTGCAGAA537 
TyrTyrSerSerGluProGlnAlaVa lAspPheLeuGluCysAlaGlu 
150155160 
GAAGCTAGAAAAAAGATTAATTCCTGGGTCAAGACTCAAACCAAAGGC585 
GluAlaArgLysLysIleAsnSerTrpValL ysThrGlnThrLysGly 
165170175 
AAAATCCCAAACTTGTTACCTGAAGGTTCTGTAGATGGGGATACCAGG633 
LysIleProAsnLeuLeuProGluGlySerValAspGly AspThrArg 
180185190195 
ATGGTCCTGGTGAATGCTGTCTACTTCAAAGGAAAGTGGAAAACTCCA681 
MetValLeuValAsnAlaValTyrPheLysGlyLys TrpLysThrPro 
200205210 
TTTGAGAAGAAACTAAATGGGCTTTATCCTTTCCGTGTAAACTCGGCT729 
PheGluLysLysLeuAsnGlyLeuTyrProPheAr gValAsnSerAla 
215220225 
CAGCGCACACCTGTACAGATGATGTACTTGCGTGAAAAGCTAAACATT777 
GlnArgThrProValGlnMetMetTyrLeuArgGluL ysLeuAsnIle 
230235240 
GGATACATAGAAGACCTAAAGGCTCAGATTCTAGAACTCCCATATGCT825 
GlyTyrIleGluAspLeuLysAlaGlnIleLeuGluLeuPro TyrAla 
245250255 
GGAGATGTTAGCATGTTCTTGTTGCTTCCAGATGAAATTGCCGATGTG873 
GlyAspValSerMetPheLeuLeuLeuProAspGluIleAlaAspVal 
26 0265270275 
TCCACTGGCTTGGAGCTGCTGGAAAGTGAAATAACCTATGACAAACTC921 
SerThrGlyLeuGluLeuLeuGluSerGluIleThrTyrAspLysLe u 
280285290 
AACAAGTGGACCAGCAAAGACAAAATGGCTGAAGATGAAGTTGAGGTA969 
AsnLysTrpThrSerLysAspLysMetAlaGluAspGluValGluV al 
295300305 
TACATACCCCAGTTCAAATTAGAAGAGCATTATGAACTCAGATCCATT1017 
TyrIleProGlnPheLysLeuGluGluHisTyrGluLeuArgSerIle 
310315320 
CTGAGAAGCATGGGCATGGAGGACGCCTTCAACAAGGGACGGGCCAAT1065 
LeuArgSerMetGlyMetGluAspAlaPheAsnLysGlyArgAlaAsn 
32 5330335 
TTCTCAGGGATGTCGGAGAGGAATGACCTGTTTCTTTCTGAAGTGTTC1113 
PheSerGlyMetSerGluArgAsnAspLeuPheLeuSerGluValPhe 
340 345350355 
CACCAAGCCATGGTGGATGTGAATGAGGAGGGCACTGAAGCAGCCGCT1161 
HisGlnAlaMetValAspValAsnGluGluGlyThrGluAlaAlaAla 
360365370 
GGCACAGGAGGTGTTATGACAGGGAGAACTGGACATGGAGGCCCACAG1209 
GlyThrGlyGlyValMetThrGlyArgThrGlyHisGlyGlyProGln 
375380385 
TTTGTGGCAGATCATCCTTTTCTTTTTCTTATTATGCATAAGATAACC1257 
PheValAlaAspHisProPheLeuPheLeuIleMetHisLysIleThr 
390 395400 
AACTGCATTTTATTTTTCGGCAGATTTTCCTCACCCTAAAACTAAG1303 
AsnCysIleLeuPhePheGlyArgPheSerSerPro 
405410 415 
CGTGCTGCTTCTGCAAAAGATTTTTGTAGATGAGCTGTGTGCCTCAGAATTGCTATTTCA1363 
AATTGCCAAAAATTTAGAGATGTTTTCTACATATTTCTGCTCTTCTGAACAACTTCTGCT1423 
ACCCACTAAATAAAAACACAGAAATAATTAGACA ATTGTCTATTATAACATGACAACCCT1483 
ATTAATCATTTGGTCTTCTAAAATGGGATCATGCCCATTTAGATTTTCCTTACTATCAGT1543 
TTATTTTTATAACATTAACTTTTACTTTGTTATTTATTATTTTATATAATGGTGAGTTTT1603 
TAAATTATTGC TCACTGCCTATTTAATGTAGCTAATAAAGTTATAGAAGCAGATGATCTG1663 
TTAATTTCCTATCTAATAAATGCCTTTAATTGTTCTCATAATGAAGAATAAGTAGGTATC1723 
CCTCCATGCCCTTCTGTAATAAATATCTGGAAAAAACATTAAACAATAGGCAAATAT ATG1783 
TTATGTGCATTTCTAGAAATACATAACACATATATATGTCTGTATCTTATATTCAATTGC1843 
AAGTATATAATGTCATAATTTCAAGACCAGCCTGGCCAACATAGCGAAACCCTACCTCCA1903 
CTAAAAATACAGAAATGAGCCGGGAGTGGTGGCA AAGTGGTGAGCACCTGTGATCCCAGC1963 
CACTGTGGAGGCCGAGGCAGGACAATCACTTGAACCCAGGAGGCGGAGGCTGCAGTGAGC2023 
TGAGATCGCTCCACTGCACTCCAGCCTGGGCAACAGAGCAAGATTCCATCTCAAAATACA2083 
TTAAAAAAAAA AACCTATCTGAGGACTCTGAAAAGTAAATGGTAGCAGATAGATTTGAGA2143 
AGGGAACTAGAACTTGAAGCACAATCTATCTGGTGCTCTTTCTTACTTTTGCTTGTTTTC2203 
TCCCAATCTTCCAGTCTGGATACAAAGGCAGCCCAATTTCTAGAAATGTATACCAGC CAT2263 
GAAGAGATAAAGCTCCAAGAGGAGATTTCTCTTTCTGGTATAAGGTATGTGTGTGTATAT2323 
GGGGGGCGATAAGGTTGGGAGTGTGAGGAATACAGAGTCGGAGAAATCCATTATTTCCAC2383 
CCTCTCTCTTGCCATTGCAACCAGAC 2409 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 415 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
MetGluAspLeuCysValAlaAsnThrLeuPheAlaLeuA snLeuPhe 
151015 
LysHisLeuAlaLysAlaSerProThrGlnAsnLeuPheLeuSerPro 
202530 
Trp SerIleSerSerThrMetAlaMetValTyrMetGlySerArgGly 
354045 
SerThrGluAspGlnMetAlaLysValLeuGlnPheAsnGluValGly 
50 5560 
AlaAsnAlaValThrProMetThrProGluAsnPheThrSerCysGly 
65707580 
PheMetGlnGlnIleGlnLysGlySerTy rProAspAlaIleLeuGln 
859095 
AlaGlnAlaAlaAspLysIleHisSerSerPheArgSerLeuSerSer 
100105 110 
AlaIleAsnAlaSerThrGlyAsnTyrLeuLeuGluSerValAsnLys 
115120125 
LeuPheGlyGluLysSerAlaSerPheArgGluGluTyrIleArgLeu 
130135140 
CysGlnLysTyrTyrSerSerGluProGlnAlaValAspPheLeuGlu 
145150155160 
CysAlaGluGluAlaArg LysLysIleAsnSerTrpValLysThrGln 
165170175 
ThrLysGlyLysIleProAsnLeuLeuProGluGlySerValAspGly 
180 185190 
AspThrArgMetValLeuValAsnAlaValTyrPheLysGlyLysTrp 
195200205 
LysThrProPheGluLysLysLeuAsnGlyLeuTyrProP heArgVal 
210215220 
AsnSerAlaGlnArgThrProValGlnMetMetTyrLeuArgGluLys 
225230235240 
LeuAsn IleGlyTyrIleGluAspLeuLysAlaGlnIleLeuGluLeu 
245250255 
ProTyrAlaGlyAspValSerMetPheLeuLeuLeuProAspGluIle 
260 265270 
AlaAspValSerThrGlyLeuGluLeuLeuGluSerGluIleThrTyr 
275280285 
AspLysLeuAsnLysTrpThrSerLysAs pLysMetAlaGluAspGlu 
290295300 
ValGluValTyrIleProGlnPheLysLeuGluGluHisTyrGluLeu 
305310315 320 
ArgSerIleLeuArgSerMetGlyMetGluAspAlaPheAsnLysGly 
325330335 
ArgAlaAsnPheSerGlyMetSerGluArgAsnAspLeuPheLeuSer 
340345350 
GluValPheHisGlnAlaMetValAspValAsnGluGluGlyThrGlu 
355360365 
AlaAlaAlaGlyThrGly GlyValMetThrGlyArgThrGlyHisGly 
370375380 
GlyProGlnPheValAlaAspHisProPheLeuPheLeuIleMetHis 
385390395 400 
LysIleThrAsnCysIleLeuPhePheGlyArgPheSerSerPro 
405410415