Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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1
MODIFIED TISSUE PLASMINOGEN ACTIVATOR
The present invention relates to fibrinolytic
factors in general, and more specifically, to modified
tissue-type plasminogen activators.
Blood coagulation is a process consisting of a
complex interaction of various blood components which even-
tually gives rise to a fibrin network or clot. Degradation
of the fibrin network can be accomplished by activation of
the zymogen plasminogen into plasmin, a serine protease
which acts directly to degrade the fibrin network. Conver-
sion of plasminogen into plasmin can be catalyzed by
tissue-type plasminogen activator (t-PA), a fibrin-specific
serine protease.
T-PA is believed to be the physiological vascular
activator of plasminogen and normally circulates as a
single polypeptide chain (Mr, 72,000). Urokinase-type
plasminogen activator (u-PA) is another member of the class
of plasminogen activators characterized as serine proteases.
U-PA is functionally and immunologically distinguishable
from t-PA.
In the presence of fibrin, t-PA is activated by
cleavage at a single site in the central region of the
molecule. The heavy chain of t-PA (two variants of Mr
40,000 and 37,000) is derived from the amino terminus,
1 X41 44 4
2
while the light chain (Mr, 33,000) is derived from the
carboxy-terminal end of the t-PA molecule.
A two-dimensional model of the potential
precursor t-PA protein has been established (Ny et al.,
PNAS 81: 5355-5359, 1984). From this model, it was deter
mined that the heavy chain contains two triple disulfide
structures known as "kringles." These kringle structures
also occur in prothrombin, plasminogen and urokinase and
are believed to be important for binding to fibrin (Ny et
al., ibid). The second kringle (K2) of t-PA is believed to
have a higher affinity for fibrin than the first kringle
(K1) (Ichinose, Takio, and Fujikawa, J. Clin. Invest. 78:
163-169 (19i~5)).
The heavy chain of t-PA also contains a "finger"
domain that is homologous to the finger domains of fibro
nectin. Fibronectin has been implicated in a variety of
biological activities, including fibrin binding, and such
biological activity has been correlated to four or five of
the nine finger domains possessed by fibronectin. The
heavy chain of t-PA also contains a growth factor-like
domain.
T-PA's light chain contains the active site for
serine protease activity, which is highly homologous to the
active sites of other serine proteases.
Native t-PA additionally comprises a pre-region
followed downstream by a pro-region, which are collectively
referred to as the "pre-pro" region. The pre-region con-
tains a signal peptide which is important for secretion of
t-PA by vascular endothelial cells (Ny et al., ibid). The
pre sequence is believed responsible for secretion of t-PA
into the lumen of the endoplasmic reticulum, a necessary
step in extracellular secretion. The pro sequence is
believed to be cleaved from the t-PA molecule following
transport from the endoplasmic reticulum to the Golgi
apparatus.
The biological activity of t-PA is substantially
enhanced in the presence of fibrin (Sherry, New Eng. J.
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3
Med. 313: 1014-1017, 1985). Unlike the less specific
plasminogen activators streptokinase and urokinase, t-PA
has relatively little serine protease activity except at
the site of the clot. It is theorized that plasminogen and
t-PA are initially bound to the fibrin clot, with the
result that enzymatic degradation of the plasminogen into
plasmin is sterically facilitated.
The use of t-PA for fibrinolysis in animal and
human subjects has highlighted several shortcomings of the
native molecule. The half-life of t-PA in vivo has been
shown to be as short as three minutes in humans (Nilsson et
al., Scand. J. Haematol. 33: 49-53, 1984). Injected t-PA
was rapidly cleared by the liver, and most of the injected
material was metabolized to low molecular weight forms
within 30 minutes. This short half-life may limit the
effectiveness of t-PA as a thrombolytic agent by
necessitating high dosages and prolonged infusion. Fuchs
et al. (Blood 65: 539-544, 1985) concluded that infused
t-PA is cleared by the liver in a process independent of
the proteolytic site, and that infused t-PA will not
accumulate in the body. Furthermore, doses of t-PA
sufficient to lyse coronary thrombi are far larger than
normal physiological levels, and lead to systemic
degradation of fibrinogen (Sherry, ibid).
Consequently, for clinical applications it would
be advantageous to employ fibrinolytic agents possessing
enhanced fibrin-binding capability, increased biological
half-life or increased solubility as compared to native
t-PA. Such agents would preferably lack structural and
functional features which may not play an active role in
fibrinolysis. For example, it would be desirable to
produce a fibrinolytic molecule which did not contain an
epidermal growth factor (EGF) domain since it has been
discovered that the EGF domain is not necessary for
fibrinolytic activity. Deletion of all or part of the
growth factor domain may also increase the solubility of
the molecule, due to the hydrophobic nature of that domain.
.. . 1 3 41 44 4
Increased solubility would permit use of smaller
(injectable) amounts of t-PA and permit faster
administration to patients. Because the active site is not
involved in the physiological clearing of t-PA, removal of
extraneous domains may also increase the half-life of the
resultant modified t-PA molecule in vivo without
inactivating it. Specific activity may also be increased,
thereby allowing the use of smaller doses. Furthermore,
enhancement of t-PA fibrin binding could be achieved by
adding additional kringle structures and/or finger domains.
Additionally, a smaller molecule may be more easily
secreted by recombinant cells.
In light of the facts that native t-PA is a
composite mosaic polypeptide and the t-PA gene comprises
multiple exons encoding the aforementioned structural and
functional domains (Ny et al., ibid), it would be desirable
to construct a DNA sequence which optimally expresses the
specific fibrinolytic activity of t-PA. Optimization of
the protein product could be accomplished by cloning only
the nucleotide sequences which encode the desired structur
al and functional properties of t-PA, while eliminating
those sequences which do not contribute to the desired
biological activity. Multiple copies of the desired
structures may also be incorporated into the optimized
proteins.
Clearly, there is a need in the art for a
fibrinolytic agent which combines the clinical efficacy of
t-PA with ease of administration and minimal undesirable
side effects. The present invention fulfills this need by
providing modified forms of t-PA which may be produced in
relatively large quantities. Through the use of
recombinant DNA technology, a consistent and homogeneous
source of modified t-PAs is provided. The modified t-PAs
can be utilized to lyse existing clots in heart attack and
stroke victims and in others where the need to lyse or
suppress the formation of fibrin matrices is
therapeutically desirable.
1341444
Briefly stated, the present invention discloses a
DNA construct containing a nucleotide sequence consisting
essentially of a first region encoding a fibrin-binding
domain, and a second region positioned downstream of the
first region, with the second region encoding a catalytic
domain for the serine protease activity of tissue-type
plasminogen activator. The sequence codes for a protein
which has substantially the same biological activity as
t-PA. The first and second regions may be derived either
from genomic clones or cDNA clones of t-PA or may be
constructed by conventional DNA synthesis techniques.
Preferably, the first region, which encodes the
fibrin-binding domain, encodes one or more kringle struc
tures. In particular, the first region encodes the K1 and
K2 kringle structures of t-PA, a duplicated K2 structure or
a single. K2 structure; or kringle structures from other
proteins may be substituted. The first region encoding the
fibrin-binding domain may additionally encode one or more
finger domains.
The second region encodes a catalytic domain
which is essentially the serine protease domain of t-PA. A
particularly preferred second region encodes the light
chain of t-PA extending from amino acid number 276 and
continuing through amino acid number 527.
In addition, the present invention discloses
expression vectors capable of directing the expression of a
protein having substantially the same biological activity
as t-PA. The vectors include a promoter which is operably
linked to a nucleotide sequence, the nucleotide sequence
consisting essentially of a first region encoding a
fibrin-binding domain, and a second region positioned
downstream from the first region, the second region
encoding a catalytic domain for the serine protease
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activity of t-PA. The sequence codes for a protein which
has substantially the same biological activity as t-PA.
A third aspect of the invention discloses
cells transfected or transformed to produce a protein
having substantially the same biological activity as t-PA.
The cells contain a DNA construct comprising a promoter
operably linked to a nucleotide sequence consisting
essentially of a first region encoding a fibrin-binding
domain and a second region positioned downstream of the
first region, the second region encoding a catalytic domain
for the serine protease activity of t-PA. The sequence
codes for a protein which has substantially the same
biological activity as t-PA.
The cells may be mammalian cells or microorgan
isms. Preferred microorganisms include bacteria, particu
larly E. coli, and eukaryotic microorganisms, particularly
the yeast Saccharomyces cerevisiae and filamentous fungi
such as As ergillus.
The present invention further provides a method
of producing a protein which has substantially the same
biological activity as t-PA. The method comprises the
steps of inserting into host cells a DNA construct which
contains a promoter operably linked to a nucleotide
sequence consisting essentially of a first region encoding
a fibrin-binding domain and a second region positioned
downstream of said first region, the second region encoding
a catalytic domain for the serine protease activity of t-PA.
The sequence codes for a protein which has substantially
the same biological activity as t-PA. The second step
involves growing the host cells in an appropriate medium,
followed by the step of isolating the protein product
encoded by the DNA construct that is produced by the host
cells. The host cells may be mammalian cells or
microorganisms. Preferred microorganisms include bacteria,
particularly E. coli, and eukaryotic microorganisms, parti-
cularly the yeast Saccharomyces cerevisiae and filamentous
fungi such as Aspergillus.
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7
Still a further aspect of the present invention
discloses proteins having substantially the same biological
activity as t-PA which consist essentially of an amino
terminal fibrin-binding domain and a carboxyl terminal
serine protease domain.
Other aspects of the invention will become
evident upon reference to the following detailed
description and attached drawingsi in which
Figure 1 illustrates the pre-pro t-PA coding
sequence constructed from cDNA and synthesized oligo-
nucleotides, together with the amino acid sequence of the
encoded protein. Numbers above the lines refer to
nucleotide position and numbers below the lines refer to
amino acid position.
Figure 2 illustrates the construction of the
vector Zem99.
Figure 3 illustrates the construction of the t-PA
expression vector pDR817.
Figure 4 shows a comparison of the amino termini
of several modified t-PA molecules described herein.
Figure 5 illustrates the construction of plasmid
pDR3002 and t-PA vectors derived from pDR3002.
Figure 6 illustrates the amino acid sequence of a
mutant t-PA protein lacking the finger and growth factor
domains, and the nucleotide sequence encoding the protein.
Figure 7 illustrates the amino acid sequence of a
mutant t-PA protein lacking the finger, growth factor and
Kringle 1 domains, together with the nucleotide sequence
encoding the protein.
Prior to setting forth the invention, it may be
helpful to an understanding thereof to set forth defini
tions of certain terms used herein.
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a
Complementary DNA or cDNA: A DNA molecule or
sequence which has been enzymatically synthesized from the
sequences present in an mRNA template.
DNA Construct: A DNA molecule, or a clone of
such a molecule, either single- or double-stranded, which
may be isolated in partial form from a naturally occurring
gene or which has been modified to contain segments of DNA
which are combined and juxtaposed in a manner which would
not otherwise exist in nature.
Plasmid or Vector: A DNA construct containing
genetic information which provides for its replication when
inserted into a host cell. Replication may be autonomous
or by integration into the host genome. A plasmid general-
ly contains at least one gene sequence to be expressed in
the host cell, as well as sequences encoding functions
which facilitate such gene expression, including promoters
and transcription initiation sites and terminators. It may
be a linear or a closed, circular molecule.
Pre-pro Region: An amino acid sequence which
generally occurs at the amino termini of the precursors of
certain proteins, and which is generally cleaved from the
protein, at least in part, during secretion. The pre-pro
region comprises, in part, sequences directing the protein
into the secretory pathway of the cell.
Domain: A three-dimensional, self-assembling
array of specific amino acids of a protein molecule, which
contains structural elements necessary for a specific
biological activity of that protein.
Fibrin-binding Domain: That portion of a protein
necessary for the binding of that protein to fibrin. In
native t-PA, the kringle structures and finger domain indi
vidually and collectively contribute to the fibrin binding.
According to the present invention, it has been found that
the EGF domain does not significantly contribute to fibrin
binding of t-PA or modified t-PAs.
Biological Activity: The function or set of
functions performed by a molecule in a biological context
141444
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( i . e. , in an organism, a cell, or an in vitro facsimile) .
Biological activities of proteins may be divided into
catalytic and effector activities. Catalytic activities of
fibrinolytic factors often involve the activation of other
proteins through specific cleavage of precursors. In
contrast, effector activities include specific binding of
the biologically active molecule to other molecules, such
as fibrin, or to cells. Effector activity frequently
augments, or is essential to, catalytic activity under
physiological conditions. Catalytic and effector
activities may, in some cases, reside in the same domain of
the protein. For native t-PA, biological activity is
characterized by the conversion, in the presence of fibrin,
of the pro-enzyme or zymogen plasminogen into plasmin,
which in turn degrades fibrin matrices. Because fibrin
acts as a cofactor in the activation of plasminogen, native
t-PA has little activity in the absence of fibrin. As used
herein the phrase "substantially the same biological
activity as t-PA" includes conversion of, in the presence
of fibrin, the pro-enzyme or zymogen plasminogen into
plasmin.
As noted above, t-PA is known to play a major
role in the degradation of fibrin clots. Because native
t-PA is a mosaic protein possessing regions within the mole-
rule which have been found to be unnecessary for the
fibrinolytic activity of t-PA, it is desirable to provide
modified t-PA molecules wherein the fibrin-binding and
serine protease activities are retained while excluding all
or part of the EGF domain and other non-fibrinolytic
functional domains from such modified t-PA. Additionally,
it would be desirable to produce modified t-PA having
enhanced fibrin-binding activity or increased in vivo
half-life.
According to the present invention, it is
preferred to produce these novel proteins through the use
of recombinant DNA technology, using cDNA clones or genomic
clones as starting materials. Suitable DNA sequences can
1 341 44 4
to
also be synthesized according to standard procedures. It
is preferred to use cDNA clones because, by employing the
full-length cDNA encoding native t-PA as starting material
for producing modified t-PA, introns are removed so that
all exons of the native t-PA are present and correctly
oriented with respect to one another. Full-length cDNA
also provides the advantage of easily generating modified
molecules by "chewing back" the t-PA cDNA from the 5' end,
thus providing a multiplicity of cDNA fragments which can
be ultimately inserted into host cells. Alternatively, the
cDNA can be used as a template for deletion or insertion of
sequences via oligonucleotide-directed mutagenesis.
Utilization of t-PA cDNA allows the convenient
enhancement of the fibrin-binding domain of native t-PA by
the insertion of additional kringle structures and finger
domains. This methodology provides a means for selecting
the optimum combination of functional domains found in
native t-PA or other proteins to provide fibrinolytic
agents with enhanced biological activity with respect to
fibrin-binding and serine protease activity.
Accordingly, the present invention provides a
method of producing novel proteins having biological
activity that is substantially the same as t-PA. Novel
proteins described herein have been shown to have a
five- to ten-fold greater in vivo half-life than native
t-PA. These novel proteins are expressed in transfected
mammalian cells, and in transformed fungi and bacteria.
A cDNA clone containing sequences encoding mature
human t-PA has been previously identified. The t-PA cDNA
sequence has been inserted into plasmid pDR1296, and this
plasmid has been introduced into E. coli JM83. This
transformant has been deposited with the American Type
Culture Collection, Bethesda, MD, and assigned Accession
No. 53347.
Strain pDR1296/JM83 was used as the source of
t-PA cDNA. Plasmid pDR1296 was isolated, and the t-PA cDNA
was excised by restriction endonuclease digestion. The
1 341 44 4
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t-PA restriction fragment was then incubated with Bal 31,
and the enzymatic reaction time was controlled in order to
produce a continuum of t-PA nucleotide sequences exhibiting
size heterogeneity. The size heterogeneity results from
the progressive shortening of the 5' terminus of the t-PA
cDNA.
The Bal 31 digested fragments were separated by
gel electrophoresis and the desired fragment size range was
selected. The fragments were eluted from the gel according
to conventional techniques.
In a second approach, part of the cDNA sequence
from pDR1296 was used as a template for oligonucleotide
directed deletion mutagenesis. By this method, sequences
encoding specific domains of native t-PA are precisely
deleted or altered.
In a third approach, the 5' coding region of a
modified t-PA sequence was constructed from synthesized
oligonucleotides and joined to the 3' region of the cDNA.
The modified t-PA fragments were then joined to
an appropriate pre-pro sequence. The pre-pro sequence of
t-PA may be isolated from cDNA or genomic libraries, or may
be constructed from synthesized oligonucleotides. Oligo
nucleotides are preferably machine-synthesized, purified,
and annealed to construct double-stranded fragments. The
resultant double-stranded fragments are ligated as
necessary to produce the pre-pro sequence. This pre-pro
sequence comprises a 105 by sequence, as depicted in Figure
1. The synthesized pre-pro sequence was then joined to the
5' termini of the modified t-PA fragments. Other pre-pro
sequences may be used, depending on the host cell type
selected. In some instances it may be desirable to use a
pre-pro sequence which is endogenous to the host species.
The pre-pro-t-PA fragments are inserted into a
suitable expression vector, which in turn is inserted into
appropriate host cells. The method of insertion will
depend upon the particular host cell chosen. Methods for
transfecting mammalian cells and transforming bacteria and
1 349 44 4
12
fungi with foreign DNA are well known in the art. Suitable
expression vectors will comprise a promoter capable of
directing the transcription of a foreign gene in a host
cell.
In some instances it is preferred that expression
vectors further comprise an origin of replication, as well
as sequences which regulate and/or enhance expression
levels, depending on the host cell selected. Suitable
expression vectors may be derived from plasmids, RNA and
DNA viruses or cellular DNA sequences, or may contain
elements of each.
Preferred prokaryotic hosts for use in carrying
out the present invention are strains of the bacteria
Escherichia coli, although Bacillus and other genera are
also useful. Techniques for transforming these hosts and
expressing foreign DNA sequences cloned in them are well
known in the art (see, for example, Maniatis et al.,
Molecular. Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1982). Vectors used for expressing foreign DNA
in bacterial hosts will generally contain a selectable
marker, such as a gene for antibiotic resistance, and a
promoter which functions in the host cell. Appropriate
promoters include the trp (Nichols and Yanofsky, Meth. in
Enzymology 101: 155, 1983), lac (Casadaban et al., J. Bact.
143: 971-980, 1980), TAC (Russell et al., Gene 20: 231-243,
1982), and phage promoter systems. Plasmids useful for
transforming bacteria include pBR322 (Bolivar et al., Gene
2: 95-113, 1977), the pUC plasmids (Messing, Meth. in
Enzymology 101: 20-77, 1983: and Vieira and Messing, Gene
19: 259-268, 1982), pCQV2 (Queen, J. Mol. Appl. Genet. 2:
1-10, 1983), and derivatives thereof.
Eukaryotic microorganisms, such as the yeast
Saccharomyces cerevisiae, or filamentous fungi including
Asperqillus, may also be used as host cells. Particularly
preferred species of Aspergillus include A. nidulans,
A. niger, A. o~~zae, and A. terreus. Techniques for
transforming yeast are described by Beggs (Nature 275:
1341444
13
104-108, 1978). Expression vectors for use in yeast
include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:
1035-1039, 1979), YEpl3 (Broach et al., Gene 8: 121-133,
1979), pJDB248 and pJDB219 (Beggs, ibid), and derivatives
thereof. Such vectors will generally comprise a selectable
marker, such as the nutritional marker TRP, which allows
selection in a host strain carrying a trill mutation.
Preferred promoters for use in yeast expression vectors
include promoters from yeast glycolytic genes (Hitzeman et
al., J. Biol. Chem. 255: 12073-12080, 1980; Alber and
Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982) or alcohol
dehydrogenase genes (Young et al., in Genetic Engineering
of Microorganisms for Chemicals, Hollaender et al., eds.,
p. 335, Plenum, New York, 1982: and Ammerer, Meth. in
Enzymology 101: 192-201, 1983). To facilitate purification
of a modified t-PA protein produced in a yeast transformant
and to obtain proper disulphide bond formation, a signal
sequence from a yeast gene encoding a secreted protein may
be substituted for the t-PA pre-pro sequence. A particu-
larly preferred signal sequence is the pre-pro region of
the MF 1 gene (Kurjan and Herskowitz, Cell 30: 933-943,
1982). Aspergillus species may be transformed according to
known procedures, for example, that of Yelton et al. (Pros.
Natl. Acad. Sci. USA 81: 1740-1747, 1984).
Higher eukaryotic cells may also serve as host
cells in carrying out the present invention. Cultured
mammalian cells, such as the BHK, CHO, and J558L cell
lines, are preferred. Tk- BHK cells are particularly
preferred. Expression vectors for use in mammalian cells
will comprise a promoter capable of directing the
transcription of a foreign gene introduced into a mammalian
cell. Particularly preferred promoters include the SV40
(Subramani et al., Mol. Cell Biol. 1:854-64, 1981) and MT-1
promoters (Palmiter et al., Science 222: 809-814, 1983).
Also contained in the expression vectors is a transcription
terminator, located downstream of the insertion site for
the DNA sequence to be expressed. A preferred terminator is
.a. 1341444
14
the human growth hormone (hGH) gene terminator (DeNoto et
al., Nuc. Acids Res. 9: 3719-3730, 1981).
For expression of mutant t-PAs in cultured
mammalian cells, expression vectors containing cloned t-PA
sequences are introduced into the cells by appropriate
transfection techniques, such as calcium phosphate-mediated
transfection (Graham and Van der Eb, Virology 52: 456-467,
1973: as modified by Wigler et al., Proc. Natl. Acad. Sci.
USA 77: 3567-3570, 1980). A DNA-calcium phosphate precipi-
tate is formed, and this precipitate is applied to the
cells in the presence of medium containing chloroquine
(100um). The cells are incubated for four hours with the
precipitate, followed by a two-minute, 15~ glycerol shock.
A portion of the cells take up the DNA and maintain it
inside the cell for several days. A small fraction of the
cells integrate the DNA into the genome of the host cell or
maintain the DNA in non-chromosomal nuclear structures.
These transfectants are identified by cotransfection with a
gene that confers a selectable phenotype (a selectable
marker). A preferred selectable marker is the DHFR gene,
which imparts cellular resistance to methotrexate (MTX), an
inhibitor of nucleotide synthesis. After the host cells
have taken up the DNA, drug selection is applied to select
for a population of cells that are expressing the
selectable marker in a stable fashion.
Coamplification as a means to increase expression
levels can be accomplished by the addition of MTX to the
culture medium, preferably by sequentially increasing the
concentration of MTX in the medium, followed by repeated
cloning by dilution of the drug-resistant cell lines.
Variations in the ability to amplify relate both to the
initial genomic configuration (i.e. extra-chromosomal vs.
chromosomal) of the cotransfected DNA sequences as well as
to the mechanism of amplification itself, in which variable
amounts of DNA rearrangements can occur. This is noticed
upon further amplification of clones previously shown to be
capable of frequent and stable coamplification. For this
15 1341444
reason, it is necessary to clone by dilution after every
amplification step. Cells which express the DHFR marker
are then selected and screened for production of t-PA.
Screening may be done by enzyme linked immunosorbent assay
(ELISA) or by biological activity assays.
Additionally, it has been observed that under
certain conditions production of t-PA has a deleterious
effect on mammalian cells in culture. This is believed to
be due in part to the plasmin (a nonspecific protease)
generated when cells which have been transfected to produce
t-PA are cultured in media containing plasminogen, a
component of serum. The t-PA produced by the transfected
cells activates the plasminogen to plasmin, which attacks
cell membranes and contributes to cell detachment. Other
proteolytic activities are also believed to be involved.
It has been found that including protease inhibitors in the
culture media blocks the activation of plasminogen and
facilitates t-PA production. A particularly preferred
protease inhibitor is aprotinin, which is included in the
culture media at a concentration of from approximately 100
units/ml to 50,000 units/ml, preferably 100 units/ml, in
medium containing plasminogen-free serum, or from approxi-
mately 1000 units/ml to 50,000 units/ml, preferably 1000
units/ml, if using normal fetal calf serum. Additional
useful protease inhibitors include tranexamic acid and
epsilon amino caproic acid, which are included in the media
at mM concentrations.
The t-PA so produced is recovered from the
cultured cells by removing the culture medium and fraction
sting it. A preferred method of fractionation is affinity
chromatography using an anti-t-PA antibody, a fibrin-celite
column or a lysine-sepharose column. Other conventional
purification methods, such as ion-exchange chromatography,
high-performance liquid chromatography or gel filtration,
may also be used.
In summary, the present invention provides
modified t-PA proteins and a method for production of
1341444
16
modified t-PA proteins having substantially the same
biological activity as native t-PA through use of stably
transfected or transformed host cells. The protein
products thus expressed are then purified from the cells or
cell growth media and assayed for biological activity. The
assay may monitor conversion of plasminogen to plasmin,
fibrin-binding affinity or plasma half-life characteristics.
Immunological assays may also be used.
To summarize the examples which follow, Example 1
discloses a t-PA cDNA clone made from mRNA from the Bowes
melanoma cell line. The cDNA was used to construct the
plasmid pDR1296 which was utilized to transform E. coli
strain JM83. The t-PA sequence of pDR1296 was then joined
to a synthesized pre-pro sequence which was constructed
from oligonucleotides. An MT-1 promoter and human growth
hormone terminator were added and the vector Zem99, as
depicted in Figure 2, was constructed.
Example 2 discloses the cloning and sequencing of
human genomic t-PA obtained from a DNA library derived from
normal liver tissue.
Example 3 discloses the preparation of t-PA
sequences having random 5' end deletions utilizing the
full-length pDR1296 t-PA clone. A TAC promoter and t-PA
pre-pro sequence were ligated to the randomly deleted t-PA
cDNA sequences. A pDR$17 construct directed the production
of t-PA fibrinolytic activity ten- to thirty-fold higher
than that obtained using a full-length t-PA clone.
Expression of the truncated sequences in cultured mammalian
cells is also disclosed.
Examples 4 and 5 describe methods of looping out
the coding sequences for the finger domain and growth
factor domain of t-PA.
Examples 6 and 7 disclose methods of looping out
the coding sequences for the finger domain, growth factor
domain and Kringle 1 structure to produce modified t-PA.
Example 8 discloses the construction of a mutant
t-PA sequence utilizing synthesized oligonucleotides.
17 1341444
Example 9 discloses a mutant sequence lacking the
growth factor domain coding sequence which is constructed
by deletion mutagenesis.
Example 10 describes the method of expressing
mutant t-PAs in transformed E. coli JM105 utilizing
bacterial expression vector pDR816 (described in Example
3).
Example 11 teaches a method of expressing mutant
t-PAs in mammalian cells by transfecting baby hamster
kidney (BHK) cells with expression vectors comprising
mutant t-PA sequences.
Example 12 describes the expression of mutant
t-PAs in S. cerevisiae cells which are transformed with a
vector comprising a mutant t-PA nucleotide sequence.
The following examples are offered by way of
illustration and not by way of limitation.
~vrnnnr ~e
Example 1 - Construction of a Full-Length t-PA Clone
The sequence of a human t-PA cDNA clone has been
reported (Pennica et al., Nature 301: 214-221, 1983). The
sequence encodes a pre-pro peptide of 32-35 amino acids
followed by a 527-530 amino acid mature protein.
A cDNA clone comprising the coding sequence for
mature t-PA was constructed using as starting material mRNA
from the Bowes melanoma cell line (Rijken and Collen, J.
Biol. Chem. 256: 7035-7041, 1981). This cDNA was then used
to construct the plasmid pDR1296. Escherichia coli strain
JM83 transformed with pDR1296 has been deposited with the
American Type Culture Collection under Accession No. 53347.
Because the pre-pro sequence was not present in
the cDNA clone pDR1296, it was constructed from synthesized
oligonucleotides and subsequently joined to the cDNA. In
the synthesized t-PA pre-pro sequence, cleavage sites for
Bam HI and Nco I were introduced immediately 5' to the
first codon (ATG) of the pre-pro sequence, and a Bgl II
(Sau 3A, Xho II) site was maintained at the 3' end of the
1 341 44 4
18
pre-pro sequence. The naturally occurring pre-pro sequence
lacks a convenient restriction site near the middle;
however, the sequence GGAGCA (coding for amino acids -20
and -19, Gly-Ala) can be altered to GGCGCC to provide a Nar
I site without changing the amino acid sequence.
To construct the pre-pro sequence, the following
oligonucleotides were synthesized using an Applied
Biosystems Model 380-A DNA synthesizer:
ZC131: 5~GGA TCC ATG GAT GCA ATG AAG AGA GGG CTC TGC
TGT GTG3~
ZC132: S~TGG CGC CAC ACA GCA GCA GCA CAC AGC AGAG3~
ZC133: S~GGC GCC GTC TTC GTT TCG CCC AGC CAG GAA ATC
CATG3~
ZC134: S AGA TCT GGC TCC TCT TCT GAA TCG GGC ATG GAT
TTC CT3
Following purification, oligomers ZC131 and ZC132
were annealed to produce an overlap of 12 base pairs
(Section 1). Oligomers ZC133 and ZC134 were similarly
annealed (Section 2).
The oligomers were mixed in Pol I buffer
(Bethesda Research Labs), heated to 65°C for five minutes,
and slowly cooled to room temperature for four hours to
anneal. Ten units of DNA polymerase I were added and the
reaction proceeded for two hours at room temperature. The
mixtures were electrophoresed on an 8% polyacrylamide-urea
sequencing gel at 1,000 volts for 2~- hours in order to size
fractionate the reaction products. The correct size
fragments (those in which the polymerase reaction went to
completion) were cut from the gel and extracted.
After annealing, Section 1 was cut with Bam HI
and Nar I and cloned into Bam HI + Nar I - cut pUC8 (Vieira
and Messing, Gene 19: 259-268, 1982; and Messing, Meth. in
Enzymology 101: 20-77, 1983). Section 2 was reannealed and
cut with Nar I and Bgl II and cloned into Bam HI + Nar (Sau
3A, Xho I - cut pUC$. Colonies were screened with the
1341444
19
appropriate labelled oligonucleotides. Plasmids identified
as positive by colony hybridization were sequenced to
verify that the correct sequence had been cloned.
Section 1 was then purified from a Bam HI + Nar I
double digest of the appropriate pUC clone. Section 2 was
purified from a Nar I + Xho II digest. The two fragments
were joined at the Nar I site and cloned into Bam HI - cut
pUC8.
The t-PA sequence of pDR1296 was then joined to
the synthesized pre-pro sequence in the following manner
(Figure 2). Plasmid pICl9R (Marsh et al., Gene 32:
481-486, 1984) was digested with Sma I and Hind III. The
on region of SV40 from map position 270 (Pvu II) to
position 5171 (Hind III) was then ligated to the linearized
pICl9R to produce plasmid Zem67. This plasmid was then
cleaved with Bgl II and the terminator region from the
human growth hormone gene (De Noto et al., Nuc. Acids Res.
9: 3719-3730, 1981) was inserted as a Bgl II - Bam HI
fragment to produce plasmid Zem86. The synthesized t-PA
pre-pro sequence was removed from the pUC8 vector by
digestion with Sau 3A. This fragment was inserted into Bgl
II-digested Zem86 to produce plasmid Zem88. Plasmid
pDR1296 was digested with Bgl II and Bam HI and the t-PA
cDNA fragment was isolated and inserted into Bgl II - cut
Zem88. The resultant plasmid was designated Zem94.
The vector, Zem99, comprising the MT-1 promoter,
complete t-PA coding sequence, and the hGH terminator was
then assembled in the following manner (Figure 2). A Kpn
I-Bam HI fragment comprising the MT-1 promoter was isolated
from MThGHlll (Palmiter et al., Science 222: 809-814, 1983)
and inserted into pUCl8 to construct Zem93. Plasmid
MThGH112 (Palmiter et al., ibid) was digested with Bgl II
and religated to eliminate the hGH coding sequence. The
MT-1 promoter and hGH terminator were then isolated as an
Eco RI fragment and inserted into pUCl3 to construct Zem4.
Zem93 was then linearized by digestion with Bam HI and Sal
I. Zem4 was digested with Bgl II and Sal I and the hGH
1 341 44 4
terminator was purified. The t-PA pre-pro sequence was
removed from the pUCB vector as a Bam HI-Xho II fragment.
The three DNA fragments were then joined and a plasmid
having the structure of Zem97 (Figure 2) was selected.
5 Zem97 was cut with Bgl II and the Xho II t-PA fragment from
Zem94 was inserted. The resultant vector is Zem99.
Example 2 - Cloning and Sequencing of Human Genomic t-PA
('l r,no
10 A genomic t-PA clone was obtained from a DNA
library derived from normal liver tissue. The library was
constructed by insertion of fetal human liver DNA fragments
into bacteriophage lambda (Lawn et al., Cell 15: 1157-1174,
1978).
15 The library was used to infect E. coli strain
LE392 (ATCC 33572) (Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1982, p.
504). An overnight culture of cells in L-broth containing
0.2~ maltose, 10 mM MgS04, and 50 ug/ml thymidine was con-
20 centrated two-fold in 10 mM MgS04. 750 u1 of the concen-
trate was plated, together with 1 u1 of the phage library
(200,000 phage/ul) on L-broth agar in a NZY amine soft agar
overlay using 22 cm x 22 cm plates. Approximately 105
colonies were obtained per plate following an overnight
incubation at 37°C. Colonies were transferred to nitro-
cellulose and the lifts were treated with 0.1 M NaOH, 1.5 M
NaCl, air-dried, and baked two hours at 80°C. Pre-hybridi-
zation and hybridization (to a full-length, nick-translated
t-PA cDNA probe) were carried out in SET buffer (SET con-
tains, per liter, 175.2 g NaCl, 72.7 g Tris, 14.8 g EDTA,
pH 8.0 with HC1) at 65°C. Filters were washed in 2 x SSC,
O.lo SDS, dried, and autoradiographed. Thirteen prelimi
nary positives were identified. Two rounds of plaque
purification (screened as above) identified nine positives
from the group of thirteen.
The nine positive genomic clones were plated on
E. coli LE392, grown overnight, and lysates were prepared.
1341444
21
The phage were purified on CsCl gradients and mapped by
hybridization to oligonucleotide probes shown in Table 1.
mnur~ i
ZC46 (5' TCG TTT ACT CTA GG3')
ZC88 (5' TGC AGC GAG CCA AGG3')
ZC89 (5' ACG TGG AGC ACA GCG3')
ZC91 (5' CCC TCC TGC TCC ACC3')
ZC94 (5' TAG GAT CCA TGG ATG CAA TGA AGA GAG GGC3')
ZC96 (5' CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC3')
ZC98 (5' TGC CCG ATT CAG AAG AGG AGC CAG ATC TTC3')
Probes ZC94, ZC96 and ZC98 will hybridize to the
signal peptide coding region; the remaining probes
hybridize to the mature peptide coding region. The nine
positive isolates were found to fall into three distinct
classes which together span the entire t-PA coding region.
Insert size was determined by digestion with Eco RI.
For each class, the clone having the largest
insert was digested with Eco RI, Bgl II, and Eco RI + Bgl
II and the restriction fragments were probed on Southern
blots (Southern, J. Mol. Biol. 98: 503-517, 1975) using
representative oligonucleotide probes. Clone number 9 was
found to contain the entire coding sequence for mature
t-PA, but not the pre-pro region. Eco RI and Bgl II-Eco RI
fragments of the insert from clone number 9 were inserted
into M13 and pUCl3 vectors for sequencing and further
analysis. The fragments were sequenced by the dideoxy
method (Sanger et al., J. Mol. Biol. 143: 161, 1983, and
Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463, 1977).
Example 3 - Preparation of t-PA Sequences Having Random 5'
End Deletions
A. Deletion of 5' Coding Sequences
Ten ug of pDR1296 were digested with either five
units of Bgl II or five units of Nar I to completion. The
1341444
22
resultant linearized DNAs were electrophoresed on a 0.7%
agarose gel. The DNA fragments were eluted from the gel
using TE buffer (10 mM Tris, pH 7.4, 5 mM EDTA) . The DNA
fragments were resuspended in 46 u1 of H20 and 12 u1 of 5x
Ba131 buffer (3M NaCl, 62.5 mM CaCl2, 62.5 mM MgCl2, 100 mM
Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0) plus 2 u1 of Ba131
(0.5 units/ l, obtained from Bethesda Research Labora-
tories). The reaction mixtures were incubated at 30°C and
u1 aliquots were removed at one minute intervals for six
10 minutes. The digested samples were combined, extracted
with phenol-CHC13, and precipitated with ethanol. The
fragment ends were filled in with DNA polymerise I (Klenow
fragment) and dNTPs. Following the fill-in reaction,
samples were digested with Xba I and separated on a 0.7%
agarose gel. Fragments smaller than 1700 by were cut out
and extracted with TE buffer. Subsequent mapping
identified 55 truncated sequences.
B. Preparation of pDR817 Expression Vector
Referring to Figure 3, the 28 by trpA terminator
(obtained from P-L Biochemicals) was ligated into pUCl8
which had been cut with Sst I and filled in using T4 DNA
polymerise to form plasmid pDR813. The 28 by terminator
fragment was similarly inserted into the filled-in Cla I
site of pICl9R to construct plasmid pDR812. Plasmid pDR540
(Russell et al., Gene 20: 231-243, 1982), which contains
the TAC promoter and lac operator (lac0), was cut with Hind
III, filled in with Klenow polymerise, and digested with
Bam HI. The resulting ~92 by fragment, comprising the TAC
promoter and lac0, was ligated into Bam HI + Sma I cut
pDR813 to form pDR814. Plasmid pDR814 was then digested
with Eco RI and Hind III and the fragment comprising the
trpA terminator, TAC promoter, and lac0 was isolated and
ligated into pDR812 which had been digested to completion
with Hind III and partially digested with Eco RI. The
resultant plasmid was designated pDR815.
1341444
23
Plasmid Zem99 (see Example 1) was digested with
Bam HI and Xba I and the fragment comprising the t-PA cDNA
(including the pre-pro sequence) was isolated. Plasmid
pDR815 was cut with Bam HI and Xba I and ligated to the
t-PA fragment to construct plasmid pDR816.
Ten ug of pDR816 were digested to completion with
five units of Bgl II and the ends filled in using DNA
polymerase I (Klenow fragment). The DNA was then digested
to completion with five units of Xba I, extracted with
phenol-CHC13, ethanol precipitated, and electrophoresed on
a 0.7% agarose gel. A 3050 by fragment (comprising pICl9R,
TAC promoter, and t-PA pre-pro sequences) was eluted from
the gel.
The 3050 by fragment and the randomly deleted
t-PA cDNA sequences were ligated overnight at 16°C.
Ligation mixtures were used to transform competent E. coli
JM83 cells. Transformed cells were plated on Ampicillin
and resistant colonies were selected and transferred to
fresh Ampicillin-containing plates in triplicate. The
plates were incubated overnight at 37°C, and colonies were
transferred to nitrocellulose filters. The filters were
placed on Ampicillin plates and incubated four hours at
37°C, then treated with CHC13 vapor for 10 minutes. The
filters were air-dried for 10 minutes and placed directly
onto a fibrin plate and incubated at 37°C overnight. The
filters were lifted off the plates and colonies capable of
causing fibrin lysis were picked for further analysis. A
duplicate set of filters was similarly processed for colony
immuno blot assay. Colonies capable of producing t-PA-like
polypeptide were identified.
One plasmid, designated pDR817, was characterized
in detail. The plasmid was digested with Bam HI and Xba I
and a 1270 by t-PA fragment was gel purified and subcloned
into Ml3mpl8 (replicative form). The DNA sequence of the
insert was determined by the dideoxy method. Results
indicated that pDR817 lacked nucleotides 192-524 (numbering
based on that of Pennica et al., ibid). It thus encodes a
~ 341 44 4
24
mature protein consisting of 416 amino acids with an amino
terminus as shown in Figure 4A. Amino acids 2-112 of
naturally occurring t-PA were deleted.
E. coli strain JM105 was transformed with pDR816
and pDR817. Untransformed cells and the transformants were
grown overnight in M9 medium supplemented with 0.4~ glucose
and 0.2~ casamino acids. Ten ml aliquots were removed from
each culture and the cells harvested. The inducer IPTG
(isopropylthiogalactoside) was added to the remainder of
the cultures to a final concentration of 1 mM. After
induction for 120 minutes and 240 minutes, 10 ml aliquots
were removed and the cells harvested. The cell pellets
were washed once with water and resuspended in 450 u1 of
20°s sucrose in 100 mM Tris, pH 8Ø The cells were lysed
by the addition of 50 u1 of 5 mg/ml lysozyme in 50 mM EDTA.
The mixtures were incubated 10 minutes at room temperature,
500 u1 of lysis buffer (0.3% Triton X-100, 150 mM Tris, pH
8.0, 0.2 M EDTA) were added, and the mixtures placed on ice
for 30 minutes. The lysates were centrifuged one hour at
35,000 rpm in a Beckman SW50 rotor. The supernatants were
removed and assayed by the fibrin lysis method. Results
(see Table 2) indicated that the pDR817 construct directed
the production of t-PA activity 10- to 30-fold higher than
that obtained using a full-length t-PA clone.
30
* tra3e-mark
1341444
TABLE 2
Fibrin Lysis
Strain Time* Activity {ug/1)
5 JM105 - 0
JM105 120 min. 0
JM105 240 min. 0
pDR816:JM105 - 7.4
10 pDR816:JM105 120 min. 5.0
pDR816:JM105 240 min. 7.4
pDR817:JM105 - 160.0
pDR817:JM105 120 min. 150.0
15 pDR817:JM105 240 min. 120.0
* Time after addition of IPTG.
(-) indicates sample taken prior to addition of IPTG.
20 Aliquots of the transformed cells and the
untransformed control were analyzed for protein production
by Western blot assay. DNA sequence analysis indicated
that the pDR817 t-PA polypeptide is approximately 100 amino
acids shorter than the polypeptide produced by pDR816
25 (Figure 4A). E. coli JM105 transformed with pDR817 has
been deposited with American Type Culture Collection under
Accession No. 53446.
C. Expression of t-PA Deletion Mutants in
Mammalian Cells
Expression vectors were constructed in the
following manner. Plasmid Zem86 {described in Example 1)
was digested with Hind III and the ends filled in using DNA
polymerase I (Klenow fragment). The linearized DNA was
then digested with EcoRI, and a 450 by fragment,
comprising the SV40 on sequence, was gel purified and
ligated to Sma I + Eco RI digested pUCl3. The resultant
1341444
26
vector was designated pDR3001. Plasmid pDR3001 was
digested with Sal I and Eco RI and the '450 by fragment,
comprising SV40 on and polylinker sequences, was gel
purified. Zem 86 was partially digested with EcoRI and
completely digested with Xho I to remove the SV40 on
sequence. The SV40 fragment from pDR 3001 was then joined
to the linearized Zem 86. The resultant plasmid was
designated pDR3002 (Figure 5).
Mammalian cell expression vectors were then
constructed. Plasmid pDR3002 was digested with Bam HI and
Xba I. The bacterial vectors described in Example 3B were
digested with Bam HI and Xba I, and the t-PA sequences are
gel purified and ligated to the linearized pDR3002. The
resultant vectors (Tab.le 3) contain expression units of
SV40 promoter - mutant t-PA sequence - hGH terminator. The
vector pDR3004 (Figure 5) comprises the mutant t-PA
sequence from pDR817. The primary translation product from
pDR3004 i.s predicted to have the sequence shown in Figure
4A. E. coli LM1035 transformed with pDR3004 has been
deposited with American Type Culture Collection under
Accession No. 53445.
TABLE 3
Summary of Sequential Deletion Mutants
Plasmid Sequence at Pre-pro Junction Domains*
pDR3004 AGA TCG TGCACC AAC K1 (partial), K2, SP
1 113114 115
Arg Ser CysThr Asn
pDR3006 AGA TCC CTG GGG AAC K1 (partial), K2, SP
1 138 139 140
Arg Ser Leu Gly Asn
._ 1 341 44 4
27
pDR3007 AGA TCC ACCAAC AGT K1 (partial), K2,
SP
1 114115 116
Arg Ser ThrAsn Trp
pDR3008 AGA TCG GGAAAC AGT K2, SP
1 176177 178
Arg Ser GlyAsn Ser
pDR3010 AGA TCG GGTGCC TCC K2 (partial), SP
1 198199 200
Arg Ser GlyAla Ser
pDR3011 AGA TCT GAGGGA ACC K2, SP
1 175176 177
Arg Ser GluGly Asn
pDR3012 AGA TCG AAGTAC AGC K1 (partial), K2,
SP
1 162163 164
Arg Ser LysTyr Ser
pDR3013 AGA TCC TGCCAG CAG GF (partial), K1,
1 62 63 64 K2, SP
Arg Ser CysGln Gln
pDR3014 AGA TCG AATGGG TCA K2, SP
1 1$4185 186
Arg Ser AsnGly Ser
pDR3016 AGA TCG GGCACC TGC GF (partial), Kl,
1 60 61 62 K2, SP
Arg Ser GlyThr Cys
* GF - growth 1 Kringle K2 Kringle
factor, - l, - 2,
K
SP - serine
protease
The described Table
vectors in 3
are
used
to
transfect cultu red mammalian cells according to
standard
1 ~414~4
28
procedures. Logarithmically growing Tk- baby hamster kid-
ney (BHK) cells were used for cotransfection of a mixture
(1:1 ratio) of expression vector DNA coding for the mouse
wild-type DHFR gene (pSV2-DHFR, disclosed by Subramani et
al., Mol. Cell. Biol. l: 854-864, 1981) and an expression
vector encoding a mutant t-PA protein. Twenty-four hours
after transfection, the cells were supplemented with
Dulbecco's modified Eagle's medium (DME) containing 10%
fetal bovine serum (depleted of plasminogen by passage over
a lysine-sepharose column), aprotinin (100 units/ml),
penicillin, and 250 mM MTX. Cells were fed with this
selective medium several times over the next 10 to 14 days.
Drug-resistant colonies were screened for t-PA activity by
the fibrin plate method. Cell lines producing active
protein at levels greater than 1 pg/cell/day were further
amplified, cloned by dilution, and scaled up for protein
isolation and characterization.
Three of the above-described deletion mutants
were chosen for further characterization. These correspond
to mutants 3016, 3004 and 3008, mutants representing
deletions into three different domains (see Table 3). A
monoclonal antibody which was capable of detecting all
three of the truncated molecules examined was used as
primary antibody for detection of truncated tPAs. The
standard ELISA protocol for detection of truncated tPAs is
outlined below.
35
1341444
29
ELISA Procedure for Truncated tPA
Plate out 100 u1 of 1 ug/ml of monoclonal antibody in
Buffer A
Buffer A = 100 mM Na2C03 pH 9.6
Incubate overnight at 4°C
wash 3x with Buffer B
Buffer B = 10 mM Na Phosphate pH 7.2; 150 mM NaCl:
0.5% Tween 20*
Block for two hours at 37°C with Buffer C
Buffer C = Buffer B + 1% BSA
Load samples in Buffer C
Incubate at 37°C for two hours
Wash 3x with Buffer B
Load 100 u1 of 4.5 ug/ml of polyclonal rabbit anti-tPA
Incubate at 37°C for one hour
Wash 3x with Buffer B
Load HRP conjugated goat anti-rabbit
Incubate 1 hour at room temperature
Wash 4x with Buffer B
Color development
Mutant proteins 3004 and 3008 were purified on a
lysine-sepharose column. Cell culture media were dialyzed
overnight against loading buffer (50 mM sodium phosphate pH
7.3, 0.1 M NaCl, 0.005% Tween 80, 0.003 M NaN3).
The dialyzed solution was loaded onto a lysine-sepharose*
column at 0.5 ml per minute. The column was washed with
loading buffer, then bound material was eluted with loading
buffer containing 0.4 M arginine. Eluate fractions were
assayed by ELISA and fibrin lysis assay.
The fibrin lysis assay is based on the method of
Binder et al. (J. Biol. Chem. 254: 1998, 1979). Ten ml of
a bovine fibrinogen solution (3.0 mg/ml in 0.036 M sodium
acetate pH 8.4, 0.036 M sodium barbital, 0.145 M NaCl, 10'4
M CaCl2, 0.02% NaN3) were added to 10 ml of a 1.5% solution
of low melting temperature agarose in the same buffer at
40°C. To this solution was added 10 u1 of bovine thrombin
(500 U/ml). The mixture was poured onto a Gelbond*agarose
* ~ra~~P-mark
1341444
support sheet (Marine Colloids) and allowed to cool. Wells
were cut in the agarose and to the wells was added 10 u1 of
the sample to be tested plus 10 u1 of phosphate-buffered
saline containing 0.1~ bovine serum albumin. Results were
5 compared to a standard curve prepared using purified tPA.
The development of a clear halo around the well indicates
the presence of biologically active plasminogen activator.
Example 4 - Loop-out of Finger and Growth Factor Domain
10 Coding Sequences
The sequences encoding the finger and growth
factor domains of t-PA were deleted from the cDNA by site-
specific mutagenesis, essentially as described by Zoller et
al., Manual for Advanced Techniques in Molecular Cloning
15 Course, Cold Spring Harbor Laboratory, 1983. Oligonucle-
otides were synthesized on an Applied Biosystems 380-A DNA
synthesizer and purified by electrophoresis on denaturing
gels.
Precise deletions were designed based on a
20 comparison of the genomic and cDNA t-PA sequences. cDNA
sequences corresponding to the exons encoding the finger
and growth factor domains were deleted.
To prepare a template for mutagenesis of the t-PA
sequence, approximately 1 ug of Zem 99 was digested with
25 one unit each of BamHI and EcoRI. The DNA fragments were
separated on a 1°s agarose gel and the 730 by BamHI-EcoRI
fragment was electro-eluted onto NA-45 DEAE membrane
(Schleicher & Schuell) as directed by the supplier. The
DNA was extracted with phenol-CHC13 and EtOH precipitated.
30 The purified fragment was then ligated to Bam HI + Eco RI
digested M13mp8 (replicative form) by incubating the two
fragments in the presence of T4 DNA ligase for twelve hours
at 12°C. The recombinant phage were transfected into
competent E. coli JM101. Phage DNA was purified from
plaques and sequenced by the dideoxy method to confirm the
presence of the correct cDNA sequence.
1341444
31
Single-stranded M13 template DNA was prepared and
site-specific mutagenesis was carried out using the
oligonucleotide ZC490 (S~TAC CAA GTG ACC AGG GCC3~) as
mutagenic primer. The universal M13 primer was used as
second primer. Twenty pmoles phosphorylated mutagenic
primer and 20 pmoles second primer were combined with one
pmole single-stranded template in 10 u1 of 20 mM Tris, pH
7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT and incubated at
65°C for ten minutes, then five minutes at room temperature
and placed on ice. Ten u1 of 20 mM Tris, pH 7.5, 10 mM
MgCl2, 2 mM ATP, 10 mM DTT containing 1 mM dNTPs, 2.5 units
Klenow polymerase, and 3.5 units DNA ligase were added to
the annealed DNA and the mixture incubated three hours at
15°C. The DNA was then transfected into competent E. coli
JM101, and the cells plated on YT agar and incubated at
37°C. Plaques were transferred to nitrocellulose and
pre-hybridized at the Tm-4°C of the mutagenic primer for
one hour in 6x SSC, lOx Denhardt's and hybridized to
32p-labeled mutagenic primer at Tm-4°C in the same solution.
After three washes at Tm-4°C, filters were exposed to X-ray
film overnight. Additional wash steps were performed at
5°C higher increments as necessary to identify mutant
plaques. The mutated inserts were sequenced by the dideoxy
method and a clone having the desired loop-out was selected.
When joined to the remainder of the t-PA coding sequence,
this sequence encodes a protein with the amino terminus
shown in Figure 4B.
Example 5 - Deletion of Finger and Growth Factor Sequences
A second strategy for deleting the finger and
growth factor sequences used as starting material the
plasmid pDR1496, which comprises the t-PA coding sequence.
S. cerevisine strain E8-11C transformed with pDR1496 has
been deposited with American Type Culture Collection under
Accession No. 20728. To prepare a template for mutagenesis
of the t-PA sequence, 1 g of pDR1496 was digested with 5
units each of Sph I and Xba I for two hours at 37°C. The
.. 1341444
32
DNA was electrophoresed on a 0.7o agarose gel and a frag-
ment of 2100 by was purified. This fragment was ligated
to Sph I + Xba I digested M13tg130 (replicative form;
obtained from Amersham; Kieny et al., Gene 26:91, 1983) to
construct M13tg130W. The recombinant phage were
transfected into E. coli JM103 and single-stranded template
DNA was prepared. Oligonucleotide-directed deletion
mutagenesis was carried out using 20 pmoles phosphorylated
mutagenic primer (sequence: 5'CGT GGC CCT GGT ATC TTG GTA
AG3') and 1 pmole template DNA in 20 mM Tris pH 7.5, 10 mM
MgCl2, 50 mM NaCl, 1 mM DTT at 65°C for 10 minutes. The
mixture was then incubated for 5 minutes at room tempera-
ture and placed on ice. Ten 1 of 20 mM Tris pH 7.5, 10
mM MgCl2, 2 mM ATP, 10 mM DTT containing 1 mM dNTPs, 2.5
units Klenow fragment and 3.5 units T4 DNA ligase were
added and the annealed DNA mixture was incubated for 3
hours at 15°C. The DNA was transfected into E. coli JM103
and the cells were plated on YT agar and incubated at 37°C.
Plaques were screened as described in Example 4. A mutant
sequence having the desired deletion of finger and growth
factor sequences was designated clone #2600. The sequence
of the encoded protein is shown in Figure 6.
Example 6 - Loop-out of Finger, Growth.Factor and Kringle 1
Coding Sequences
Sequences encoding the finger, growth factor, and
Kringle 1 sequences were deleted from the cloned cDNA in a
manner analogous to the deletion described in Example 4.
The loop-out precisely joined codons for amino acids 4 and
176 of mature t-PA, resulting in a deletion of DNA
sequences corresponding to the exons encoding the finger,
growth factor and Kringle 1 regions (Figure 4C).
The "'730 by BamHI-EcoRI t-PA fragment comprising
the pre-pro and 5' end of the mature sequence from zem 99
was prepared and cloned in M13mp19 (replicative form).
Single-stranded template DNA was prepared and mutagenesis
carried out using oligonucleotide ZC722 (5'ACT GTT TCC CAC
.. 1 341 44 4
33
TTG GTA3') and the M13 universal primer. Mutant plaques
were screened and sequenced to identify clones having the
desired mutation.
Example 7 - Deletion of Finer, Growth Factor and Kringle 1
Coding Sequences
The finger, growth factor and Kringle 1 sequences
were deleted in a second mutagenesis procedure using the
single-stranded M13tg130W template described in Example 5.
Mutagenesis was carried out as described in Example 5 using
an oligonucleotide primer having the sequence 5'GCA GTC ACT
GTT TCC TTG GTA AG3'. Mutant plaques were screened as
previously described. A clone having the correct deletion
was designated #2700. The sequence of the encoded protein
is shown in Figure 7.
Example 8 - Construction of a Mutant t-PA Sequence
Comprising Synthesized Oliqonucleotides
The 5' portion of a DNA sequence encoding a
mature t-PA with a deletion for the finger, growth factor,
and Kringle 1 regions is constructed from synthesized
oligonucleotides. The resultant fragment is then joined to
the 3' cDNA and pre-pro sequences. The following
oligonucleotides are used:
ZC636: 5'GAT CTT ACC AAG TGG GAA ACA GTG ACT GCT ACT TTG
GGA ATG GGT CAG3'
ZC637: 5'TAG GCT GAC CCA TTC CCA AAG TAG CAG TCA CTG TTT
CCC ACT TGG TAA3'
ZC638: 5'CCT ACC GTG GCA CGC ACA GCC TCA CCG AGT CGG GTG
CCT CCT GCC TCC CGT GG3'
ZC639: 5'AAT TCC ACG GGA GGC AGG AGG CAC CCG ACT CGG TGA
GGC TGT GCG TGC CAC GG3'
The four oligonucleotides were separately
phosphorylated using T4 kinase. Oligonucleotides ZC636 and
ZC637 are annealed under conditions described in Example 1
1341444
34
with an overlap of 44 bp. Oligonucleotides ZC638 and ZC639
are similarly annealed with an overlap of 49 bp.
To construct a mammalian cell expression vector
containing the mutant t-PA sequence, Zem99 is digested to
completion with Bgl II and partially digested with Eco RI
to remove the 600 by 5' portion of the t-PA gene. The
fragment comprising the vector, t-PA pre-pro and 3' t-PA
sequences is gel purified and joined, in a three-part
ligation, to the paired oligonucleotides. The
amino-terminal sequence of the encoded protein is shown in
Figure 4C.
Example 9 - Deletion of Growth Factor Coding Sequence
A mutant sequence lacking the growth factor domain
coding sequence was constructed by deletion mutagenesis
using oligonucleotide ZC820 (S~GTA GCA CGT GGC CCT GGT TTT
GAC AGG CAC TGA GTG3~) and the M13 template described in
Example 6. This deletion joined the codons for amino acids
49 and 88 of mature t-PA (Figure 4D). A correct clone was
identified by sequencing and designated M13mp19-820.
Replicative form DNA was prepared and digested with PvuII
and HindIII. A 713 by fragment comprising the mutated t-PA
sequence was isolated and ligated to SmaI and HindIII cut
pUCl8. The ligation mixture was transformed into E. coli
HB101. A correct clone was identified and designated
pUCl8-820.
Exam 1e 10 - Expression of Mutant t-PAs in Bacteria
For expression in transformed bacterial cells,
mutant t-PA sequences described above are removed from
their respective M13 vectors by digesting replicative form
DNA to completion with Bgl II and partially with Eco RI.
The mutant t-PA sequences are purified by standard
procedures.
The bacterial expression vector pDR816 (described
in Example 3) is digested with Bgl II and Xba I, and the
fragment comprising the pICl9R, TAC promoter, t-PA pre-pro,
1 341 44 4
and trpA terminator sequences is purified. The 3' t-PA
coding sequence is purified from an Eco RI + Xba I digest
of pDR816.
The three fragments are joined in a triple
5 ligation to construct bacterial expression vectors
containing the entire mutant t-PA sequences.
Similarly, the phosphorylated, annealed oligonu-
cleotide pairs described in Example 8 are inserted in
pDR816 which has been digested to completion with Bgl II
10 and partially digested with Eco RI to remove the 600 by 5'
portion of the mature t-PA sequence.
The resultant vectors are used to transform
E. coli JM105. Transformed cells are grown in M9 medium
supplemented with 0.4o glucose and 0.2~ casamino acids.
15 Cells are harvested and lysed, and the cell debris was
removed by centrifugation. The supernatants are assayed
for t-PA activity by fibrin lysis assay and for protein
production by Western blot assay.
20 Example 11 - Expression of Mutant t-PAs in Mammalian Cells
The mutant sequences descr ibed above in Examples
4, 5, 6, 7 and 9 were inserted into mammalian cell
expression vectors comprising the SV40 or MT-1 promoter and
the DHFR selectable marker. The resultant expression
25 vectors were cotransfected into Tk- BHK cells, drug
selection was applied, and transfected cell lines were
selected, expanded for scale-up and protein production and
characterization.
The mutant sequences were removed from clones
30 #2600 and #2700 as Bgl II-Apa I fragments and were inserted
into Zem99 (Example 1). The resultant vectors were
cotransfected with pSV2-DHFR DNA into Tk- BHK cells, as
described in Example 3C.
Plasmid pUCl8-820 was digested with EcoRI and
35 BamHI and the mutant t-PA sequence (620 bp) was inserted
into pDR3002. The resultant vector, designated p820, was
transfected into Tk- BHK cells, as previously described.
.. ' 34~ 444
36
The mutant protein #2600 was purified on a 2.6 x
20 cm column of monoclonal antibodies immobilized on
Sepharose~(Pharmacia). Media from transfected BHK cells
were applied to the column at a rate of 200 ml/hr at 4°C.
The column was equilibrated with 0.1 M Tris-HC1 buffer
pH7.5, containing 0.5 M NaCl and 20 KIU/ml of Aprotinin.
The column was washed with 1000 ml of the above buffer and
the t-PA was eluted with the same buffer containing 5 M
KSCN. The t-PA fractions were concentrated by
ultra-filtration to a volume of 5m1 and loaded on a column
(2.6 x 90 cm) of Sephacryl S-200~T(Pharmacia) equilibrated
with 50 mM Tris-HCl buffer pH7.5, containing 1.5 M KSCN and
0.5 M NaCl. The column was developed with the same buffer
at a rate of 25 ml/hr. The t-PA fractions were
concentrated by ultrafiltration and subjected to gel
filtration on a column of Sephadex G-25~(PD10, Pharmacia)
equilibrated with 1 M ammonium bicarbonate. The resultant
purified protein was lyophilized in the presence of
mannitol.
Media containing mutant t-PA 2700 were applied to
a column (5 x 20 cm) of zinc-chelating Sepharose
(Pharmacia) equilibrated with 50 mM Tris-HC1 buffer pH 7.5,
containing 1 M NaCl and 20 KIU/ml of Aprotinin at a rate of
400 ml/hr. at 4°C. The column was washed with 2000 ml of
the same buffer. The t-PA was eluted by gradually
increasing the concentration of imidazole from 0 to 50 mM
in the same buffer. The t-PA fractions were directly
applied to a column (2.6 x 20 cm) of concanavalin A coupled
Sepharose 4B (Pharmacia) equilibrated with 10 mM sodium
phosphate buffer pH 7.5, containing 1M NaCl at a rate of 30
ml/hr. The column was washed with the same buffer. The
t-PA was eluted by gradually increasing the concentration
of ~-methylamannoside from 0 to 0.4 M on 10 mM sodium
phosphate buffer pH 7.5, containing 1M NaCl, 20 KIU/ml of
Aprotinin and 2 M KSCN. The t-PA fractions were
concentrated by ultra-filtration to about 5 ml and loaded
on a column (2.6 x 90 cm) of Sephacryl S-200 equilibrated
~- !~Y'C7C.~~ ~f~~~c
37 1 341 44 4
with 50 mM Tris-HC1 pH 7.5, containing 1.5 M KSCN and 1M
NaCI. The column was developed with the same buffer at a
rate of 25 ml/hr and the t-PA fractions were pooled,
concentrated, desalted and lyophilized as described above.
Plasma clearance of the 2600 and 2700 mutant t-PA
molecules was tested in rats. Male Sprague Dawley rats
(230 g - 270 g body weight) were injected with 0.4 mg/kg
body weight of 125z_labelled mutant t-PA or authentic
recombinant t-PA purified from transfected BHK cells.
Injection was via the femoral vein. Blood samples (0.5 ml)
were withdrawn from the jugular vein and assayed for t-PA
protein by a sandwich ELISA using affinity purified
polyclonal. rabbit antibody. Results, shown in Table 4,
indicate that the mutant proteins have a plasma half-life
up to five times that of authentic t-PA.
TABLE 4
Plasma Half-Life of Mutant t-PAs
Protein ~ Phase (minutes) ~ Phase (minutes)
Authentic t-PA 1.7 40
2600 10 210
2700 9.6 ~ 84
Mutant proteins 3008 and 2600 were purified and
assayed for in vivo half-life. Subconfluent 75 cm2 flasks
containing cells producing mutant protein 3008, 2600 or
authentic t-PA (control) were washed in 10 ml
methionine-free Dulbecco's MEM containing 100 U/ml
penicillin, 100~4g/ml streptomycin and 10 ~ 9/m1 aprotinin.
The cells were incubated in 10 ml of the same medium
containing 1 mci 35S-methionine (New England Nuclear) for
20 hours at 37°C. The supernatant was centrifuged at 2000
rpm for 10 minutes and stored at -20°C. Non-radioactive
t-PA's were produced from cell layers in 1200 cm2 trays
1 341 444
38
(NUNC) maintained in Dulbecco's containing 100 U/ml
penicillin, 100~1.(g/ml streptomycin and 100~Gtg/ml aprotinin.
Approximately 400 ml medium was harvested. NaCl and Tween
80* were added to the combined cell culture media to
concentrations of 1M and 0.2~, respectively. The pH was
adjusted to 7.5. After filtration on a 0.45~t m filter the
mutants were purified using immunosorbent chromatography on
monoclonal antibodies.
The purified t-PA mutants were found to be
predominantly in the single-chain form. The specific
activities were determined by the fibrin plate assay and
are shown in Table 5.
TABLE 5
Protein Specific Activity (IU/mg protein)
authentic.t-PA 400,000
3008 590,000
2600 380,000
In vivo half-life of the mutant proteins 3008 and
2600 was determined. Female Wistar rats were anaesthetized
and catheters were placed in the jugular vein for
intravenous administration and in the carotid artery for
sampling. Heparin (1 mg/kg body weight) was given i.v. ten
minutes before administration of the test solution. Blood
samples of approximately 2501 were taken before and 2, 3,
4, 6 and 8 minutes after the administration of 0.25-0.5 ml
test solutions. Radioactivity in plasma was determined by
liquid scint illation and elimination half-lives were
calculated by linear regression using a one compartment
model. Half-lives (mean or 3-5 experiments) were: native
t-PA, 2.3 min.: 3008, 12 min. and 2600, 17 min.
* trade-mark
1 341 44 4
39
Example 12 - Expression of Mutant t-PAs in Yeast
For expression in yeast, the mutant t-PA
sequences are excised from the bacterial vectors described
in Example 10 as Bgl II-Xba I fragments.
Plasmid pDR1496 is a yeast expression vector
comprising the S. cerevisiae TPI promoter (Alber and
Kawasaki, J. Mol. Appl. Genet. 1:419-434, 19$2), the S.
cerevisiae MF 1 signal sequence (Kurjan and Herskowitz,
Cell 30: 933-943, 1982: and U.S. Patent 4,546,082), t-PA
coding sequence, and the S. cerevisiae TPI terminator
(Alber and Kawasaki, ibid). S, cerevisiae strain E8-llc
transformed with pDR1496 has been deposited with American
Type Culture Collection under Accession No. 20728. The
plasmid is isolated from the transformant by standard
methods. The t-PA sequence is removed from pDR1496 by
partial digestion with Bgl II and Xba I. The 12.2 kb
vector fragment is purified and ligated to the Bgl II-Xba I
mutant t-PA sequence.
Expression vectors are then used to transform S.
cerevisiae strain E8-llc. Cells are grown at 30° in -leu
medium supplemented with 2~ glucose and 0.1 M potassium
hydrogen phthalate, pH 5.5. Cells are removed during log
phase growth, harvested by centrifugation, disrupted, and
the lysates assayed for t-PA activity.
From the foregoing it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the spirit
and scope of the invention. Accordingly, the invention is
not limited except as by the appended claims.
