1340 97'
METAL~L:OSPRCYrEINASE INHIBITOR SEQUENCE RECONBINANT VEC~R
SYSTEM FOR USING SAME AND REC1~I~IBINANT-DNA METHOD FOR THE
MANUFACTURE OF SAME
BACK;GROUND OF THE INVE,'NTION
Endogenc>us proteolytic enzymes serve to degrade invading
organisms, antigen-antibody complexes and certain tissue proteins which
are no longer necessary or useful to the on~anism. In a normally
functioning Organ7.STil, proteolytic enzymes are produced in a limited
quantity and are recfiulated in part through specific inhibitors.
Metalloproteinases are enzymes present in the body which are
often involved in the degradation of connective tissue. While some ,
connective tissue degradation is necessary for normal functioning of an
organism, an excess of connective tissue degradation occurs in several
disease states and _Ls believed to be attributable, at least in part, to
excess metalloprote:~_nase. It is believed that metalloproteinases are at
least implicated in periodontal disease, corneal and skin ulcers,
rheumatoid arthriti:~ and the spread of cancerous solid tumors.
These diseases generally occur in areas of the body which
contain a high proportion of collagen, a particular form of connective
tissue. An examination of ;patients with these diseases of connective
tissue has revealed an excessive breakdown of the various components of
connective tissues, including collagen proteoglycans and elastin.
Therefore, it has been deduced that an excessive concentration of a
particular metallopz~oteinas~=_, for example collagenase, proteoglyconase,
gelatinase, and certain elastases, may cause or exacerbate the connective
tissue destruction associated with the aforerr~ntioned diseases.
In the normal state, the body possesses metalloproteinase
inhibitors which bir..d to met=alloproteinases to effectively prevent these
enzymes from acting on thei~_ connective tissue substrates. Specifically,
in a healthy organism, metalloproteinase inhibitors are present in
concentrations sufficient to interact with metalloproteinases to an
extent which allows sufficient quantities of metalloproteinase to remain '
active while binding the excess metalloproteinase so that the
~i
134pg71
-2-
connective tissue damages seen in the various diseases does not
occur.
It is postulated that one immediate cause of the con-
nective tissue destruction present in the foregoing disease
states is an imbalance i.n the relative metalloproteinase/metallo-
proteinase inhibitor concentrations. In these situations, either
due to an excessive amount of active metalloproteinase or a defi-
ciency in the amount of active metalloproteinase inhibitor, the
excess metalloproteinase~ is believed to cause the connective tis-
sue degradation responsible for causing or exacerbating the dis-
ease. It is postulated that, by treating persons with connective
tissue diseases with metalloproteinase inhibitors, the
degradative action of the excess metalloproteinase may be cur-
tailed or halted. Therefore, particular metalloproteinase inhib-
itors of specific interest to the present inventors are collage
nase inhibitors because it is believed that these inhibitors
would be pharmaceutically useful in the treatment or prevention
of connective tissue diseases.
The exi:>tence of metalloproteinase and metallopro-
teinase inhibitors has been discussed in the scientific litera-
ture. For exampls~, Sellers et al., Biochemical And Biophysical
Research Communications 87:581-587 (1979), discusses isolation of
rabbit bone collactenase inhibitor. Collagenase inhibitor iso-
lated from human :;kin fi'broblasts is discussed in Stricklin and
Welgus, J.B.C. 25Et:12252-12258 (1983) and Welgus and Stricklin,
J.B.C. 258:12259-1.2264 (1983). The presence of collagenase in-
hibitors in naturally-occurring body fluids is further discussed
in Murphy et al., Biochem. J. 195:167-170 (1981) and Cawston _et
al., Arthritis and Rheumatism, _27:285 (1984). In addition, met-
alloproteinase inhibitors are discussed by Reynolds et al. in
Cellular Interactions, D.ingle and Gordon, eds., (1981). Although
these articles characterize particular, isolated metallopro-
teinase inhibitors and discuss, to some extent, the role or
potential role of metalloproteinases in connective tissue disease
treatment and speculate on the ability of metalloproteinase
inhibitors to counteract this destruction, none of these re-
searchers had previously been able to isolate a portable DNA
~ 340 97 ~
-3-
sequence capable of directing intracellular production of metal-
loproteinase inhibitors or to create a recombinant-DNA method for
the production of these :inhibitors.
Surprisingly, 'the present.inventors have discovered a
portable DNA sequence capable of directing the recombinant-DNA
synthesis of metalloproteinase inhibitors. These metallopro-
teinase inhibitors are biologically equivalent to those isolated
from human skin fibroblaat cultures. The metalloproteinase in-
hibitors of the present :invention, prepared by the recombinant-
DNA methods set forth herein, will enable increased research into
prevention and treatment of metalloproteinase-induced connective
tissue diseases. Zn add:ition, the metalloproteinase inhibitors
of the present invention are useful in neutralizing metallopro-
teinases, including the excess metalloproteinase associated with
disease states. Therefor e, it is believed that a cure for these,,
diseases will be developed which will embody, as an active ingre-
dient, the metalloproteinase inhibitors of the present invention.
Furthermore, the metalloproteinase inhibitors of the present
invention are capable of interacting with their metalloproteinase
targets in a manner which allows the development of diagnostic
tests for degradative connective tissue diseases using the newly
discovered inhibitors.
The recombinant metalloproteinase inhibitors discussed
herein interact stoichiornetrically (i.e., in a 1:1 ratio) with
their metalloproteinase targets. In addition. these metallopro-
teinase inhibitors are hE~at resistant, acid stable, glycosylated,
and exhibit a high isoelEactric point.
SUMMARY OF THE INVENTION
The present invention relates to metalloproteinase in-
hibitors and a recombinant-DNA method of producing the same and
to portable DNA sequences capable of directing intracellular pro-
duction of the metalloproteinase inhibitors. Particularly, the
present invention relates to a collagenase inhibitor, a recombi-
nant-DNA method for producing the same and to portable DNA se-
quences for use in the rEacombinant method. The present invention
also relates to a series of vectors containing these portable DNA
sequences.
1 340 97 ~
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One object of tree present invention is to provide a
metalloproteinase inhibitor, which can be produced in sufficient
quantities and purities to provide economical pharmaceutical
compositions which posses metalloproteinase inhibitor activity.
An additional object of the present invention is to provide
a recombinant-DNA method for the production of these metalloproteinase
inhibitors. The recombinant metalloproteinase inhibitors produced by
this method are biologically equivalent to the metalloproteinase
inhibitor isolable from human skin fibroblast cultures.
To facilitate the recombinant-DNA synthesis of these
metalloproteinase inhibitors, it is a further object of the present
invention to provide portable DNA sequences capable of directing
intracellular production of metalloproteinase inhibitors. It is also
an object of the present invention to provide cloning vectors
containing these portable sequences. These vectors are capable of.~
being used in recombinant systems to produce pharmaceutically useful
quantities of metalloproteinase inhibitors.
Additional objects and advantages of the invention will be
set forth in part i:n the description which follows, and in part will
be obvious from the description or may be learned from practice of the
invention. The objects and advantages may be realized and attained by
means of the instrumentalities and combinations particularly pointed
out in the appended claims.
To achieve the objects and in accordance with the purposes
of the present invention, metalloproteinase inhibitors are set forth,
which are capable o:E stoichiometric reaction with metalloproteinases.
These metalloproteinase inhibitors are remarkably heat resistant, acid
stable, glycosylated, and exhibit a high isoelectric point.
Furthermore, these tnetalloproteinase inhibitors are biologically
equivalent to those inhibitors isolated from human skin fibroblast
cultures.
To furthe:= achieve the objects and in accordance with the
purposes of the present invention, as embodied and broadly described
herein, portable DNA sequences coding for metalloproteinase inhibitors
are provided. Theses sequences comprise
s
y
1340971
-5-
nucleotide sequences capable of directing intracellular produc-
tion of metalloproteinase inhibitors. The portable sequences may
be either synthetic sequences or restriction fragments ("natural"
DNA sequences). In a preferred embodiment, a portable DNA se-
quence is isolated from a human fibroblast cDNA library and is
capable of directing intracellular production of a collagenase
inhibitor which is biologically equivalent to that inhibitor
which is isolable from a human skin fibroblast culture.
The coding strand of a first preferred DNA sequence
which has been discoverEad has the following nucleotide sequence:
20 30 40 50 60
GTTGTTGCTG TGGCTGATAG CC:CCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG
70 80 90 100 110 120
ACGGCCTTCT GCAATTCCGA CC:TCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAG~C
130 140 150 160 170 180
AACCAGACCA CCTTATACCA GC:GTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC
190 200 210 220 230 240
CAAGCCTTAG GGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC
250 260 270 280 290 300
TGCGGATACT TCCACAGGTC CC:ACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG
310 320 330 340 350 360
CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC
370 380 390 400 410 420
TTAGCTCAGC GCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG
430 440 450 460 470 480
TTTCCCTGTT TATCCATCCC CTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC
490 500 510 520 ~ 530 540
CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCGG
i
134097 1
-6-
550 560 570 580 590 600
GAGCCAGGGC TGTGCACCTG GC:AGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGGA
610 620 630 640 650 660
GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA
670 680 690 700
ATGAAATAAA GAGTTACCAC CC:AGCAAAAA AAAAAAGGAA TTC
The nucleotides represented by the foregoing abbreviations are
set forth in the Detailed Description of the Preferred Embodi-
ments.
A second prefe:rrred DNA sequence has been discovered
which has an additional nucleotide sequence 5' to the initiator
sequence. This sequence, which contains as the eighty-second
through four-hundred-thirty-second nucleotides nucleotoides 1
through 351 of the first. preferred sequence set forth above, has
the following nucleotides sequence:
20 30 40 50 60
GGCCATCGCC GCAGAT~~CAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTT
70 80 90 100 110 120
GACCCCTGGC TTCTGC,ATCC TGITTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCACC
130 140 150 160 170 180
TGTGTCCCAC CCCACC~~ACA .Gp,CC;GCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAAG
190 200 210 220 230 240
TTCGTGGGGA CACCAG,AAGT CF,ACCAGACC ACCTTATACC AGCGTTATGA GATCAAGATG
250 260 270 280 290 300
ACCAAGATGT ATAAAGGGTT CC'.AAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC
310 320 330 340 350 360
ACCCCCGCCA TGGAGA'uTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAG
370 380 390 400 410 420
TTTCTCATTG CTGGAAAACT GC'.AGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTG
_.' : ~ a
134097 ~
_7_
430
GCTCCCTGGA AC
A third preferred DNA sequence which incorporates the
5' region of the second preferred sequence and the 3' sequence of
the first preferred sequence, has the following nucleotide se-
quence:
20 30 40 50 60
GGCCATCGCC GCAGATCCAG CGiCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTT
70 80 90 100 110 120
GACCCCTGGC TTCTGC.4TCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCACC
1.30 140 150 160 ~ 170 180
TGTGTCCCAC CCCACCCACA GA.CGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAAG
190 200 210 220 230 240
TTCGTGGGGA CACCAGi~AGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGATG
250 260 270 280 290 300
ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC
310 320 330 340 350 360
ACCCCCGCCA TGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAG
370 380 390 400 410 420
TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTG
430 440 450 460 470 480
GCTCCCTGGA ACAGCCiCGAG CTTAGCTCAG CGCCGGGGCT TCACCAAGAC CTACACTGTT
490 500 510 520 530 540
GGCTGTGAGG AATGCAC:AGT GT'TTCCCTGT TTATCCATCC CCTGCAAACT GCAGAGTGGC
550 560 570 580 590 600
ACTCATTGCT TGTGGAC:GGA CC.AGCTCCTC CAAGGCTCTG AAAAGGGCTT CCAGTCCCGT
I
- 1340971
_8_
610 620 630 640 650 660
CACCTTGCCT GCCTGCc;TCG GGAGCCAGGG CTGTGCACCT GGCAGTCCCT GCGGTCCCAG
670 680 690 700 710 720
ATAGCCTGAA TCCTGC(:CGG AGTGGAAGCT GAAGCCTGCA CAGTGTCCAC CCTGTTCCCA
730 740 750 760 770 780
CTCCCATCTT TCTTCCGGAC AATGAAATAA AGAGTTACCA CCCAGCAA.AA AAAAAAAGGA
Currently, for expression of the instant met-
alloproteinase inhibitors in animal cells, the inventors most
prefer a method which utilizes a fourth preferred DNA sequence.
The coding strand of this sequence reads as follows:
20 30 40 50 60
GGCCATCGCC GCAGATC'.CAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTT
70 80 90 100 110 120
GAGCCCCTGG CTTCTGGCAT CC'CGTTGTTG CTGTGGCTGA TAGCCCCCAG CAGGGCCTGC
130 140 150 160 170 180
ACCTGTGTCC CACCCCA.CCC ACAGACGGCC TTCTGCAATT CCGACCTCGT CATCAGGGCC
190 200 210 220 230 240
AAGTTCGTGG GGACACCAGA AG9t'CAACCAG ACCACCTTAT ACCAGCGTTA TGAGATCAAG
250 260 270 280 290 300
ATGACCAAGA TGTATAAAGG GTTCCAAGCC TTAGGGGATG CCGCTGACAT CCGGTTCGTC
310 320 330 340 350 360
TACACCCCCG CCATGGAGAG TGTCTGCGGA TACTTCCACA GGTCCCACAA CCGCAGCGAG
370 380 390 400 410 420
GAGTTTCTCA TTGCTGGAAA ACTGCAGGAT GGACTCTTGC ACATCACTAC CTGCAGTTTC
430 440 450 460 470 480
GTGGCTCCCT GGAACAGCCT GAGCTTAGCT CAGCGCCGGG GCTTCACCAA GACCTACACT
490 500 510 520 530 540
GTTGGCTGTG AGGAATGCAC AGTGTTTCCC TGTTTATCCA TCCCCTGCAA ACTGCAGAGT'
J.
_9_ 1 3 4 0 9 7 1
550 560 570 580 590 600
GGCACTCATT GCTTGTGGAC GGACCAGCTC CTCCAAGGCT CTGAAAAGGG CTTCCAGTCC
610 620 630 640 650 660
CGTCACCTTG CCTGCCTGCC TCGGGAGCCA GGGCTGTGCA CCTGGCAGTC CCTGCGGTCC
670 680 690 700 710 720
CAGATAGCCT GAATCCTGCC CGGAGTGGAA GCTGAAGCCT GCACAGTGTC CACCCTGTTC
730 740 750 760 770 780
CCACTCCCAT CTTT~~TTCCG GACAATGAAA TAAAGAGTTA CCACCCAGCA
GGAATTC
To facilitate identification and isolation of natural
DNA sequences for use in the present invention, the inventors
have developed ;3 human skin fibroblast cDNA library. This li-
brary contains the genetic information capable of directing a
cell to synthesize the metalloproteinase inhibitors of the pres-
ent invention. Other natural DNA sequences which may be used in
the recombinant DNA methods set forth herein may be isolated from
human genomic libraries.
Additionally, portable DNA sequences useful in the pro-
cesses of the present invention may be synthetically created.
These synthetic DNA sequences may be prepared by polynucleotide
synthesis and sequencing techniques known to those of ordinary
skill in the art..
Additionally, to achieve the objects and in accordance
with the purposes of the present,invention, a recombinant-DNA
method is disclosed which results in microbial manufacture of the
instant metalloproteinase inhibitors using the portable DNA se-
quences referred to above. This recombinant DNA method com-
prises:
(a) preparation of a portable DNA sequence
capable of directing a host microorga-
nism to produce a protein having
metallo:proteinase inhibitor activity,
preferably collagenase inhibitor
activity;
s
a
134097 ~
-lo-
(b) cloning i:he portable DNA sequence into a
vector capable of being transferred into
and replicating in a host microorganism,
such vector containing operational ele-
ments for the portable DNA sequence;
(c) transfers-ing the vector containing. the
portable DNA sequence and operational
elements into a host microorganism capa-
ble of expressing the metalloproteinase
inhibitor protein;
(d) culturing the host microorganism under
conditions appropriate for amplification
of the vector and expression of the in-
hibitor; and
(e) in either order: ,
(i) harvesting the inhibitor; and
(ii) cau~~ing the inhibitor to assume an
active, tertiary structure whereby
it possesses metalloproteinase in-
hibitor activity.
To further accomplish the objects and in further accord
with the purposes of they present invention, a series of cloning
vectors are provided comprising at least one of the portable DNA
sequences discussed above. In particular, plasmid pUC9-F5/237P10
is disclosed.
It is understood that both the foregoing general de-
scription and the following detailed description are exemplary
and explanatory only and. are not restrictive of the invention, as
claimed.
The accompanying drawing, which is incorporated in and
constitutes a part of this specification, illustrates one embodi-
ment of the invention anl, together with the description, serves
to explain the principles of the invention.
B;EtIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a partial restriction mad of the plasmid
pUC9-F5/237P10.
_11_ 1 3 4 0 9 7 1
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Reference will now be made in detail to the presently
preferred embodiments of the invention, which, together with the
drawing and the following examples, serve to explain the princi-
ples of the invention.
As noted above, the present invention relates in part
to portable DNA sequences capable of directing intracellular pro-
duction of metalloprote:inase inhibitors in a variety of host
microorganisms. "Portable DNA sequence" in this context is in-
tended to refer either to a synthetically-produced nucleotide se-
quence or to a restriction fragment of a naturally occuring DNA
sequence. For purposes of this specification, "metalloproteinase
inhibitor" is intended t=o mean the primary structure of the pro-
tein as defined by the codons present in the deoxyribonucleic ,
acid sequence which dirEacts intracellular production of the amino
acid sequence, and which may or may not include post-transla-
tional modifications. 7:t is contemplated that such post-transla-
tional modifications include, for example, glycosylation. It is
further intended that the term "metalloproteinase inhibitor"
refers to either the form of the protein as would be excreted
from a microorganism or the methionyl-metalloproteinase inhibitor
as it may be present in microorganisms from which it was not ex-
creted.
In a preferred embodiment, the portable DNA sequences
are capable of directing intracellular production of collagenase
inhibitors. In a particularly preferred embodiment, the portable
DNA sequences are capable of directing intracellular production
of a collagenase inhibitor biologically equivalent to that previ-
ously isolated from human skin fibroblast cultures. By "biologi-
cally equivalent," as used herein in the specification and
claims, it is meant that an inhibitor, produced using a portable
DNA sequence of tile present invention, is capable of preventing
collagenase-induced tissue damage of the same type, but not nec-
essarily to the same degree, as a native human collagenase inhib-
itor, specifically that native human collagenase inhibitor
isolable from human skin fibroblast cell cultures.
,~,;~.i
. 1340971
-12-
A first. preferred portable DNA sequence of the present
invention has a nucleotide sequence as follows:
20 30 40 50 60
GTTGTTGCTG TGGCTGATAG CCCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG
70 80 90 100 110 120
ACGGCCTTCT GCAATTCCGA CCTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC
130 140 150 160 170 180
AACCAGACCA CCTTATACCA GCGTTATGAG ATCAAGATGA CCAAGATGTA TA.AAGGGTTC
190 200 210 220 230 240
CAAGCCTTAG GGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC
250 260 270 280 290 300
TGCGGATACT TCCACA.GGTC Cc:ACAACCGC AGCGAGGAGT TTCTCATTGC TGGAA.AAC:TG
310 320 330 340 350 360
CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC
370 380 390 400 410 420
TTAGCTCAGC GCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG
430 440 450 460 470 480
TTTCCCTGTT TATCCATCCC C7CGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC
490 500 510 520 530 540
CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCGG
550 560 570 580 590 600
GAGCCAGGGC TGTGCACCTG GC:AGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGGA
610 620 630 640 650 660
GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA
670 680 690 700
ATGAAATAAA GAGTTACCAC CC:AGCAAAAA AAAAAAGGAA TTC
S
1340971
-13-
wherein the following nucleotides are represented by the abbrevi-
ations indicated below.
Nucleotides Abbreviation
Deoxyadenylic acid A
Deoxyguany~.ic acid G
Deoxyc~rtidylic acia C
Thymid;rlic acid T
A second preferred portable DNA sequence of the present
invention has thEa following nucleotide sequence:
20 30 40 50 60
GGCCATCGCC GCAGA7:'CCAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTT
70 80 90 100 110 120
GACCCCTGGC TTCTGC:ATCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCACC
130 140 150 160 170 180
TGTGTCCCAC CCCACC:CACA GACGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAAG
190 200 210 220 230 240
TTCGTGGGGA CACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGATG
250 260 270 280 290 300
ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC
310 320 330 340 350 360
ACCCCCGCCA TGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAG
370 380 390 400 410 420
TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTG
430
GCTCCCTGGA AC
In this second preferred sequence, an open reading frame exists
from nucleotides 1 through 432. The first methionine of this
reading frame is encoded by nucleotides by 49 .through 51 and is
the site of translation initiation. It should be noted that the
amino acid sequence prescribed by nucleotides 49 through 114 is '
s
r~ ,
-14- 1 3 4 0 9 7 1
not found in the mature metalloproteinase. It is believed that this
sequence is the :Leader peptide of the human protein..
A third preferred portable DNA sequence has the nucleotide
sequence:
20 30 40 50 60
GGCCATCGCC GCAGATCCAG CGCCCAGAGA GACACCACAC AACCCACCAT GGCCCCCTTT
70 80 90 100 110 120
GACCCCTGGC TTCTGCATCC T~~TTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCACC
130 140 150 160 170 180
TGTGTCCCAC CCCACC.'CACA G:~.CGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAAG
190 200 210 220 230 240
TTCGTGGGGA CACCAC~AAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGA'~'G
250 260 270 280 290 300
ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC
310 320 330 340 350 360
ACCCCCGCCA TGGAGA.GTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAG
370 380 390 400 410 420
TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTG
430 440 450 460 470 480
GCTCCCTGGA ACAGCCTGAG CTTAGCTCAG CGCCGGGGCT TCACCAAGAC CTACACTGTT
490 500 510 520 530 540
GGCTGTGAGG AATGCACAGT G7.'TTCCCTGT TTATCCATCC CCTGCAAACT GCAGAGTGGC
550 560 570 580 590 600
ACTCATTGCT TGTGGACGGA CC'AGCTCCTC CAAGGCTCTG AAAAGGGCTT CCAGTCCCGT
610 620 630 640 ' 650 660
CACCTTGCCT GCCTGCCTCG GGAGCCAGGG CTGTGCACCT GGCAGTCCCT GCGGTCCCAG
X. r.
_15_ 1 3 4 0 9 7 1
670 680 690 700 710 720
ATAGCCTGAA TCCTGC:CCGG AGTGGAAGCT GAAGCCTGCA CAGTGTCCAC CCTGTTCCCA
730 740 750 760 770 780
CTCCCATCTT TCTTCfGGAC A~ATGAAATAA AGAGTTACCA CCCAGCAAAA P,AAAAAAGGA
This third sequence contains the 5' nontranslated region of the
second preferred sequence and the 3' region of the first pre-
ferred sequence. It is envisioned that this third preferred se-
quence is capable of directing intracellular production of a met-
alloproteinase analogous; to a mature human collagenase inhibitor
in a microbial or mammalian expression system.
Currently, for' expression of the instant met-
alloproteinase in~hibitor~s in animal cells, the inventors most
prefer a method which utilizes a fourth preferred DNA sequence.
The coding strand of this sequence reads as follows:
20 30 40 50 60
GGCCATCGCC GCAGAT(:CAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTT
70 80 90 100 110 120
GAGCCCCTGG CTTCTGGCAT CC'I'GTTGTTG CTGTGGCTGA TAGCCCCCAG CAGGGCCTGC
130 140 150 160 170 180
ACCTGTGTCC CACCCCACCC ACAGACGGCC TTCTGCAATT CCGACCTCGT CATCAGGGCC
190 200 210 220 230 240
AAGTTCGTGG GGACACC.AGA AGTCAACCAG ACCACCTTAT ACCAGCGTTA TGAGATCAAG
250 260 270 280 290 300
ATGACCAAGA TGTATAAi~GG GTTCCAAGCC TTAGGGGATG CCGCTGACAT CCGGTTCGTC
310 320 330 340 350 360
TACACCCCCG CCATGGAGAG TGTCTGCGGA TACTTCCACA GGTCCCACAA CCGCAGCGAG
370 380 390 400 410 420
GAGTTTCTCA TTGCTGGp,AA ACTGCAGGAT GGACTCTTGC ACATCACTAC CTGCAGTTTC
430 440 450 460 470 480
GTGGCTCCCT GGAACAGCCT GAG<:TTAGCT CAGCGCCGGG GCTTCACCAA GACCTACACT
~r
-16-
1340971
490 500 510 520 530 540
GTTGGCTGTG AGGA,ATGCAC AGTGTTTCCC TGTTTATCCA TCCCCTGCAA ACTGCAGAGT
550 560 570 580 590 600
GGCACTCATT GCTT(3TGGAC GGACCAGCTC CTCCAAGGCT CTGAAAAGGG CTTCCAGTCC
610 620 630 640 650 660
CGTCACCTTG CCTGCCTGCC TCGGGAGCCA GGGCTGTGCA CCTGGCAGTC CCTGCGGTCC
670 680 690 700 710 72p
CAGATAGCCT GAATC:CTGCC CGGAGTGGAA GCTGAAGCCT GCACAGTGTC CACCCTGTTC
730 740 750 760 770 780
CCACTCCCAT CTTTC:TTCCG GACAATGAAA TAAAGAGTTA CCACCCAGCA A,~~A.AAAAAAA
It mu~;t be borne in mind in the practice of the present
invention that the alteration of some amino acids in a protein
sequence may not affect the fundamental properties of the pro-
tein.' Therefore, it is also contemplated that other portable DNA
sequences, both those capable of directing intracellular produc-
tion of identical amino acid sequences and those capable of
directing intracellular production of analogous amino acid se-
quences which also possess metalloproteinase inhibitor activity,
are included within then ambit of the present invention.
It is contemplated that some of these analogous amino
acid sequences will be substantially homologous to native human
metalloproteinase inhiY~itors while other amino acid sequences,
capable of functioning as metalloproteinase inhibitors, will not
exhibit substantial homology to native inhibitors. By "substan-
tial homology," as used herein, is meant a degree of homology to
a native metalloproteinase inhibitor in excess of 50%, preferably
in excess of 60%, preferably in excess of 80%. The percentage
homology as discussed Herein is calculated as the percentage of
amino acid residues found in the smaller of the two sequences
that align with identical amino acid residues in the sequence
being compared when four gaps in a length of 100 amino acids may
be introduced to assist. in that alignment as set forth by
Dayhoff, M.O. in Atlas of Protein Sequence an_d Structure Vol. 5,
p. 124 (1972), National. Biochemical Research Foundation,
Washington, D.C.
X34097 ~
-17-
As noted above, the portable DNA sequences of the pres-
ent invention may be synthetically created. It is believed that
the means for synthetic creation of these polynucleotide se-
quences are generally known to one of ordinary skill in the art,
particularly in light of the teachings contained herein. As an
example of the current state of the art relating to. poly-
nucleotide synthesis, one is directed to Matteucci, M.D. and
Caruthers, M.H., in J. Am. Chem. Soc. 103: 3185 (1981) and
Beaucage, S.L. and Caruthers, M.H. in Tetrahedron Lett. _22: 1859
(1981).
Additionally, the portable DNA sequence may be a frag-
ment of a natural_ sequence, i.e., a fragment of a polynucleotide
which occurred in nature and which has been isolated and purified
for the first time by the present inventors. In one embodiment,
the portable DNA sequence is a restriction fragment isolated from
a cDNA library. In this preferred embodiment, the cDNA library
is created from ruuman skin fibroblasts.
In an alternative embodiment, the portable DNA sequence
is isolated from a human genomic library. An example of such a
library useful in this embodiment is set forth in Lawn et al.
Cell 15: 1157-1174 (1978),
As also noted above, the present invention relates to a
series of vectors, each containing at least one of the portable
DNA sequences described herein. It is contemplated that addi-
tional copies of the portable DNA sequence may be included in a
single vector to increase a host microorganism's ability to pro-
duce large quantities of the desired metalloproteinase inhibitor.
In add ition, the cloning vectors within the scope of
the present invent=ion may contain supplemental nucleotide se-
quences preceding or subsequent to the portable DNA sequence.
These supplementa~_ sequences are. those that will not interfere
with transcription of the portable DNA sequence and will, in some
instances as set 1'orth more fully hereinbelow, enhance transcrip-
tion, translation, or the ability of the primary amino acid
structure of the resultant metalloproteinase inhibitor to assume
an active, tertiary form. '
-18- ~ 3 4 0 9 7 ~
A preferred vector of the present invention is set
forth in Figure 1. This vector, pUC9-F5/237P10, contains the
preferred nucleotide sequence set forth above. Vector
pUC9-F5/237P10 is present in the C600/pUC9-FS/237P10 cells on de-
posit in the American Type Culture Collection in Rockville,
Maryland under Accession No. 53003.
A preferred nucleotide sequence encoding the metallo-
proteinase inhibitor is identified in Figure 1 as region A.
Plasmid pUC9-FS/?37P10 also contains supplemental nucleotide se-
quences preceding and subsequent to the preferred portable DNA
sequence in region A. These supplemental sequences are identi-
fied as regions F3 and C, respectively.
In alternate :preferred embodiments, either one or both
of the preceding or subsequent supplemental sequences may be re-
moved from the vector o:E' Fig. 1 by treatment of the vector with
restriction endonucleasEas appropriate for removal of the supple-
mental sequences. The supplemental sequence subsequent to the
portable DNA sequence,' identified in Fig. 1 as region C, may be
removed by treatment of the vector with a suitable restriction
endonuclease, preferabl~~ H~iAI followed by reconstruction of the
3' end of region A using synthetic oligonucleotides and ligation
of the vector with T-4 DNA ligase. Deletion of the supplemental
sequence preceding the portable DNA sequence, identified as re-
gion B in Fig. 1, would be specifically accomplished by the meth-
od set forth in E:Xample 2.
In preferred embodiments, cloning vectors containing
and capable of expressing the portable DNA sequence of the pres-
ent invention cons=ain various operational elements. These "oper-
ational elements," as discussed herein, include at least one pro-
moter, at least one Shine-Dalgarno sequence, at least one
terminator codon. Preferably, these "operational elements" also
include at least one operator, at least one leader sequence, and
for proteins to be exported from intracellular space, at least
one regulator and any ot)ner DNA sequences necessary or preferred
for appropriate transcription and subsequent translation of the
vector DNA.
X34097'
-19-
Additional embodiments of the present invention are en-
visioned as emplo~ring other known or currently undiscovered
vectors which would contain one or more of the portable DNA se-
quences described herein. In particular, it is preferred that
these vectors have some or all of the following characteristics:
(1) possess a minimal number of host-organism sequences; (2) be
stable in the desired host; (3) be capable of being present in a
high copy number i.n the desired host; (4) possess a regulatable
promoter; (5) have at least one DNA sequence coding for a se-
lectable trait present on a portion of the plasmid separate from
that where the portable 17NA sequence will be inserted; and (6) be
integrated into the vecto r.
The following, noninclusive, list of cloning vectors is
believed to set forth vectors which can easily be altered to meet
the above-criteria and are therefore preferred for use in the
present invention. Such alterations are easily performed by
those of ordinary skill in the art in light of the available lit-
erature and the te,achings~ herein.
TABLE I
HOST Vectors Comments
E. coli pUCB Many selectable replicons
pUC9 have been characterized.
pBR322 Maniatis, T. et al. (1982),
pGW7 Molecular Clonin ~ A
placIq Laboratory Manual, Cold
pDPB Spring Harbor Laboratory.
BACILLUS pUB110 Genetics and Biotechnology
B. subtilis p~5A0501 of Bacilli, Ganesan and
B. amyloliquefaciens p;5A2100 Hoch, eds., 1984, Academic
B. stearothermophilus p13D6 Press.
p13D8
p'.~ 12 7
1 340 97 ~
-20-
PSEUDOMONAS RSF1010 Some vectors useful in
P. aeruginosa Rms149 broad host range of gram-
P. putida pKT209 negative bacteria including
RK2 Xanthomonas and
Agrobacterium.
pSa727
CLOSTRIDIUM pJUl2 Shuttle plasmids for E.
C. perfringens pJU7 coli and C.
perfringens
pJUlO construction ref. Squires,
pJUl6 C. et al. (1984) Journal
pJUl3 Bacteriol. 159:465-471.
SACCHAROMYCES YEp24 Botstein and Davis in
S. cerevisiae YIpS Molecular Biology of the .
YRpl7 Yeast Saccharomyces,
Strathern,. Jones, and
Broach, eds., 1982, Cold
Spring Harbor Laboratory.
It is to be understood that additional cloning vectors may now
exist or will be <9iscovered which have the above-identified prop-
erties and are the refore suitable for use in the present inven-
tion. These vectors are also contemplated as being within the
scope of the disclosed series of cloning vectors into which the
portable DNA sequences may be introduced, along with any neces-
sary operational elements, and which altered vector is then in-
cluded within the scope of the present invention and would be
capable of being used in the recombinant-DNA method set forth
more fully below.
In addition to the above list, an E. coli vector sys-
tem, as set forth in Example 2, is preferred in one embodiment as
a cloning vector. Moreover, several vector plasmids which auton-
omously replicate in a broad range of Gram Negative bacteria are
preferred for use as cloning vehicles in hosts of the genera
Pseudomonas. The:;e are described by Tait, R.C:, Close, T.J.,
Lundquist, R.C., fiagiya, M., Rodriguez, R.L., and Kado, C.I. in
Biotechnology, Ma~~, 1983, pp. 269-275; Panopoulos, N.J. in
134097 ~
- 21 -
Genetic Engineering_in the Plant Sciences, Praeger Publishers, New York,
New York, pp. 163-185, (1981); and Sakaguchi, K. in Gl~rrent Topic in
Microbiology and Imnn~nology 96:31-45, (1982).
One particularly preferred construction employs the plasmid
RSF1010 and derivatives thereof as described by Bagdasarian, M.,
Bagdasarian, M.M., C:oleman, S., and Timmis, K.N. in Plasmids of Medical
Environmental and Ccm~nercia:l Importance, Timmis, K.N. and Puhler, A.
eds., Elsevier/North Holland Biomedical Press, (1979). The advantages of
RSF1010 are that it is relatively small, high copy number plasmid which
is readily transformed into and stably maintained in both E. coli and
Pseudomonas species. In this system, it is preferred to use the Tac
expression system as described for Escherichia, since it appears that the,
E. coli trp promoter is readily recognized by Pseudomonas RNA polymerase
as set forth by Sakaguchi, K. in Clzrrent Topics in Microbiology and
Immunology 96:31-45 (1982) and Gray, G.L., McKeown, K.A., Jones, A.J.S.,
Seeburg, P.H., and Heyneker, H.L. in Biotechnology Feb. 1984, pp. 161-
165. Transcriptional activity may be further maximized by requiring the
exchange of the prompter with, e.g., an E. coli or P. aeruginosa trp
promoter.
In a preferred e~~nbodiment, P. aeruginosa is transformed with
vectors directing the synthesis of the metalloproteinase inhibitor as
either an intracellu:Lar product or as a product coupled to leader
sequences that will ssffect its processing and export from the cell. In
this embodiment, there leader sequences are preferably selected from the
group consisting of )seta-lactainase, OmpA protein, the naturally
occurring human signal peptide, and that of carboxypeptidase G2 from
Pseudomonas. Translation may be coupled to translation initiation for any
of the E. coli proteins as described in Example 2, as well as to
initiation sites for any of the highly expressed proteins of the host to
cause intracellular expression of the metalloproteinase inhibitor.
.~~" . ~
4
~y'~.
134097 1
- 22 -
In those cases vvhere restriction minus strains of a host
Pseudomonas species are not available, transformation efficiency with
plasmid constructs isolated from E. coli are poor. Therefore, passage of
the Pseudomonas cloning vector through an r- m+ strain of another species
prior to transformation of 'the desired host, as set forth in Bagdasarian,
M., et al., Plasmids of Medical, Environmental and Commercial Importance,
pp. 411-422, Timmis and Puh:Ler eds., Elsevier/North Holland Biomedical
Press (1979), is de:;ired.
Furthern~~re, a preferred expression system in hosts of the
genera Bacillus involves us:Lng plasmid pUB110 as the cloning vehicle. As
in other host vector systema, it is possible in Bacillus to express the
metalloproteinase inhibitor: of the present invention as either an ,
intracellular or a secreted protein. The present embodiments include both
systems. Shuttle vectors that replicate in both Bacillus and E. coli are
available for constructing and testing various genes as described by
Dubnau, D., Gryczan, T., Corrtente, S., and Shivakumar, A.G. in Genetic
Encrineering, Vol. 2, Setlow and Hollander eds., Plenum Press, New York,
New York, pp. 115-131, (198C)). For the expression and secretion of
metalloproteinase inhibitors from B. subtilis, the signal sequence of
alpha-amylase is preferably coupled to the coding region for the
metalloproteinase inhibitor. For synthesis of intracellular
metalloproteinase irihibitor, the portable DNA sequence will be
translationally coupled to the ribosome binding site of the alpha-amylase
leader sequence.
Transcription of either of these constructs is preferably
directed by the alpha-amylase promoter or a derivative thereof. This
derivative contains ~=he RNA polymerase recognition sequence of the native
alpha-amylase promoter but incorporates the lac operator region as well.
Similar hybrid promot=ers constructed from the penicillinase gene promoter
and the lac operator have been shown to function in Bacillus hosts in a
regulatable fashion as set forth by Yansura, D.G. and Henner in Genetics
and Biotechnology of Bacilli, Ganesan, A.T. and Hoch,
1340 g7 ~
- 23 -
J.A., eds., Academic: Press, pp. 249-263, (1984). The lacI gene of lacIq
would also be included to effect regulation.
One pre=ferred construction for expression in Clostridium is
in plasmid pJZTl2 described loy Squires, C. H. et al in J. Bacteriol.
159:465-471 (1984), transfo:cmed into C. perfrinc~ens by the method of
Heefner, D. L. et al.. as described in J. Bacteriol. 159:460-464 (1984).
Transcription is directed b~~ the promoter of the tetracycline resistance
gene. Translation is coupled to the Shine-Dalgarno sequences of this
same tetr gene in a inner ~;trictly analogous to the procedures outlined
above for vectors suitable for use in other hosts.
Maintenance of foreign DNA introduced into yeast can be
effected in several ways (Botstein, D., and Davis, R. W., in The
Molecular Bioloctv of the Yeast Saccharomyces, Cold Spring Harbor ,
Laboratory, Strathern, Jones and Broach, eds., pp. 607-636 (1982). One
preferred expression system for use with host organisms of the genus
Sacchammyces harbors the anticollagenase gene on the 2 micron plasmid.
The advantages of th~= 2 micron circle include relatively high copy number
and stability when i:ztroduced into cir° strains. These vectors
preferably incorporave the replication origin and at least one antibiotic
resistance marker from pBR322 to allow replication and selection in E.
coli. In addition, t=he plasmid will preferably have 2 micron sequences
and the yeast LEU2 ge=ne to serve the same purposes in LEU2 mutants of
yeast.
The regi:~latable promoter from the yeast GAL1 gene will
preferably be adapted to direct transcription of the portable DNA
sequence gene. Tram>lation of the portable DNA sequence in yeast will be
coupled to the leader sequen~~e that directs the secretion of yeast alpha-
factor. This will cause fozm~tion of a fusion protein which will be
processed in yeast arid result in secretion of a metalloproteinase
inhibitor. Alternatively, a methionyl-metalloproteinase inhibitor will
be translated for inclusion within the cell.
134pg7 ~
-24-
It is anticipated that translation of mRNA coding for
the metalloproteinase inhibitor in yeast will be~ more efficient
with the preferred codon usage of yeast than with the sequence
present in pUCB-Fic, as identified in Example 2, which has been
tailored to the prokaryotic bias. For this reason, the portion
of the 5' end of the portable DNA sequence beginning at the
Tth111I site is preferably resynthesized. The new sequence fa-
vors the codons most frequently used in yeast. This new sequence
preferably has the following nucleotide sequence:
HgiA:I
S' GAT CCG TGC ACT TGT GTT CCA CCA CAC
GC ACG 'FGA ACA CAA GGT GGT GTG
CCA CAA ACT GCT TTC TGT AAC TCT GAC C
GGT GTT TGA (:GA AAG ACA TTG AGA CTG GA 3'
As will be seen from an examination of the individual
cloning vectors and systems contained on the above list and de-
scription, various. operational elements may be present in each of
the preferred vectors of the present invention. It is contem-
plated any additional ope rational elements which may be required
may be added to these vec tots using methods known to those of or-
dinary skill in the art, particularly in light of the teachings
herein.
In practice, ii. is possible to construct each of these
vectors in a way that al7Lows them to be easily isolated, assem-
bled, and interchanged. This facilitates assembly of numerous
functional genes from connbinations of these elements and the
coding region of the metalloproteinase inhibitor. Further, many
of these elements will beg applicable in more than one host.
At least one origin of replication recognized by the
contemplated host microorganism, along with at least one select-
able marker and at least one promoter sequence capable of
initiating transcription of the portable DNA sequence are contem-
plated as being included in these vectors. It is additionally
contemplated that the vectors, in certain preferred embodiments,
will contain DNA sequences capable of functioning as regulators
("operators"), and other DNA sequences capable of coding for
_ .
1340971
-25-
regulator proteins. In preferred vectors of this series, the
vectors additionally contain ribosome binding sites, transcrip-
tion terminators and leader sequences.
These regulators, in one embodiment, will serve to pre-
vent expression of the portable DNA sequence in the presence of
certain environmental conditions and, in the presence of other
environmental conditions, allow transcription and subsequent ex-
pression of the protein coded for by the portable DNA sequence.
In particular, it. is preferred that regulatory segments be in-
serted into the vector such that expression of the portable DNA
sequence will not. occur in the absence of, for example, isopro-
pylthio- ~ -d-gal<ictoside. In this situation, the transformed mi-
croorganisms containing the portable DNA may be grown to a de-
sired density pr:Lor to initiation of the expression of the
metalloproteinasEa inhibitor. In this embodiment, expression of
the desired protease inhibitor is induced by addition of a sub-
stance to the microbial environment capable of causing expression
of the DNA sequence after the desired density has been achieved.
Additionally. it is preferred that an appropriate se-
cretory leader sequence be present, either in the vector or at
the 5' end of they portable DNA sequence, the leader sequence
being in a position which allows the leader sequence to be imme-
diately adjacent.to the initial portion of the nucleotide se-
quence capable oi= directing expression of the protease inhibitor
without any intervening transcription or translation termination
signals. The presence of the leader sequence is desired in part
for one or more of the following reasons: 1) the presence of the
leader sequence nnay facilitate host processing of the initial
product to the mature recombinant metalloproteinase inhibitor; 2)
the presence of t:he lea~aer sequence may facilitate purification
of the recombinant metalloproteinase inhibitors, through
directing the met:allopr~oteinase inhibitor out of the cell
cytoplasm; 3) the presence of the leader sequence may affect the
ability of the recombinant metalloproteinase inhibitor to fold to
its active structure through directing the metalloproteinase in-
hibitor out of the cell cytoplasm. ,
-26- 1 3 4 0 9 7 1
In particular, the leader sequence may direct cleavage
of the initial translation product by a leader peptidase to re-
move the leader sequence and leave a polypeptide with the amino
acid sequence which has the potential of metalloproteinase inhib-
itory activity. In some species of host microorganisms, the
presence of the appropriate leader sequence will allow transport
of the completed protean into the periplasmic space, as in the
case of E. coli. In the case of certain yeasts and strains of
Bacillus and Pseudomonas, the appropriate leader sequence will
allow transport of the protein through the cell membrane and into
the extracellula.r medium. In this situation, the protein may be
purified from ex.tracel:Lular protein.
Thirdly, in the case of some of the metalloproteinase
inhibitors prepared by the present invention, the presence of the
leader sequence may be necessary to locate the completed protein
in an environment where it may fold to assume its active struc-
ture, which structure possesses the appropriate metalloproteinase
activity.
Additional operational elements include, but are not
limited to, ribosome-b;Lnding sites and other DNA sequences neces-
sary for microbial expression of foreign proteins. The opera-
tional elements as disc ussed herein can be routinely selected by
those of ordinary skil:L in the art in light of prior literature
and the teachings contained herein. General examples of these
operational elements are~set forth in B. Lewin, Genes, Wiley &
Sons, New York (1983).
Various examples of suitable operational elements
may be found on the vectors discussed above and may be elucidated
through review of the publications discussing the basic charac-
teristics of the aforementioned vectors.
In one preferred embodiment of the present invention,
an additional DNA sequence is located immediately preceding the
portable DNA sequence which codes for the metalloproteinase in-
hibitor. The additional DNA sequence is capable of functioning
as a translational coupler, i.e., it is a DNA sequence that
encodes an RNA which serves to position ribosomes immediately
adjacent to the ribosome binding site of the metalloproteinase
inhibitor RNA with which it is contiguous. ,
1 340 97 ~
-27-
Upon synthesis and/or isolation of all necessary and
desired component parts of the above-discussed cloning vectors,
the vectors are assembled by methods generally known to those of
ordinary skill in the ari:. Assembly of such vectors is believed
to be within the duties and tasks performed by those with ordi-
nary skill m the art an<i, as such, is capable of being performed
without undue experiments tion. For example, similar DNA se-
quences have been ligate<i into appropriate cloning vectors, as
set forth in Schoner et al., Proceedings of the National Academy
of Sciences U.S.A., 81:5403-5407 (1984).
In construction of the cloning vectors of the present
invention, it should additionally be noted that multiple copies
of the portable DNA sequcance and its attendant operational ele-
ments may be inserted ini_o each vector. In such an embodiment;
the host organism would produce greater amounts per vector of the
desired metalloproteinase inhibitor. The number of multiple
copies of the DNA sequence which may be inserted into the vector
is limited only by the ability of the resultant vector, due to
its size, to be transferred into and replicated and transcribed
in an appropriate host microorganism.
Additionally, .it is preferred that the cloning vector
contain a selectable marker, such as a drug resistance marker or
other marker which causes expression of a selectable trait by the
host microorganism. In a particularly preferred embodiment of
the present invention, the gene for ampicillin resistance is in-
cluded in vector p~UC9-F5,~237P10.
Such a drug resistance or other selectable marker is
intended in part to facilitate in the selection of transformants.
Additionally, the presence of such a selectable marker on the
cloning vector may be of use in keeping contaminating microorga-
nisms from multiplying in the culture medium. In this embodi-
ment, such a pure culture of the transformed host microorganisms
would be obtained by culturing the microorganisms under condi-
tions which require the :induced phenotype for survival.
It is noted that, in preferred embodiment, it is also
desirable to reconstruct the 3' end of the coding region to allow
.,.,
1,340 97 1
-28-
assembly with 3' non-translated sequences. Included among these
non-translated s~equence~s are those which stabilize the mRNA or
enhance its transcription and those that provide strong tran-
scriptional terminatior,~ signals which may stabilize the vector as
they are identified by Gentz, R., Langner, A., Chang, A.C.Y.,
Cohen, S.H., and Bujard,,. H. in Proc. Natl. Acad. Sci. USA
78:4936-4940 (19;91).
This invention also relates to a recombinant-DNA method
for the producti~~n of metallproteinase inhibitors. Generally,
this method includes:
(a) p:ceparation of a portable DNA sequence
c.3pable of directing a host microorga-
nism to produce a protein having ,
m~stallop~roteinase inhibitor activity;
(b) c:Loning the portable DNA sequence into a
vector capable of being transferred into
and replicating in a host microorganism,
such vector containing operational ele-
ments for the portable DNA sequence;
(c) transferring the vector containing the
portable DNA sequence and operational
elements into a host microorganism capa-
b:Le of expressing the metalloproteinase
inhibitor protein;
(d) culturing the host microorganism under
conditions appropriate for amplification
o:E the vector and expression of the in-
h:Lbitor; and
(e) in either order:
(:i) harvesting the inhibitor; and
(:ii) causing the inhibitor to assume an
active, tertiary structure whereby
it possesses metalloproteinase in-
hibitor activity.
In thia method, the portable DNA sequences are those ,
synthetic or naturally-occurring polynucleotides described above.
-29- 1 3 4 0 9 7 1
In a preferred embodiment of the present method, the portable DNA
sequence has the nucleotide sequence as follows:
20 30 40 50 60
GTTGTTGCTG TGGCTGA'rAG CCf.CAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG
70 80 90 100 110 120
ACGGCCTTCT GCAATTC~~GA CC'rCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC
130 140 150 160 170 180
AACCAGACCA CCTTATA~CA GCCiTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC
190 200 210 220 230 240
CAAGCCTTAG GGGATGCCGC TGP~CATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC
250 260 270 280 ' 290 300
TGCGGATACT TCCACAGGTC CCFvCAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG
310 320 330 340 350 360
CAGGATGGAC TCTTGCACAT CAC'.TACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC
370 380 390 400 410 420
TTAGCTCAGC GCCGGGGCTT CAC'.CAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG
430 440 450 460 470 480
TTTCCCTGTT TATCCATCCC CTC4CAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC
490 500 510 520 530 540
CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCGG
550 560 570 580 590 600
GAGCCAGGGC TGTGCACCTG GCAGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGGA
610 620 630 640 650 660
GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA
670 680 690 700
ATGAAATAAA GAGTTACCAC CCAGCAAAAA AAAAAAGGAA TTC
134097 1
- 30 -
The vectors contemplated as being useful in the present
method are those described above. In a preferred embodiment, the cloning
vector pUC9-F5/237P10 is used in the disclosed method.
The vect~~r thus obtained is then transferred into the
appropriate host microorganism. It is believed that any microorganism
having the ability t.o take up exogenous DNA and express those genes and
attendant operational elements may be chosen. It is preferred that the
host microorganism k~e an an,~erobe, facultative anaerobe or aerobe.
Particular hosts which may 'oe preferable for use in this method include
yeasts and bacteria. Specific yeasts include those of the genus
Saccharomyces, and especial=Ly Saccharomvces cerevisiae.
Specific bacterua include those of the genera Bacillus and
Escherichia and Pseizd~nas. Various other preferred hosts are set forth
in Table I, s-upra. In other, alternatively preferred embodiments of the
present invention, E,acillus subtilis. Escherichia coli or Pseudomonas
aerucrinosa are selecaed as the host microorganisms.
After a host organism has been chosen, the vector is
transferred into the=_ host organism using methods generally known by those
of ordinary skill in the art. Examples of such methods may be found in
Advanced Bacterial Genetics by R. W. Davis et al., Cold Spring Harbor
Press, Cold Spring Harbor, Pdew York, (1980). It is preferred, in one
embodiment, that the transformation occur at low temperatures, as
temperature regulation is contemplated as a means of regulating gene
expression through the use of operational elements as set forth above.
In another embodiment, if osmolar regulators have been inserted into the
vector, regulation of the :salt concentrations during the transformation
would be required to insure' appropriate control of the synthetic genes.
If it is contemplated that the recombinant metalloproteinase
inhibitors will ultimately be expressed in yeast, it is preferred that
the cloning vector first beg transferred into Escherichia coli, where the
vector would be allowed to replicate and from which the vector would be
obtained and purified after amplification. The vector would then be
transferred into tr.e yeast for ultimate expression of the
metalloproteinase inhibitor.
,.
1340871
-31-
The host microorganisms are cultured under conditions
appropriate for the expression of the metalloproteinase inhib-
itor. These conditions are generally specific for the host or-
ganism, and are readily determined by one of ordinary skill in
the art, in light of the published literature regarding the
growth conditions for such organisms, for example Bergey's Manual
of Determinative Bacteriology, 8th Ed., Williams & Wilkins Compa-
ny, Baltimore, Maryland..
Any conditions. necessary for the regulation of the ex-
pression of the D.NA sequence, dependent upon any operational ele-
ments inserted into or F>re$ent in the vector, would be in effect
at the transformation and culturing stages. In one embodiment,
the cells are grown to a, high density in the presence of appro-
priate regulatory conditions which inhibit the expression of the,
DNA sequence. When optimal cell density is approached, the envi-
ronmental conditions are altered to those appropriate for expres-
sion of the portable DNA sequence. It is thus contemplated that
the production of the metalloproteinase inhibitor will occur in a
time span subsequent to the growth of the host cells to near
optimal density, and that the resultant metalloproteinase inhib-
itor will be harvested at some time after the regulatory condi-
tions necessary for its expression were induced.
In a preferred embodiment of the present invention, the
recombinant metal7.oproteinase inhibitor is purified subsequent to
harvesting and pr3.or to assumption of its active structure. This
embodiment is preferred as the inventors believe that recovery of
a high yield of re-folded protein is facilitated if the protein
is first purified. However, in one preferred, alternate embodi-
ment, the metalloproteinase inhibitor may be allowed re-fold to
assume its active structure prior to purification. In yet
another preferred, alternate embodiment, the metalloproteinase
inhibitor is caused to assume its re-folded, active state upo n
recovery from the cultur:Lng medium.
In certain circumstances, the metalloproteinase inhib-
itor will assume its proper, active structure upon expression in
the host microorganism and transport of the protein through the ,
134Q971
-32-
cell wall or memt~rane or into the periplasmic space. This will
generally occur if DNA coding for an appropriate leader sequence
has been linked to the I)NA coding for the recombinant protein.
The preferred metalloprotienase inhibitors of the present inven-
tion will assume their mature, active form upon translocation out
of the inner cell membrane. The structures of numerous signal
peptides have been published, for example by Marion E.E. Watson
in Nuc. Acid Res. 12:515-5164, 1984.
It is intendec9 that these leader sequences,
together with portable DNA, will direct intracellular production
of a fusion protean which will be transported through the cell
membrane and will have the leader sequence portion cleaved upon
release from the cell.
In a preferred embodiment, the signal peptide of the
E.coli OmpA protein is used as a leader sequence and is~located.~
in a position contiguous with the portable DNA sequence coding
for the metalloproteines~e inhibitor structure.
Additionally preferred leader sequences include those
of beta-lactamase, carbo:Kypeptidase G2 and the human signal pro-
tein. These and other leader sequences are described.
If the metalloproteinase inhibitor does not assume its
proper, active structure,, any disulfide bonds which have formed
and/or any noncovalent interactions which have occurred will
first be disrupted by denaturing and reducing agents, for exam-
ple, guanidinium chlorides and ~ -mercaptoethanol, before the
metalloproteinase inhibitor is allowed to assume its active
structure following dilution and oxidation of these agents under
controlled conditions.
The transcription terminators contemplated herein serve
to stabilize the vector. In particular, those sequences as de-
scribed by Gentz _et. al., in Proc. Natl. Acad. Sci. USA _78:
4936-4940 (1981), are contemplated for use in the present
invention.
It is to be understood that application of the teach-
ings of the present: invention to a specific problem or environ-
ment will be within the capabilities of one having ordinary skill
in the art in light. of the teachings contained herein. Examples
1340971
-33-
of the products of the present invention and representative processes
for their isolation and manufacture appear in the following examples.
EXAMPLES
EXAMPLE 1
Preparation of Poly A+ RNP, from HEF-SA Fibroblasts
HEF-SA cells were grown to near confluence in 75 cm2 T-
flasks. Cells were washed twice in Dulbecco's phosphate buffered
saline solution and harve:~ted by the addition of 2 ml of lOmM Tris, pH
7.5 containing 1% w/v SDS (obtained from BDH Chemicals, Ltd., Poole,
England), 5mM EDTA and 20 ug/ml protease K (obtained from Boehringer
Mannheim Biochemica.ls, Indianapolis, Indiana). Each flask was
subsequently washed with an additional milliliter of this same
solution.
The pooled aliquots from the cell harvest were made to 70
ug/ml in protease K and incubated at 40°C for 45 minutes. The ,
proteolyzed solution was brought to a NaCl concentration of 150 mM by
the addition of 5 M stock and subsequently extracted with an equal
volume of phenol: chloroform 1:1. The aqueous phase was reextracted
with an equal volume of chloroform. Two volumes of ethanol were added
to the aqueous phase and incubated overnight at -20°C. The
precipitated nucleic acids were recovered by centrifugation at 17,500
TM
xg for 10 minutes i:n a Beckman J2-21 centrifuge, Beckman Instruments,
Palo Alto, California, and were redissolved in 25 ml of 0.1% w/v SDS.
This solution was a~3ain extracted with an equal volume of chloroform.
The aqueous phase w~~s added to two volumes of cold ethanol and kept at
-20°C for 2 hours. The precipitate was collected by centrifugation at
10,000 xg for 15 minutes a:nd redissolved in 10 ml of 1 mM Tris, 0.5 mM
EDTA, 0.1% SDS, pH '7.5. R1VA was precipitated from this solution by
the addition of 10 rnl of 4 M LiCl, 20 mM NaoAc, pH 5.0 and incubated
at -20°C for 18 hours. The precipitate was again recovered by
centrifugation and washed twice with 2 M LiCl before redissolving in 1
mM Tris, 0.5 mM EDTA, 0.1% SDS, pH 7.5. This solution was stored at
-70°C.
1340971
-34-
Chromatography on Oligo dT Cellulose
Total cellular RNA prepared as above was ethanol pre-
cipitated and redissolved in 0.5 M NaCl. Five ml of RNA at 0.45
mg/ml were applied to a 1 ml column of washed type VII oligo dT
cellulose (obtained from PL Biochemicals, Milwaukee, Wisconsin).
The column was then washed with 10 ml of 0.5 M NaCl and eluted
with 2.0 ml of sterile H20. The eluted poly(A+) fraction of RNA
was ethanol precipitated and dissolved to give a 1 mg/ml solution
in 1 mM Tris, 0.1 mM EDTA, pH 8Ø This was stored at -70°C.
cDNA Synthesis
Poly(A+) RNA Hras primed with oligo dT (obtained from PL
Biochemicals, Milwaukee, Wisconsin) to serve as a template for
cDNA synthesis by AMV reverse transcriptase (obtained from Life
Sciences, Inc., St. Petersburg, Florida). Following the synthe-
sis reaction, the RNA was hydrolyzed by the addition of 0.1 voi~
ume of 3 N NaoH and incubated at 67°C for 10 minutes. The solu-
tion was then neutralized and the cDNA purified by gel filtration
rM
chromatography on Biogel A 1.5 (obtained from BioRad
Laboratories, Rictunond, California) in a 0.7x25 cm column in a 10
mM Tris, 5 mM EDTA, and 1% SDS, pH 7.5 solution. Fractions con-
taining cDNA were pooled and concentrated by ethanol precipita-
tion. The cDNA was dG trailed and purified by gel filtration
using the procedure set forth above. Second strand synthesis was
primed with oligo dC and polymerized in an initial reaction with
the large (Klenow) fragment of DNA polymerase (obtained from
Boehringer Mannheim). Following second strand synthesis, _E. coli
DNA polymerase I (obtain<~d from Boehringer Mannheim) was added
and incubation continued to form blunt ends. The double stranded
cDNA was again purified by chromatography. EcoRI restriction
sites within the cDNAs were modified by the action of EcoRI
methylase, obtained from New England Biolabs, Beverly, Mas-
sachusetts. The cDNA wa~~ again purified and ligated to synthetic
EcoRI linkers. Finally, the ends were then trimmed with the
endonuclease and t'he cDNA, purified by gel filtration. This DNA
was ligated into a unique EcoRI site in lambda'gtl0 DNA packaged
in vitro and used to infect _E. coli strain hflA according to the
method set forth b:~~ Huynh, T.V., Young, R.A., and Davis, R.W., in
1~4097~
- 35 -
DNA Cloning Technirn;~es, A Practical Approach (ed. Glover, D.M.) (IRL
Press Oxford), in pz~ess. Approximately 25,000 recombinants were
amplified in this manner.
Screenincr
Recombinant-phage-containing sequences of interest were
selected by their preferential hybridization to synthetic oligo-
nucleotides encoding portions of the primary structure of the desired
metalloproteinase inhibitor, hereinafter referred to as FIBAC. These
portions of the protein seq~zence correspond in part to those set forth in
the published literature by Stricklin, G.P. and Welgus, H.G., J. Biol.
Chem. 258: 12252-12258 (198:3). Recombinant phage were used to infect E.
coli strain hflA and plated at a density of approximately 2x103 pfu/150
mm petri dish. Phao~e were blotted onto nitrocellulose filters (BA85,
Schleicher & Schuell Inc., Keene, New Hampshire), and DNA was denatured
and fixed essentially as described by Benton and Davis in Science
196:180-182 (1979).
Using that procedure, the filters were treated sequentially
for 10-15 minutes each in 0,.5 M NaCl, then 1.0 M Tris, 1.5 M NaCl pH 8.0,
and finally submerged in 2x SSPE. (2x SSPE is 0.36 M NaCl, 20 mM NaHzP04,
2 mM EDTA pH 7.4). Filters were blotted dry and baked 75°-80°
for 3-4
hours. Duplicate filters were made of each plate. Filters were
prehybridized for 1-3 hours at 37° in 5x SSPE containing O.lx SET,
0.150
NaPPi, and lx Denhardts solutions. Filters were then hybridized for 72
hours at 37° in this same solution containing 5x105cpm/ml of 5' end-
labeled 51-mer oligonucleotide specific activity approximately 106
cpm/pmole). Following hybridization, filters were washed six times in 5x
SSPE containing O.lx SET and 0.05% sodium pyro-phosphate at 37°,
then
three times in 2x SSPE at 21.°. These were then blotted dry and
autorad_iographed on .Kodak'"' YP.R-5 film at -70° with a Kodak
lightening-
plus intensifying screen. ~~ignals clearly visible from duplicate filters
were used to pick ph~ge for plaque purification. Filter preparations and
hybridization procedires for plaque purification steps were the same as
above.
.;
1340971
- 36 -
The washing procedure was simplified to 6 changes of 2x SSPE at
37°. Six
isolates purified by repetit=ive plating were then arranged on a single
lawn of _E. coli strain C600 for testing with subsequent probes.
Preferential hybridization of the 17-mer to each of the
isolates (as opposed to control plaques) was observed under a condition
identical to that u:~ed for ;plaque purification. Probe C was used in a
similar test, except: that the SSPE concentration during hybridization was
reduced to 4x. Aga__n, each of the isolates demonstrated stronger
hybridization to the probe than did control plaques.
Phag_e Purification <rnd cDNA Characterization
Quantities of each of the six isolated phage were made by the
plate stock technique and purified by serial CsCl block gradient ,
centrifugation. DNi~ was extracted from these by dialysis against 50%
forn~amide as described by Davis, R.W., Botstein, D., Roth, J.R., in A
Manual for Genetic l~gineering~ Advanced Bacterial Genetics, 1980, Cold
Spring Harbor L~abor,~tory. DNA from each of the isolates was digested with
EcoRI and the produ~~ts were: analyzed by agarose gel electrophoresis. The
insert from one of the larder clones, lambda FIBACT"' 5, was found to lack
internal sites for SalI, Hi.ndIII, BamHI, and EcoRI. The cDNA insert was
released from lambda FIBAC 5 DNA and the lambda arms digested by co-
digesting with these four enzymes. The fragments were then ethanol-
precipitated and ligated into the EcoRI site of plasmid pUC9 without
further purification. There plasmids _were then used to transform E. coli
strain JM83. Transforn~ants were selected on ampicillin containing
plates. Plasmids from several trans-formants were purified and
characterized on tr.e basis of the EcoRI digestion products. One was
selected which had an insert co-migrating with the insert from lambda
FIBAC 5 on agarose gel electrophoresis. This plasmid has been named
pUC9-F5/237P10.
Mapping and Subclonin
The insert in pUC9-F5/237P10 was mapped with respect to
internal PstI site:>. Double digests with EcoRI and Pst demonstrated
three internal Pstl recognition sites. The entire insert
:"'~ ,
-37- 1 3 4 0 9 7 1
and the component. pieces were subcloned into M13 bacteriophage
mpl9 and mpl8, respectively. Sequencing of the pieces was per-
formed by the dic9eoxynucleotide method described by Sanger et al.
in Sanger, F., Nicklen, S., and Coulson, A.R., Proc. Natl. Acad.
Sci. USA 74:5463--5467 (1977), ,
The sequence of the DNA insert from pUC9-F5/237P10
showed an open rEaading frame which encodes the primary structure
of a mature fibroblast collagenase inhibitor biologically equiva-
lent to that isol.able from human skin fibroblasts. The salient
features of the :sequence are:
(1) The insert is flanked by EcoRI restric-
tion sites and by G/C and A/T homo-
polymeric tracts consistent with the
cloning methodology;
(2) The coding strand is presented in the 5'
to 3' convention with poly C at the 5'
en.d and poly A at the 3' end, again con-
sistent with the techniques employed;
(3) If' the first G in the sequence GTTGTTG
i~runediately adjacent to the 3' end of
the poly C tract is considered as
nucleotide 1, then an open reading frame
is presented which encodes the primary
structures of the mature human fibroblast
collagenase inhibitor beginning at
nucleotide 34 and continuing through
nucleotide 585;
(4) The termination codon TGA at nucleotides
586 through 588 defines the carboxy ter-
minus of the translation product which
is the same as that of the mature pro-
tein;
(5) Nucleotides 1 through 33 define an amino
acid sequence which is not found in the
primary structure of the processed pro-
tein, but: which is probably a portion of
:.
_38_ 1 3 4 0 9 7 1
a leader. peptide characteristic of se-
creted proteins;
(6) The three internal PstI sites have as
their first base nucleotides 298, 327,
and 448;
(7) There is a single recognition sequence
for the restriction enzyme Tth111I
beginning at nucleotide 78; and
(8) There is. a single recognition sequence
for the restriction endonuclease NcoI
b,eginnin.g at nucleotide 227.
The sequence of nucleotides 1 through 703 and restriction site
analysis are shovan.
# SITES FRAGMENTS FRAGMENTS ENDS
ACC 1 (GTVWAC) 1 214 495 (69.8) 214 709
214 (30.2) 1 214
ALU 1 (AGCT) 4 35B 358 (50.5) 1 358
363 124 (17.5) 482 606
48.2 119 (16.8) 363 482
6015 103 ( 14 . 5 ) 606 709
( 0.7) 358 363
AVA 1 (CQCGPG) 1
53Ei 536 (75.6) 1 536
173 (24.4) 536 709
AVA 2 ( GGRCC ) 3
25i' 257 (36.2) 1 257
47f 220 (31.0) 257 477
572 137 (19.3) 572 709
95 (13.4) 477 572
BBV 1 (GCTGC) :l
269 440 (62.1) 269 709
269 (37.9) 1 269
.,~.:r<.
.~
1340971
-39-
# SI'T'ESFRAGM ENTS FRAGMENTS ENDS
BST N1(CCRGG) 3
344 344 (48.5) 1 344
544 200 ( 28. 344 544
2 )
55;~ 152 (21.4) 557 709
13 ( 1.8) 544 557
DDE 1 (CTNAG) 4
lBEi 344 (48 365 709
. 5
)
355 186 (26.2) 1 186
360 169 (23.8) 186 355
365 5 ( 0.7) 360 365
5 ( 0.7) 355 360
ECO R1(GAATTC) 1
69E3 698 ( 98 1 698
. 4
)
11 ( 1.6) 698 709
FNU4H 1 (GCNGC)2
19E. 440 ( 62 269 709
. 1
)
269 196 (27.6) 1 196
73 (10.3) 196 269
FOK 1 (GGATG) 4
192 274 (38.6) 435 709
204 192 (27.1) 1 192
303 132 (18.6) 303 435
435 99 (14.0) 204 303
12 ( 1.7) 192 204
HAE 2 (PGCGCQ) 1
368. 368 (51.9) 1 368
341 (48.1) 368 709
134pg71
-40-
# SI'.CESFRAGMENTS FRAGMENTS ENDS
HAE 3 (GGCC) 3
30 616 (86.9) 93 709
63 30 ( 4.7) 30 63
93 30 ( 4.2) 63 93
30 ( 4.2) 1 30
HGI A1 (GRGCRC)
1
55:! 552 (77.9) 1 552
157 (22.1) 552 709
HHA 1 (GCGC) 1
369 369 (52.0) 1 369
~
340 (48.0) 369 709
HINC 2 (GTQPAC) 1
118 591 (83.4) 118 709
118 (16.6) 1 118
HINF 1 (GANTC):2
308 308 (43.4) 1 308
587 279 (39.4) 308 587
122 (17.2) 587 709
HPA 2 (CCGG) 4
207 224 (31.6) 372 596
372 207 (29.2) 1 207
596 165 (23.3) 207 372
654 58 ( 8.2) 596 654
55 ( 7.8) 654 709
HPH 1 (GGTGA) 2.
380 380 (53.6) 1 380
519 190 (26.8) 519 709
139 (19.6) 380 519
MBO 2 (GAAGA) 1
650 650 (91.7) 1 650 '
59 ( 8.3) 650 709
~~j ;
~u
w. 134097 1
-41-
# SITES FRAGM ENTS FRAGMENTS ENDS
MNL (CCTC) 5
1
S:l 193 (27.2) 81 274
274 174 (24.5) 535 709
406 132 (18.6) 274 406
486 81 (11.4) 1 bl
5 3!i 80 ( 11 406 486
. 3
)
49 ( 6.9) 486 535
MST (CCTNAGG)1
2
18!i 524 ( 73 185 709
' . 9
)
185 (26.1) 1 185
NCI (CCSGG) 2
1
37a 372 (52.5) 1 372
59!i 223 (31.5) 372 595
114 (16.1) 595 709
NCO (CCATGG) 1 '
1
22',7 482 (68.0) 227 709
227 (32.0) 1 227
NSP B2 (CVGCWG) 1
197 512 (72.2) 197 709
197 (27.8) 1 197
PST 1 (CTGCAG) 3
298 298 (42.0) 1 298
32T 261 (36.8) 448 709
448 121 (17.1) 327 448
29 ( 4.1) 298 327
SAU 1 (CCTNAGG) 1
185 524 (73.9) 185 709
185 (26.1) 1 185
SAU 3A (GATC) :l ,
150 559 (78.8) 150 709
,r.'
1340971
-42-
# SITES FRAGMENTS FRAGMENTS ENDS
150 (21.2) 1 150
SAU96 1 (GGNCC) 5
29 220 (31.0) 257 477
'92 165 (23.3) 92 257
257 137 (19.3) 572 709
477 95 (13.4) 477 572
572 63 ( 8.9) 29 92
29 ( 4.1) 1 29
SCR F1 (CCNGG)5
344 344 (48. 1 344
5 )
3'72 172 (24. 372 544
3 )
544 114 (16.1 595 709
)
5li7 38 ( 5.4) 557 595
5!~5 28 ( 3.9) 344 372
13 ( 1.8) 544 557
SFA N1 (GATGC)1
1<.~3 516 (72.8) 193 709
193 (27.2) 1 193
TTH111 1 1
(GACNNNGTC) 79 630 (88.9) 79 709
79 (11.1) 1 79
The following appear:
do not
AAT 2 AFL2 AFL 3 AHA 3
APA 1 ASU2 AVA 3 AVR 2
B.AL1 BAMH1 BCL 1 BGL 1
BGL 2 BIN1 BSSH BST E2
1
CfR 1 CLA1 ECO R5 FNUD
2
G;DI2 HAE1 HGA 1 HGI C1
H3I D1 HGIJ2 HIND HPA 1
3
KIPN1 MLU1 MST 1 NAE 1
Ni~R1 NDE1 NRU 1 NSP C1
PVU 1 PW 2 RRU 1 RSA 1
SAC 1 SAC2 SAL 1 SMA 1
1340971
-43-
SNA 1 SPH 1 STU 1 TAQ 1
XBA 1 XHO 1 XHO 2 XMA 3
XMN 1
20 30 40 50 60
GTTGTTGCTG TGGCTGATAG C:CCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG
SH
AA
UE
13
70 80 90 100 110 120
ACGGCCTTCT GCAAT'TCCGA C:CTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC
H T M SH H
A T N AA I
E H L UE N'
3 1 1 13 2
130 140 150 160 170 180
AACCAGACCA CCTTA'rACCA G'~CGTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC
S
A
U
A
190 200 210 220 230 240
CAAGCCTTAG GGGATGCCGC T'GACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC
SD FS FN F H A N
AD OF ITS O P C C
UE KA 1:JP K A C O
11 11 :l2 1 2 1 1
250 260 270 280 290 300
TGCGGATACT TCCACAGGTC CCACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG
A B M P
V B N S
A V L T
2 1 1 1
~~T~ .1
1340971
-44-
310 320 330 340 350 360
CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC
F H P B D A D
O I S S D L D
K N T T E (1 E
1 1 1 1 1 1 1
370 380 390 400 410 420
TTAGCTCAGC GCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG
A D HH N H M
L D AH C P N
U E EA I H L
1 1 21 1 1 1
430 440 450 460 470 480
TTTCCCTGTT TATCCATCCC CTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC
F P A
O S V
K T A
1 1 2
490 500 510 520 530 540
CAGCTCCTCC AAGGCTCTGA AP~AGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCGG
A M H MA
L N P NV
U L H LA
1 1 1 11
550 560 570 580 590 600
GAGCCAGGGC TGTGCA~CCTG GC'.AGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGGA
B H B A H NH
S G S V I CP
T I 'T A N IA
1 1 1 2 1 12
1340 97 ~
-45-
610 620 630 640 650 660
GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA
A M H
L 8 P
U O A
1 2 2
670 680 690 700
ATGAAATAAA GAGTTA.CCAC CCAGCAAAAA AAAAAAGGAA TTC
E
C
O
i
EXAMPLE 2-EXPRESSION OF COLLAGENESE INHIBITOR IN E. COLI
In this Example, a preferred method of coupling a pre-
ferred portable DNA sequence to the 5' end of the cloned cDNA is
set forth. This involv<as making a nucleolytic cleavage at a
specified point within the coding sequence and reconstructing the
desired portion of the (:oding sequence by means of synthetic
oligonucleotides in a manner that allows its excision and recom-
bination (i.e., by incorporating useful restriction sites).
Trimming the 5' end of the coding region will be
accomplished by synthesizing both strands of the DNA extending
from the Tth111I site in the 5' direction and ending in a BamHI
overhang. This synthet~~c oligonucleotide, referred to as FIBAC
A, has the following features:
(1) Codon selection has been biased toward those most
frequently found in the genes of highly expressed
bacterial. proteins;
(2) A methionine codon from which to initiate transla-
tion has been provided immediately upstream from
the cyste~ine which begins the coding region of,
human processed FIBAC;
(3) The spacing of the BamHI site to the methionine
codon is such that when cloned into pUCB, the
coding region of FIBAC will be in-frame with the
5' end o~: the beta-galactosidase gene;
L.
-46- 1 3 4 0 9 7 1
(4) An in-frame stop codon and Shine Dalgarno sequence
are also presented. Translation of this frame for
t:he amino terminal portion of the beta-
<~alactosidase is terminated at the TAA codon, and
translation of FIBAC should be initiated at the
following ATG;
(5) <:odons have been selected to create a HgiAI site
beginning with the G in the FIBAC initiation
c:odon; and
(6) There is a PvuI site separated by one base from
t:he 3' end of the BamHI sequence.
The structure of FIHAC A is
GA TCC: GCG A'TC GGA GTG TAA GAA ATG TGC ACT
G. CGC T,AG CCT CAC ATT CTT TAC ACG TGA
TGC GTT CCG ~~CG CAT CCG CAG ACT GCT TTC
ACG CP,A GGC GGC GTA GGC GTC TGA CGA AAG
TGC AP,C TCT ~3AC C
ACG TT'G AGA CTG GA
FIHAC A is synthesized using the ABI DNA synthesizer
(Foster City, California) as a series of four component
oligonucleotides.
Component ol:igonucleotide FA1 is:
GATCC GCGAT CGGAG TGTAA GAAAT GTGCA CTTGC
Component ol:igonucleotide~FA2 is:
GGAACG CAAGT GCACA TTTCT TACAC TCCGA TCGCG
Component oligonucleotide FA3 is:
GTTC CGCCG CATCC GCAGA CTGCT TTCTG CAACT CTGAC C
Component ol3.gonucleotide FA4 is:
AGGTC AGAGT TGCAG AAAGC AGTCT GCGGA TGCGG C
The remainder of the coding portion of the FIBAC gene
is isolated as the 3' Tth111I to EcoRI fragment generated by a
double digest of pUC9-P'S/237P10 with these enzymes.
a
.w
1340971
-47-
A synthetic linker is made to couple the 3' end of the
Tth111I to EcoRI fragment to a Sall site. These oligonucleotides
will be designed to recreate the SalI site and destroy the EcoRI
site. The linker is comprised of the oligonucleotides linker A1
and linker A2.
Linker A1 is: AATTGGCAG
Linker A2 is: TCGACTGCC
These oligonuc:Ieotides and oligonucleotides FA1-FA4 are
kinased separately and annealed in equal molar ratios with the
Tth111I to EcoRI 3' end of the cDNA and BamHI/SalI cut mpl9RF
DNA. The ligated DNA is used to transfect JM105. Plaques are
picked by their color in the presence of IPTG and X-gal and by
hybridization to oligonucleotide FA2. Several positive plaques
are to be sequenced. Tt~:ose containing the designed sequence are
subcloned into BamHI/Sal.I digested pUCB. Translation of the
FIBAC gene in this construct is coupled to translation initiated
for beta-galactosidase. This expression vector is referred to as
pUCB-Fic.
Coupling tran~olation of FIHAC to translation initiated
for other highly expressed proteins is similarly arranged. For
example, a portion of the OmpA gene which contains the Shine-
Dalgarno and initiator methionine sequences has been synthesized.
This sequence encodes the entire signal peptide of OmpA protein
and had convenient restriction sites, including those for EcoRI,
EcoRV, Pvul, and StuI.
The sequence of the sene~e strand is:
20 30 40 50 60
GAATTCGATA TCTCGT'rGGA GF~TATTCATG ACGTATTTTG GATGATAACG AGGCGCAAAA
E T E F M H
C A C O N H
O Q O K L A
1 1 5 1 1 1
,1~ ~~ ,i
134097 ~
-48-
70 80 90 100 110
AATGAAAAAG ACAGCTATCG CGATCGCAGT GGCACTGGCT GGTTTCGCTA CCGTA
A NF PS
L RN V.A
U UU UU
1 12 lA
120 130
GCGCA GGCCTCTGGT AAAAGC'rT
H S H M HA
H T A N IL
A U E L NU
1 1 3 1 31
This sequence is hereinafter referred to as OmpA lead-
er. Coupling the trans:Lation of FIBAC to OmpA is accomplished,t~y
cutting the pUCB-Fic wii_h PvuI and SalI and isolating the coding
region. This, together with the EcoRI to PvuI fragment isolated
from OmpA leader, will tie cloned into EcoRI/SalI-cut pUCB. As in
the prior example, transcription is driven by the lac promoter
and regulated by the lac I gene product at the lac operator.
This FIBAC expression vector is referred to as pUCB-F/OmpAic.
To effect the translocation of FIBAC across the inner
cell membrane, an appropriate leader sequence is added to the
amino terminus of FIBAC. The protein thus produced will be
translocated and processed to yield the mature form.
To effect such a translocation, a FIBAC gene encoding
the signal peptide of the E. coli OmpA protein continuous with
the structural region of FIBAC is created. This particular FIBAC
gene necessitates having' in frame stop codons at the 5' end of
the FIBAC coding region changed. To accomplish this, the portion
of the 5' coding region from pUCB-Fic that extends from the HgiAI
site to the NcoI cite is isolated. Upstream sequences are
resynthesized as ,3 linker having cohesive ends from BamHI and
HgiAI and containing an internal StuI site. This is synthesized
as two oligonucleotides, linker B1 and linker 82.
Linker 131 is: GATCCCAGGCCTGCA.
Linker 132 is: GGCCTGG
r
134097 ~
Linker:: B1 and B2 are kinased separately and annealed
in equal molar ratios with the HgiAI to NcoI fragment described
above and BamHI/rfcoI cut pUCB-Fic. The resulting construct has
the coding sequence of FIBAC in frame with the translation of the
amino terminus of beta-galactosidase. Translation of this se-
quence forms a fusion protein with FIBAC. This plasmid is
referred to as pUC8-Ff.
Attaching the OmpA leader sequences to the coding re-
gion of FIBAC is accomp:Lished by ligating EcoRI/StuI cut pUCB-Ff
with an excess of the purified EcoRI to StuI fragment of OmpA
leader. Following transformation, plasmids from several colonies
will be characterized by hybridization. Those that have incorpo-
rated the OmpA leader fragment are characterized further to veri-
fy the structure. This plasmid, pUCB-F OmpAl, will direct the
synthesis of a fusion protein beginning in the signal peptide o'f
the E. coli OmpA protein and ending in human FIBAC. The signals
present in the OmpA portion of the protein effect the protein's
export from the cytopla~~m and appropriate cleavage from the pri-
mary structure of FIBAC.
If the efficiency of expression were to be compromised
by the sequence of the leader peptide or its combination with
FIBAC either at t',he protein or at the nucleic acid level, the
gene could be altered to encode any of several known E. coli
leader sequences.
Transcription of all of the genes discussed is effected
by the lac promoter. As in the case of initiation sites for
translation, the promoter and operator region of the gene may be
interchanged. FIBAC may also be expressed from vectors incor-
porating the lambda PL promoter and operator (OL), and the hybrid
promoter operator,, Tac as described in Amann, E., Brosius, J.,
and Ptashne, M. Gene _25:167-178 (1983).
Excision of those portions of the gene
including ribosome binding site structural region and 3' non-
translated sequences and insertion in alternate vectors contain-
ing the PL or Tac promoter makes use of the unique restriction
sites that flank these structures in pUCB-F/OmpAic and
pUCB-F/OmpAl. In:;ertion of the EcoRI to SalI fragment from
I
. ...,
-5~- ~ 3 4 ~ 9 7
either into similarly digested plasmid pDP8 effects transcription of
these genes directed by i:he lambda PI, promoter. Transcriptional
regulation would k>e temp<~rature sensitive by merit of the cI857
mutation harbored on thia same plasmid.
Putting simila~_ gene fragments into the transcription unit
of the Tac promoter will be accomplished by first isolation the EcoRv
to SalI fragment. This, together with the synthetic Tac promoter
sequence which is flanked by BamHI and PvuII sites and which contains
the lac operator will be inserted into the BamHI to SalI sites of
pBR322 or preferably der=ivatives. The derivatives in this case refer
to constructs containing either the lacI gene or the Iq gene.
Expression of FIBAC in host microorganisms other than
Escherichia is considered. Yeast and bacteria of the genera Bacillus,
Pseudomonas, and Clostridium may each offer particular advantages.
The processes outlined above could easily be adapted to others.
In general, expression vectors for any microorganism will
embody features analogous to those which we have incorporated in the
above mentioned vectors of E. coli. In some cases, it will be
possible to simple move t:he specific gene constructs discussed above
directly into a vector compatible with the new host. In others, it
may be necessary or desirable to alter certain operational or
structural elements of the gene.
EXAMPLE 3
The human collagenase inhibitor may be readily purified
after expression in a variety of microbes. In each case, the spectrum
of contaminant proteins will differ. Thus, appropriate purification
steps will be selected from a variety of steps already known to give a
good separation of the human collagenase inhibitor from other proteins
and from other procedures which are likely to work.
If the inhibitor is not secreted from the microbes, it may
form inclusion bodies inside the recombinant microbes. These bodies
are separated from other proteins by differential centrifugation after
disruption of the cells with a French Press. The insoluble inclusion
bodies are solubil.ized in 6 M guanidine
W.
-51- 1340971
hydrochloride or 8 M urea, and the inhibitor protein is more com-
pletely solubilized by reaction of its cysteines with sodium
sulfite. At any time subsequent to this step, the cysteines are
converted back to their reduced form with dithiothreitol. Once
the inhibitor protein is solubilized from inclusion bodies,
immunoaffinity chromatography using antibodies raised against the
unfolded inhibitor are used for purification before refolding.
The inhibitor can be refolded according to the protocol
mentioned in Example 6, infra. After refolding of the inhibitor,
or if the inhibitor is :secreted from the microbes, purification
from other proteins is accomplished by a variety of methods.
Initial steps include ul.trafiltration through a SO K dalton cut-
off membrane or ammonium sulfate fractionation. Other useful
methods include, but are not limited to, ion-exchange
chromatography, gel filtration, heparin-sepharose chromatography,
reversed-phase chromatography, or zinc-chelate chromatography.
All of these step;s have been successfully used in purification
protocols. Addit:ional.high resolution steps include hydrophobic
interaction chrom~3tography or immunoaffinity chromatography.
After purification, the metalloproteinase inhibitor is preferably
at least 90-95$ pure.
FY~MD1-F d
Purification of Human Collagenase Inhibitor from Human
Amniotic Fluid
Human annniotic-fluid obtained from discarded
amniocentesis samples was pooled and 6 liters were subjected to
ultrafiltration through a 100 kD MW cutoff filter, obtained from
Millipore Corporation, in a Millipore Pellicon Cassette System.
The eluate was concentrated through a 10 kD cutoff filter,
TM
obtained from Millipore Corporation, then through an Amicon PM-10
membrane. Aliquots (10 ml) of concentrated amniotic fluid were
eluted through a 2.5 x 100 cm column of Ultrogel AcA54, obtained
from LKB Corporation, which was equilibrated with pH 7.6, 0.05 M
hepes, 1 M sodium chloride, 0.01 M calcium chloride, and 0.02$
sodium azide (all chemicals were obtained from~Sigma Chemical
Company). Fractions containing the inhibitor were collected and
pooled, dialyzed against pH 7.5, 0.025 M Hepes buffer containing
1340971
-52-
0.01 M calcium chloride and 0.02% sodium azide, and loaded onto a
TM
1.5 x 28 cm heparin-Sepharose CL-6B (obtained from Pharmacia,
Inc.) column equilibrated with the same buffer. This column was
rinsed with 1 liter of ~:he above buffer and eluted with a linear
gradient of 0-0.3 M sodium chloride. The fractions from the
largest peak of inhibitor activity, eluting at about 0.1-0.15 M
sodium chloride, were pooled, concentrated to 1 ml, and loaded
rM
onto a Synchropak rp-8 reverse phase HPLC column equilibrated
with 0.05% trifluoroacet_ic acid (Aldrich Chemical Company). The
column was eluted with a linear gradient of 0-40% acetonitrile
(J. T. Baker Chemical Company) at 1/2% per minute. All fractions
were immediately dried in a Savant speed-vac concentrator to re-
move acetonitrile, and redissolved in pH 7.5, 0.1 M Hepes before
assay. The inhibitor eluted between 32-38% acetonitrile. Frac-
tions containing the inhibMtor were pooled, and 100 ul aliquots~
were eluted over a Bio-rad biosil-TSK 250 HPLC gel filtration
column. The pooled peaka of inhibitor activity contained 0.1 mg
of inhibitor, which was over 95% pure as judged by SDS- poly-
acrylamide gel electrophoresis.
FY~MDT.F S
Purification of Human Fibroblast Collagenase Inhibitor
from Human Embryonic Skin Fibroblast Serum-Free Medium
Human embryonic skin fibroblasts were grown in serum-
free tissue culture medium. Ten liters of this medium were col-
lected, dialyzed <igainst pH 7.5, 0.02 M hepes buffer containing
0.02% sodium azide and 0.01 M calcium chloride, and applied to a
2.8 x 48 cm column of heparin-sepharose CL-6B (Pharmacia, Ine.)
equilibrated with the same buffer. The column was rinsed with 2
liters of this bu1'fer and was then eluted with linear gradient of
0-0.3 M sodium chJ.oride contained in this buffer. The fractions
obtained were tested for the presence of inhibitor by their abil-
ity to inhibit human fibroblast collagenase. The fractions cor-
responding to the peak of activity were those obtained near 0.15
M sodium chloride. These fractions were concentrated to about 5
ml by ultrafiltrat:ion through an Amicon YM10 filter and the con-
centrate was applied in four separate runs to a 250 x 4.1 mm
Synchropak rp-8 reverse phase HPLC column, equilibrated with 1% '
s
_~ 'S:..
134097 ~
- 53 -
trifluoroacetic acid. The column was eluted with a 0-60% linear gradient
of acetonitrile in 0.1% trif:luoroacetic acid. The gradient was run at
1/2o acetontrile per minute. The inhibitor eluted in two sharp peaks
between 26-29% acetonitrile. All fractions were immediately dried in a
Savant speed-vac concentrator, redissolved in pH 7.5, 0.1 M Hepes, and
assayed. At least 1.2 mg of: collagenase inhibitor was recovered, which
was 90-95% pure. This material gives single band when run on a 17.50
reducing SDS gel. After cax-boxymethylation of the cysteines and elution
through the same RP-8 column under identical conditions, the inhibitor is
suitably homogenous for protein sequencing.
EXAMPLE 6
It is contemplated that the human collagenase inhibitor can
be readily refolded into its native structure from its denatured state
after expression of its gene in a microbe and separation of the
collagenase inhibitor from most of the other proteins produced by the
microbe. By analogy to the conditions necessary for the refolding of
other disulfide-containing proteins as set forth by Freedman, R. B. and
Hillson, D. A., in "Formation of Disulfide Bonds," in: The Enzymology of
Post-Translational N.odificat-ion of Proteins, Vol. 1, R. B. Freedman and
H. C. Hawkins, eds., pp. 158-207 (1980), refolding of the human
collagenase inhibitor should occur in solutions with a pH of 8.0 or
greater. At this pH, the c;~steines of the protein are partially ionized,
and this condition is neces:~ary for the attainment of native disulfide
bond pairings. 'I'he inhibitor concentration should be relatively low,
less than 0.1 mg/ml, to min_~mize the formation of intermolecular
disulfide-linked aggregates which will interfere with the refolding
process.
Since th~~ stabil.ity of the refolded (native) disulfide bonded
structure relative to the enfolded (reduced) structure depends on both
the solution oxidation-reduction potential and the concentrations of
other redox-active molecule,, it is conter~lated that the redox potential
should be buffered with a rc=_dox buffer giving a potential equivalent to a
reduced: oxidized c~lutathione ratio of 10. The preferred concentration
range of reduced glutathione would be 0.1-1.0 mM. At higher
concentrations, mixed disulfides will form with
-54- 1 3 4 0 9 7 1
protein, reducing t:he yield of the refolded (native) structure. The
relative stabilities of the unfolded protein and the native structure,
and thus the rate and yie:Ld of refolding, will also depend on other
solution variables, such as the pH, temperature, type of hydrogen-ion
buffer, ionic strength, and the presence or absence of particular
anions or cations as discussed in Privalov, P. L., "Stability of
Proteins, Small Globular 1?roteins," in Advances in Protein Chemistry,
Vol. 33, pp. 167-2 .6, (19'79) .
These conditions vary for every protein and can be
determined experimentally.. It is contemplated that addition of any
molecule that strongly prefers to bind the native (as opposed to the
unfolded) structure, and which can be readily separated afterwards
from the native (refolded) protein, will increase not only the yield
but the rate of re-folding. These molecules include monoclonal
antibodies raised against the native structure, and other proteins
which tightly bind the native collagenase inhibitor, such as the
mamalian enzymes collagenase or gelatinase.
Example 7
The second preferred sequence as set forth herein,
i.e.,
20 30 40 50 60
GGCCATCGCC GCAGATCC'AG CGCC:CAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTT
H F XB HH N S
A N HI AH C A
E U ON EA O U
3 1 21 21 1 1
70 80 90 100 110 120
GACCCCTGGC TTCTGCAT'CC TGT7.'GTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCACC
B SF S H
S FO A A
T AK U E
1 11 1 3
i...
A
-55-
1 34n g7 ~
130 140 150 160 170 180
TGTGTCCCAC CCCACCCACA GA.CGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAAG
H T M SH
A T N AA
E H L UE
3 1 1 13
190 200 210 220 230 240
TTCGTGGGGA CACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGATG
H S
I A
N U
2 A
250 260 270 280 290 300
ACCAAGATGT ATAAAGCiGTT CC.AAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC
SD FS FN F H A
AD OF NS O P C
UE KA UP K A C
11 11 12 1 2 1
310 320 330 340 350 360
ACCCCCGCCA TGGAGAGTGT CT~~CGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAG
N A B M
C V B N
0 A V L
1 2 1 1
370 380 390 400 410 420
TTTCTCATTG CTGGAAAACT GC,AGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTG
P F H P
S O I S
T K N T
1 1 1 ~ 1
4
~.~~'7
-56- ~ 3 4 0 9 7 ~
430
GCTCCCTGGA AC
B
S
T
1
has the following restriction sites:
SITES FRAGMENTS FRAGMENTS ENDS
ACC 1 (GTVWAC)
:l
295 295 (68.3) 1 295
137 (31.7) 295 432
AVA 2 (GGRCC)
1.
338 338 (78.2) 1 338
94 (21.8) 338 432
BBV 1 (GCTGC)
1
350 350 (81.0) 1 350
82 (19.0) 350 432
BIN 1 (GGATC)
1
14 418 (96.8) 14 432
14 ( 3.2) 1 14
BST N1 (CCRGG)
2
65 360 (83.3) 65 425
425 65 (15.0) 1 65
7 ( 1.6) 425 432
DDE 1 (CTNAG)
1
1340971
-57-
SITES FRAGMENTS FRAGMENTS ENDS
267 267 (61.8) 1 267
165 (38.2) 267 432
FNU4H 1 (GCNGC)
3
8 269 (62.3) 8 277
277 82 (19.0) 350 432
350 73 (16.9) 277 350
8 ( 1.9) 1 8
FOK 1 (GGATG)
c4
76 197 (45.6) 76 273
273 99 (22.9) 285 384
285 76 (17.6) 1 76
384 48 (11.1) 384 432
12 ( 2.8) 273 285
HAE 2 (PGCGCQ)
1
19 413 (95.6) 19 432
19 ( 4.4) 1 19
HAE 3 (GGCC)
1 258 (59.7) 174 432
51 60 (13.9) 51 111
111 50 (11.6) 1 51
144 33 ( 7.6) 111 144
174 30 ( 6.9) 144 174
1 ( 0.2) 1 1
HHA 1 (GCGC)
1
20 412 (95.4) 20 432
20 ( 4.6) 1 20 ,
_:.s~~.
~ 34~ 97 ~
-58-
SITES FRAGMENTS FRAGMENTS ENDS
HINC 2 (GTQPAC)
:L
199 233 (53.9) 199 432
199 (46.1) 1 199
HINF 1 (GANTC)
1.
389 389 (90.0) 1 389
43 (10.0) 389 432
HPA 2 (CCGG)
1
288 288 (66.7) 1 288
144 (33.3) 288 432
MNL 1 (CCTC)
2
162 193 (44.7) 162 355
355 162 (37.5) 1 162
77 (17.8) 355 432
MST 2 (CCTNAGG)
1
266 266 (61.6) 1 266
166 (38.4) 266 432
NCO 1 (CCATGG)
2
47 261 (60.4) 47 308
308 124 (28.7) 308 432
47 (10.9) 1 47
NSP B2 (CVGCWG)
1
278 278 (64.4) 1 278
154 (35.6) 278 432
1340 97 ~
-59-
# SITES FRAGMENTS FRAGMENTS ENDS
PST 1 (CTGCAG)
2
3751 379 (87.7) 1 379
40E1 29 ( 6.7) 379 408
~4 ( 5.6) 408 432
SAU 1 (CCTNAGG)
1
266~ 266 (61.6) 1 266
166 (38.4) 266 432
SAU 3A (GATC)
:2
14 217 (50.2) 14 231
231 201 (46.5) 231 432
14 ( 3.2) 1 14
SAU96 1 (GGNCC)
S1 165 (38.2) 173 338
110 94 (21.8) 338 432
173 63 (14.6) 110 173
338 59 (13.7) 51 110
51 (11.8) 1 51
SCR F1 (CCNGG)
2
65 360 (83.3) 65 425
425 65 (15.0) 1 65
7 ( 1.6) 425 432
SFA N1 (GATGC)
75 199 (46.1) 75 274
274 158 (36.6) 274 432
75 (17.4) 1 75
-6a- ~ 3 4 0 9 7 ~
# S:LTES FRAGMENTS FRAGMENTS ENDS
STY 1 (CCRRGG)
2
X47 261 (60.4) 47 308
308 124 (28.7) 308 432
47 (10.9) 1 47
TTH111 1 (GACNNNGTC)
1
1E~0 272 (63.0) 160 432
160 (37.0) 1 160
XHO 2 (PGATCQ)
1
13 419 (97.0) 13 432
.,
13 ( 3.0) 1 13
The following not
do appear:
AAT 2 AFL 2 AFL 3 AHA 2
AHA 3 ALU 1 APA 1 ASU 2
AVA 1 AVA 3 AVR 2 BAL 1
BAM H1 BAN 1 BAN 2 BCL 1
BGL 1 BGL 2 BSM 1 BSP 1286
nSSH BST E2 CFR 1 CLA 1
1
ECO R1 ECO RS FNUD 2 GDI 2
HAE 1 HGA 1 HGI A1 HGI C1
HGI D1 HGI J2 HIND 3 HPA 1
HPH 1 KPN 1 MBO 2 MLU 1
MST 1 NAE 1 NAR 1 NCI 1
NDE 1 NHE 1 NOT 1 NRU 1
NSP C1 PW 1 PW 2 RRU 1
RSA 1 SAC 1 SAC 2 SAL 1
SCA 1 SMA 1 SNA 1 SNA B1
SPE 1 SPH 1 SSP 1 STU 1
TAQ 1 XBA 1 XHO 1 XMA 3
XMN 1
_....y
r P.
1340971
-61-
The salient features of this cDNA are:
1. The coding strand is presented in the 5'
to 3' convention with the polyC tract at
the 5' sand.
2. If the :First G in the sequence GGC CAT
CGC CGC is considered as nucleotide 1,
then an open reading frame exists from
nucleotide 1 through nucleotide 432,
which is the 3' end of this partial
cDNA.
3. The fir~~t methionine in this reading
frame is encoded by nucleotides 49
through 51 and represents the initiation
site of translation.
4. The amino acid sequence prescribed by
nucleotides 49 through 114 is not found
i;z the primary structure of the mature
protein, but it is the sequence of the
leader peptide of human protein.
5. Tile sequence of nucleotides 82 through
432 is identical to the sequence of
nucleotides numbered 1 through 351 in
the insert from the first preferred se-
quence of Example 1.
6. The amino .acid sequence of the mature
protein displays two consensus sequences
for sugar attachment. These sequences,
-N-Q-T- prescribed by nucleotides 202
through 210 and -N-R-S- prescribed by
nucleotides 346 through 354, are amino
acid residues 30 through 32 and 78
through 80, respectively, in the mature
protein. Both sites are glycosylated in
the human inhibitor protein.
FYLMD1.F Q
A series of expression vectors have been constructed
which direct the transcription and translation of the FIBAC gene
in E.~coli.
134pg71
-H2-
A. Expression Vector pFib51
The first of these constructions is a derivative of
plasmid pUCB which contains the coding region of the human
fibroblast collagenase inhibitor ("FIBAC") gene arranged in such
a way as to allow its transcription to be directed and regulated
by the lac promoter and opc>rator. This, construct, pFib5l, was
made with minor modification of the procedure outlined in Example
Two for the assembly of expression vector pUCB-Fic.
Trimming of t:he S' end of the coding region was
effected by isolating that piece of DNA extending from the HaeIII
site at nucleotid a 93 through the 3' EcoRI site beginning at
nucleotide 698. Reconstruction of the 5' end was accomplished as
described except that oligonucleotides FA3 and FA4 were each
lengthened by 12 bases to extend the reconstruction to the HaeIII
recognition site. Hence, the structure of FIBAC A' was create:
GA Tt:C GCG ATC GGA GTG TAA GAA
G CGC TAG CCT CAC ATT CTT
ATG TC3C ACT TGC GTT CCG CCG CAT
TAC AC:G TGA ACG CAA GGC GGC GTA
CCG CP~G ACT GCT TTC TGC AAC TCT
GGC GTC TGA CGA AAG ACG TTG AGA
GAC CTG GTG ATC AGG G
CTG GA.C CAC TAG TCC C
The salient features of FIBAC A' have remained as described in
Example 2.
A synthetic linker was made to couple the 3' end of the
HaeIII to EcoRI fragment to a SalI site. These oligonucleotides
were designed to recreai:e the SalI site and to destroy their
EcoRI site. In addition, the linkers were modified from the
original description to include an internal K~nI site. The new
linker is comprised of t:he oligonucleotides "modified-linker A1"
and "modified-linker A2,."
Modified-linker A1 is: AATTGGTACCAG
Modified-linker A2 is: TCGACTGGTACC
r
1340971
-63-
Ligation into M13 mpl9, cloning, and selection were es-
sentially as described in previous Examples. The coding region of
FIBAC was removect from a clone with the designed sequence by di-
gestion with BamFiI and HindIII and subcloned into these restric-
tion sites in pUC'8. The resulting plasmid is pFib5l. In this
~lasmid, the transcription of the FIBAC gene is directed by the
lac promoter. Translation of methionyl FIBAC is coupled to
translation initiated for beta-galactosidase.
B. Exyressioia Vector pFib55
Creation of the Hc~iAI restriction site at the 5' end of
the mature FIBAC coding sequence would allow the entire FIBAC
coding sequence to be portable via a H~CiAI/SalI, KpnI, or HindIII
double digestion in plasmid pFib5l, except for another H~iAI site
at position 552 within the coding sequence. This restriction
sitetwas removed using in vitro oligonucleotide-directed, site-'
specific mutagenesis essentially as described by Zoller and Smith
in Methods in Enzymology 100:468.
Single-stranded DNA isolated from a derivative of
bacteriophage mpl8 containing the met-FIBAC gene translationally
coupled to lacZ as described above was annealed to the synthetic
oligonucleotide GGGCTTTGCACCTGGCAG. This oligonucleotide anneals
to the FIBAC coding region across the H~CiAI site with a single
mismatch. The resultant. DNA was then incubated with the Klenow
fragment of E. coli DNA polymerase, T4 DNA ligase and a mixture
of all four deoxy-nucleotidetriphosphates.
The cov,alently-closed, double-stranded phage DNA thus
obtained was used to tra.nsfect E. coli strain JM107. Plaques
were assayed for the presence of the mutant sequence by their hy-
bridization to the mutagenic oligonucleotide shown at~ove at 58°C
in 6x SSC.
The selected clone had the leu codon CTT replacing CTG
at amino acid position 173. The coding region from this clone
was excised as a l3amHI to HindIII fragment and ligated into a
similarly-digested pUC8. The resulting plasmid, pFib55, has all
of the features o:E pFib51 as well as a more easily mobilized
FIBAC coding region.
...
97
-64-
C. Expression Vector pFib56
Alternate methods of translational coupling to beta-
galactosidase or any E., coli protein can be similarly con-
structed. For one embodiment, a clone has been designed which is
similar from a regulatp.on of expression point of view to plasmid
pFib51 (i.e., FIBAC translationally coupled to lacZ in pUCB), but
which uses an alternate translational coupler. To create pFib56,
the following pieces of: DNA were synthesized:
EcoRI end CAI end
' A A T T C C .)1 A G G. A G A A A T A A A T G T G C A 3 '
H~iAI end EcoRI end
5' C A T T T A 't T T C' T C C T T G G 3'
This double-stranded EcoRI/Hc~iAI fragment was then combined with
the ~AI/HindII:L FIBAC coding sequence and plasmid pUCB that had
been digested wit=h EcoRI and HindIII and ligated. The resulting
plasmid has been called pFib56. When this plasmid is transformed
into E. coli strain JM107, the strain, JM107/pFib56, can be in-
duced to express even more methionyl FIBAC than JM107/pFib51 or
JM107/pFib55. From this, it has been concluded that the transla-
tional coupler in pFib5~6 is more efficient than that in pFib5l.
D. ~>ressio:n Vectors pFiblO and pFibll
To direct the expressed FIBAC to the periplasmic space
of E. coli, a leader peptide is added to the amino terminus of
FIBAC. The leader peptide will effect the transport of the fu-
sion protein out of the inner cell membrane. Cellular processing
removes the signal peptide and yields the mature form of FIBAC.
Two signal sequences have been separately fused to
FIBAC for this purpose. They are the leader peptides of _E. coli
ompA and phoS gene products. Both om~AL-FIBAC and phoSL-FIBAC
fusion proteins contain signals to direct them to be located in
the periplasm of E. colil and to allow proteolytic processing of
the fusion to ompA or phoS leader fragments and native FIBAC.
Plasmid pFiblO is a derivative of pUCB into which the
coding sequence for the om~AL-FIBAC fusion protein has been in-
serted. In addition, some 5' nontranslated sequences from the
ompA gene are included. Transcription of this plasmid is
e:~.
1340971
-65-
directed by the lac promoter/operator of pUCB. Translation is
initiated at the methionine codon beginning the ompA leader se-
quence and uses. the Slzine-Dalgarno sequence found in the ompA
gene.
The plasmid was constructed by liga,ting the FIBAC
coding sequence contained on a HgiAI/HindI~I fragment of pFit~55
together with synthetic oligonucleotides encoding the ompA leader
peptide and with EcoR:L/HindIII-digested pUCB. The coding strand
of the synthesized oligonucleotide is:
o-~-'~i.
EcoRI end
5' A A T T C G A T A T C T C G T T G G A G A T A T T C A T G A C
G T A T T T T G G Fv T G A T A A C G A G G C G C A A A A A A T
PvuI
G A A A A A G A C A, G C T A T C G C G A T C G C A G T G G C A
C T G G C T G G T T' T C G C T A C C G T A G C G C A G G C C T
G C A 3'
~iAI end
The s;rnthesis was accomplished as four oligonucleo-
tides, two for each of the strands. Together, the double-
stranded DNA features:
(a) An EcoRI cohesive end at the 5' end and a HgiAI
cohesive end at the 3' end of the coding strands;
(b) An open reading frame encoding the leader peptide
of the ompA gene product which is in frame with
t:he FIB.~C gene when ligated at the HgiAI site;
(c) Dfon-coding sequences 5' to the translated portion
which contain the ribosome binding site normally
found in the ompA gene; and
(d) ~l,n inte:rnal, unique PvuI site.
Plasmi.d pFib:ll is a derivative of plasmid pKK223-3 and
is identical to pFiblO except that its pUC8 portion has been re-
placed by the 4550 by EcoRI/HindIII fragment of plasmid pKK223-3.
In this construct, transcription is directed and regulated by the
hybrid tac promo~ter/ope rator. Additionally, this plasmid con-
tains a transcriptiona:l terminator which may stabilize the ,
plasmid in a high expression system.
> _-.
~~~097'
-66-
E. Expression Vector pFibl3
In plasmid pfibl3, the 5' non-coding region of the
o_mpAL sequence has been eliminated and the om~AL-FIBAC fusion
gene is translationallh coupled directly to the N-terminal por-
tion of the lacZ gene as it appears in plasmid pUCB. This has
been accomplished by li.gating the.EcoRI/PvuI (3200 bp) fragment
of plasmid pFiblO to a synthetic oligonucleotide shown here.
EcoRI end
5' A A T T C C A A G G A G A A A T A A A T G A A A A A G A C A G C
T A T C G C G A T 3'
PvuI end
PvuI end
5' C G C G A T A G C T G T C T T T T T C A T T T A T T T C T C C T
T G G 3'
EcoRI end
F. ~~ression Vector pFib31
The ~hoS gene of E. coli codes for the phosphate bind-
ing protein and has been described and sequenced by Surin, B.D.
et al., in J. Bacteriol. 157:772-778 (1984),
This protein i.s periplasmic, with a
25-amino-acid leader sequence that directs it to that compart-
ment. The leader sequence is proteolytically removed during the
translation process to leave only mature phoS protein in the
periplasm. We have synthesized the phoS leader sequence as two
double-stranded DNA fragments with EcoRI and CAI ends as shown:
EcoRI/ClaI
EcoRI end
5' A A T T C A T G A A A G T T A T G C G T A C C A C C G T C G C
A A C T G T T G T C G C C G C G A C C T T A T 3'
ClaI end
ClaI end
5' C G A T A A G G T C G C G G C G A C A A C A G T T G C G A C G
G T G G T A C G C A ~,~ A A C T T T C A T G 3'
EcoRI end
-67- ~ 34~ 97 1
ClaI/HgiAI
ClaI end HgiAI end
5' C G A T G A G T G C 'T T T C T C T G T G T T T G C G T G C A 3'
HgiAI end ClaAI.e.nd
5' C G C A A A C A C A G A G A A A G C A C T C A T 3'
These fragments were ligated together at a ClaI site internal to
the phoS leader. These fragments were simultaneously combined
with the HgiAI/HindIII 1~IBAC coding fragment described above and
plasmid pKK223-3 that had been digested with EcoRI and HindIII.
The resulting plasmid has been called pFib3l.
RXAMDf.R 4
EXPRESSION OF THE FIBAC GENE IN E. COLI
Three methods have been employed to qualitatively de-.
termine the amount and !'orm of FIBAC produced by _E. coli cells
harboring the plasmids described above. They are
(1) specific reaction of FIBAC antibody to _E. coli
proteins produced following induction of the FIBAC
gene resolved by polyacrylamide-SDS-
el~~ctrophoresis and subsequently bound to
ni~~rocellulose paper (western blotting);
(2) labeling of E. coli proteins with 35S-cysteine,
35;x-methionine, or 35S04 following induction of
the FIBAC gene; and
(3) inspection of polyacrylamide SDS gels containing
E. coli proteins and FIBAC without antibody cou-
pling or radioactive-labeling.
These mEathods have allowed not only comparison of the
amounts of FIHAC produced by each strain, but also purification
of the FIHAC folly>wing expression without the need for functional
metalloproteinase inhibition. All of the plasmids discussed in
Example 8 have been expressed in the background of _E. coli strain
JM107. The expre~;sion of FIBAC in E. coli has so far been
greater in those systems designed to transport the protein out-
side of the inner cell membrane. This is possibly due to degra-
dation of the expressed ;protein in the cytoplasm.
~,: '( ..
-68-
Processing om.~aL-FIBAC fusion protein to yield the ma-
ture form of FIBAC has been found to be partially dependent on
the phase of growth of the cells. Cells induced with IPTG in
early log phase of growth accumulate a mixture of processed and
unprocessed FIBA~" while cell cultures induced in late log phase
accunn~late only processed PIBAC. Strains expressing the phoSL-
FIBAC fusion appear to process the protein completely independent
of growth phase.
All of the expression vectors described above in Exam-
ple 8 have ampic:illin resistance as the selectable marker. For
the purposes of production, it might be preferable to have a
tetracycline resistance marker. One plasmid generally useful as
an expression vector has been constructed with a tetracycline re-
sistance marker. This plasmid is a derivative of pKK223-3 in
which the truncated tetracycline resistance gene has been re- '"
placed with fully functional tetr gene adapted from pBR322.
EXAMPLE 10
PURIFJ:CATION OF FIBAC EXPRESSED IN E. COLI
The recombinant human co.lleagenase inhibitor (FIBAC)
has been purified! from E. coli strain JM107 transformed with
plasmid pFibll. In this strain, JM107/pFibll, FIBAC can be made
to accumulate as an insoluble aggregate. In this example, the
conditions for growth o:E the cells, induction of FIBAC gene ex-
pression, cell harvesting, and purification of FIBAC from the in-
soluble fraction of a total cell lysate are described. The same
protocol may also be used to substantially enrich Fibac from the
soluble fraction of total cell lysate.
A. Insoluble Fraction
Luria broth containing ampicillin at a concentration of
100 ug/ml was innoculatc~d with an overnight culture of
JM107/pFibll to an initp.al OD600 of 0.15-0.20. The shake flask
cell culture was allowed to grow at 37°C to an OD600 of 1.5, at
which time the culture medium was supplemented with IPTG to a
final concentration of 0.5 mM. Incubation at 37°C was then con-
tinued for 2.5 to 3 hours. The cell culture was rapidly cooled
to 4°C in an ice bath and the cells harvested by centrifugation.
A one-liter culture growrn as described above yielded on the
. 9 _.
1340971
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average 3 gr of cells (wet weight). The cells were washed once
in cold lysis buffer (50 mM MES, pH 6.0, 4 mM EDTA) and then
resuspended in th.e lysi;s buffer to a final concentration of 0.26
g cells (wet weight)/ml. The cell suspension was frozen at -70°C
until further processinc3.
Total ceell lysate was prepared by passing the cell sus-
TM
pension two times throuc3h a French pressure cell (SLM-Aminco
model #FA-079 fitted wii:.h piston #FA-073 and operated at 20,000
psti, SLM Instruments, 'Inc., Urbana, Illinois). The resultant
cell lysate was incubatE~d with 10 ug of DNase I/gr of cells (wet
weight) for 2-3 hours on ice. The cell lysate was then divided
into small aliquots and stored frozen at -70°C until further pro-
cessing.
A cell lysate supernatant and cell lysate pellet frac-
tion were obtained by centrifugation of a 5 ml portion of cell~~
lysate for 30 minutes ai: 4°C in an Eppendorf micro centrifuge.
The resultant pellet was washed twice with 3 ml of 50 mM
Tris-HC1, pH 8.0, 4 mM EDTA, 50 mM DTT. The supernatants from
these washes were poolec! and saved for further analysis. The
washed pellet was then solubilized by resuspension in 3 ml of 50
mM MES, pH 6.0, 4 mM EDTA, 50 mM DTT, 10 M urea, and incubated at
room temperature for 15 minutes. Should protein carbamylation
occur due to urea solubi.lization the side reaction could be
quenched by the addition of a one hundred fold excess of a suit-
able nucleophile over total protein amino groups. The resultant
solution was clarified by centrifugation for 15 minutes at 4°C in
an Eppendorf micro centrifuge. The supernatant contained essen-
tially all of the protein from the solubilization procedure and
was saved for further analysis. The remaining small pellet con-
sisted mostly of cell wall debris and few proteins (none of which
were FIBAC). This was discarded.
The identification of FIBAC in the various fractions
prepared as described above was accomplished by SDS-PAGE and
probing of western blots with anti-FIBAC antibodies. SDS-gel
analysis of total cell l.ysate protein obtained from IPTG-induced
JM107/pFibll shows the presence of a protein band of approxi-
mately 20,000 dalton apparent molecular mass that is absent in '
134087 ~
_70_
the gel pattern From a cell Lysate of non-included JM107/pFibll.
The 20,000 Da protein and a faster migrating band, presumably a
degradation product of FIBAC, react with anti-FIBAC antibodies in
the western blot analysis. The IPTG induction-dependent presence
of this protein, the molecular weight, and the reactivity with
anti-FIBAC antibodies suggest that the protein represents the ex-
pressed recombinant FIB,AC. Analysis of the cell lysate
supernatant fraction obtained from IPTG-induced JM107/pFibll and
the cell lysate pellet wash revealed little FIBAC. The bulk of
the FIBAC was found, however, in the urea-DTT solubilized cell
lysate pellet fraction. This was interpreted to mean that, upon
IPTG induction, the FIBAC accumulates an insoluble fraction and
can be isolated in a substantially purified form .from the washed
cell lysate pellet.
The urea-DTT solubilized cell lysate pellet was used~as
the starting material for the CM-chromatography. A 1.2 ml ali-
quot of solubilized cel.'L lysate pellet (16 mg protein) was di-
luted to 25 ml with cold CM-buffer (50 mM MES, pH 6.0, 6 M urea,
14 mM 2-ME). The samplsa was then applied to a carboxymethyl cel-
lulose column (25x130 mnn) previously equilibrated with CM-buffer
at 4°C. After sample application, the column was washed with
CM-buffer until the A28C1 returned to baseline. Adsorbed protein
was eluted with a linear sodium chloride gradient (0-200 mM) in
CM-buffer. Total gradient volume was 400 ml, flow rate was
26 ml/hr, and 5 ml fractions were collected. The "flow-through"
fraction (CM-FT) and ths: peak fractions 58-61 were pooled and
analyzed by SDS-PAGE. 9fie electrophoretic and immunological
analysis of these fractions revealed that the recombinant FIBAC,
including some degraded FIBAC, eluted at approximately 120 mM
NaCl without any other detectable proteins. Under the
chromatographic conditions employed, most non-FIBAC proteins did
not adsorb to the CM-column and were found in the "flow-through"
fraction.
The amount of CM-purified FIBAC obtained in this proce-
dure was estimates to represent 1.3% of the total cell protein.
Bradford protein assays were used for quantitation throughout the
isolation and purification procedure.
1340971
-71-
Two ml (100 ug) of purified FIBAC in 50 mM MES, 6 M
urea, 14 mM 2-mercaptoethanol were concentrated to 200 ul by
Centricon centrifugation and adjusted to pH 8.5 by addition of
2 M Tris-HC1, pH 8.5 to a final Tris concentration of 0.5 M. The
cysteine residuee~ of FIBAC were then carboxymethylated using
3H-iodoacetic acid. The alkylation reaction mixture was desalted
by reverse phase HPLC. The modified FIBAC eluted at 30$
acetonitrile and was collected for further analysis. An aliquot
of the modified F'IBAC isolated from the HPLC was subjected to
SDS-PAGE analysis.. Comparison of the CM-purified FIBAC that was
used for the cart~oxymetlzyla'tion (starting material) and the FIBAC
after alkylation and HPLC desalting showed that the modified
FIBAC migrated slightly slower in the SDS gel than the non-
modified FIBAC. This is not an unusual observation, particularly
in view of the substantial number of modified cysteines present,
in FIBAC.
The carboxymethylated FIBAC was then applied to an
Applied Biosystems (model 470A) gas-phase protein sequencer (Fos-
ter City, CA) for automatic Edman degradation, and the amino acid
sequence for the first :>.4 residues was identified. The
sequencing data for the first 6 cycles of the Edman degradation
are shown in the table below. The data clearly establish that
the N-terminal amino acid sequence of the purified FIBAC
(C-T-V-P-P...) is identical to that previously determined for na-
tive FIBAC. It is thene:fore concluded that pFibll properly pro-
cesses the recombinant f'IBAC by cleaving the ompA-FIBAC fusion
protein at its ala-cys junction to produce the mature form of the
FIBAC protein.
N-TERMINAL AMINO ACID SEQUENCE ANALYSIS OF PURIFIED
RECOMBINANT FIBAC
(cysteine residues were labeled with 3H-iodoacetic acid prior to
sequencing of the protein)
~34097~
-72-
CYCLE 3H--CPM PTH-AA IDENTIFICATION
1 11470 CYS
2 385 THR
3 12 fi 90 CYS
4 :3 3 9 VAL
1.45 PRO
6 :!55 PRO
B. Soluble fraction
Because of the additional contaminants in the starting
material, the procedures discussed herein does not initially
result in a homogeneous. preparation of FIBAC. However, the pro-
cedure does provide sufficient purification to allow refolding of
FIBAC to its native cor,~formation. Subsequent purification steps
may then be used to complete the isolation procedure.
In this example, the cell growth, induction, harvest
ing, and preparation of a total cell lysate were as in the previ-
ous example. In order to demonstrate the ability of the present
procedure to purify FIBAC from any fraction of the cell lysate,
the original cent=rifugal fractionation of the homogenate was
omitted. Instead, the total cell lysate was made to 10 M urea, 4
mM EDTA, 50 mM D'.CT, 50 mM MES, pH 6.0 and incubated at 22°C for
minutes. The solution was then centrifuged for 15 minutes in
an Eppendorf microcentrifuge at 4°C. The pellet was discarded
and the supernatant diluted with CM-buffer (50 mM MES, pH 6.0,
6 M urea, 14 mM :>.-mercaptoethanol) and chromatographically frac-
tionated on carboxymethyl cellulose as in the previous example.
FIBAC eluted at approximately 120 mM salt along with some de-
graded FIBAC and several immunologically non-related
contaminants. Estimation of the purity of FIBAC by SDS-PAGE
analysis showed !.t to be greater than 50 percent of the total
protein in this fraction. At this level of purity, it is possi-
ble to refold they FIBAC to its native conformation using the
refolding procedure below. The refolded FIBAC is fully function-
al as a metalloprotease inhibitor and may be further purified by
anion exchange chromatography.
Anion e~xchang~e chromatography was effected on a column
TM
10x100 mm of What.man DE~-52 (Whatman Inc., Clifton, New Jersey)
, ,
1340971
-73-
equilibrated in 600 mM urea, SO mM Tris, pH 9.6. The solution
containing the impure, refolded FIBAC was titrated to a pH of 9.6
by drop-wise addition of 5 N NaOH and applied to the DEAE cellu-
lose. Analysis of the flow-through fraction demonstrated that
the FIBAC was not retained. The immunologically non-related
contaminants bound to t:he matrix and were thereby removed from
the solution. The flo4r-through fractions can be concentrated on
a CM-cellulose column as shown below.
The flow-through fraction from the anion exchange col-
umn was titrated to pH 7.5 by the addition of 5 N HC1. This so-
lution was applied to a CM-cellulose column (25x130 mm) previous-
ly equilibrated with 600 mM urea and 50 mM Tris, pH 7.5. The
column was washed with this same buffer until no protein could be
spectrally observed to elute. The FIBAC was then eluted with the
above buffer made to 250 mM in NaCL. The protein peak was poo~,ed
and dialyzed to equilibrium against 50 mM Tris, pH 7.5.
Electrophoret.ic, immunological, and functional assays
of the resulting FIBAC demonstrate an active, refolded col-
lagenase inhibitor of greater than 90% purity. The only de-
tectable contaminant is a FIBAC degradation product as in the pu-
rification from the cell lysate pellet. Because of the identical
amino terminal sequence of this contaminant and its apparent mo-
lecular mass, it has been concluded that a proteolytic clip has
been made close to the carboxy terminus. This material can be
removed in further purification steps (e. g., higher resolution
ion exchange chromatography or affinity chromatography of the
refolded FIBAC).
EXAMPLE 11
CONSTRUCTION OF A YEAST FIBAC EXPRESSION CLONE
Another organism in which gene expression and protein
export vectors have been constructed is the yeast Saccharomyces
cerevisiae. The yeast alpha-factor is a mating hormone which. is
produced intrace:Llularly and exported to the growth medium. A
single peptide sequence directs this transport. The FIBAC coding
sequence has been cloned into a yeast expression vector to create
a fusion of the breast alpha-factor leader sequence to FIBAC. A
construct was first made as a derivative of p~S385. Plasmid
pGS385 has the following features:
1340971
-74-
(a) I:t contains portions of the yeast alpha-factor
gene including the promoter, leader peptide,
polyadenylation signal, and transcriptional termi-
nation aignals;
(b) T'he portion of the alpha-factor gene between the
two most distant HindIII sites has been deleted,
creating a unique HindIII site;
(c) It contains a unique SalI site 3' to the HindIII
site; and
(d) It has t:he pBR322 origin of replication and
ampicill.in resistance gene to allow replication
and selection in E. coli.
The plasmid F>GS385 was digested with HindIII and SalI.
The HindIII site defines the carboxy terminus of the alpha-factor
leader sequence. The ~;ynthetic octanucleotide 5'-AGCTTGCA-3' was
used to bridge the alpha-factor C-terminus to the N-terminus of
FIBAC. The alpha-factor transcription-translation sequences
drive gene expression in this vector. The entire alpha-factor-
FIBAC fusion was then removed by digestion with EcoRI and in-
serted into plasmid YIPS, a derivative of plasmid pBR322, con-
tains the yeast ura3 gene. This plasmid is suitable for use as
an expression vector following digestion at a unique StuI site in
ura3 and transformation into S. cerevisiae to direct integration
of the entire plasmid into the chromosomal ura3 locus. Such
integrants of an alpha-FIBAC fusion derivative of YiP5 have been
obtained and herE~ produced and secreted immunoreactive material
as determined by colony screening techniques.
FYnMDT.F 1 7
EXPRESSION OF RECOMBINANT FIBAC IN ANIMAL CELLS
Two expression systems for the production of FIBAC in
animal cells are proposed. The first incorporates the SV40 late
promoter to direct transcription in COS-1 cells. This expression
system is primarily useful for studying the expression, protein
synthesis, post-t:ranslational modification, and transport of
FIBAC in COS-1 cells. .Although the system is~rapid and conve-
nient, it is limited to the COS-1 monkey cell line. The SV40 ex-
pression plasmid will be a derivative of pJC119, the construction
''~:Y~ a
134097 ~
- 75 -
of which is described in derail by Sprague, J., Condra, J.H., Arnheiter,
H. and Lazzarini, R.A., in J. Virol. 45:773-781 (1983). The complete
FIBAC coding region, including the naturally-occurring signal sequence,
has been assembled from the partial cDNA clones. The NcoI site coinciding
with the initiator methionine for the leader peptide will be linked via a
short synthetic olic~onucleot~ide to the unique XhoI site in PJC119. The
entire FIBAC coding region <~nd 3' nontranslated sequences are inserted at
this site where transcription is directed by the SV40 late promoter. The
plasmid would thus contain 3V40 origin of replication, the pBR322 origin
of replication and the ampicillin resistance selectable marker.
The second system would be preferred for production of FIBAC
in animal cells because it will result in the stable and continuous ,
expression of FIBAC from a human cell line. This vector will be a
derivative of pBPV5~:-1, described by Florkiewicz, R.Z., Smith, A.,
Bergmann, J.E., and Rose, J.K. in Cell Biol. 97:1381-1388 (1983). This
plasmid features the bovine papilloma virus origin of replication, pBR322
origin of replicatic>n, beta-lactamase gene, and the 69o transforming
fragment of BPV DNA. The S'J40 origin of replication and the SV40 early
promoter will be cloned into this plasmid. Sequences from pBR322 that
interfere with the replication of the vector in human cells will be
excluded. As with the previous plasmid, the entire coding portion of the
FIBAC cDNA will be inserted so as to direct its transcription by the SV40
early promoter.
It is expected that purification of FIBAC from the medium
following expression and secretion from these cells will be possible
essentially as described previously in Examples 4 and 5.
EXAMPLE 13
REFOLDING FIBAC
Two assays have been used to monitor the refolding of FIBAC.
Both assays measure the apps=_arance of the functional capacity of FIBAC as
its native structure:. The :First assay is an inhibition assay which
measures the inhibitory efff=_ct of the
W ~
134097 ~
-76-
sample on the ability o:E human fibroblast collagenase to degrade
14C-Labeled collagen. 'the second assay is a modified ELISA which
measures the binding of the refolded FIBAC to human collagenase.
The collagenase binding ELISA is the primary assay by
which FIBAC activity was detected. Here, collagenase is coated
overnight at 4°C in 96-well Immulon II plates (1.0 ug./ml in 50 mM
Tris, pH 8.2, 5 mM CaCl~~; 100 ul per well). After the wells are
blocked for 45 minutes with 150 ul/well of 3% BSA in washing
buffer (50 mM Tris, pH 7.5, 5 mM CaCl2. 0.02% Tween-20), varying
dilutions of FIBAC standards or unknown samples diluted in
blocking buffer are pipe~tted into the wells (100 ul/well). Fol-
lowing a 45-minute incubation period (37°C), the wells are washed
three times with washing buffer. Affinity-purified rabbit and
anti-FIBAC is added to the wells (diluted 1/100, 100 ul/well) and
incubated at 37°C for 45 minutes. The wells are again washed and
alkaline phosphat.ase-conjugated goat anti-rabbit IgG (Sigma, di-
luted 1/1000 in washing buffer) is then added to the wells
(100 ul/well). Following a one-hour incubation period at 37°C,
the wells are waslZed a last time and alkaline phosphatase sub-
strate (Sigma #104-105, 1 mg/ml in 10% diethanolamine, 100 mM
MgCl2, pH 9.8) is added to the wells. Color development is moni-
TM
tored at 495 nm uaing a Titertek Multiskan MC ELISA reader (Flow
Laboratories). N<itive FIBAC serves as a standard curve against
which unknown samples may be quantitated.
In the collagenase inhibition assay, 14C-labeled guinea
pig skin collagen pellets (25 ul/pellet = 2100 cpm) is digested
with 50 microliters of trypsin-activated collagenase (approxi-
mately 75 ug/ml in 50 mM Tris, pH 7.5, 10 mM CaCl2), which re-
leases 14C into solution. After incubating the pellets for 1-3
hours (depending on the rate of digestion), the reaction is
stopped by adding 100 ul of Tris buffer and centrifuging for
minutes at 10,000 rpm. The supernatant is then pipetted into
scintillation vials containing 3 mls of scintillation fluid and
counted. Preincubation of the collagenase with 50 ul of varying
dilutions of standard or purified FIBAC prior to adding the solu-
tion to the collagen pellet should inhibit digestions of the col-
lagen and the subsequent release of 14C into solution. The
'34087 ~
_7,_
quantitation of inhibitory activity of an unknown sample depends
on the amount of active collagenase used in the assay. From
this, the activity of the unknown sample may be calculated by
assuming a 1:1 molar ratio between inhibitor and enzyme.
Using these assays, the efficiency of the refolding
process with respect to protein concentration, oxidised
gluthathione concentration, pH, and temperature has been exam-
ined. While all combinations of these parameters have not been
exhaustively examined, a procedure has been developed which
allows efficient renaturation.
The refolding of FIBAC depends greatly on the concen-
tration of FIBAC at which refolding is performed. Under
oxidizing conditions, dp~lute solutions prevent the formation of
interchain disulfides, which eventually lead to the precipitation
of aggregates. ~ "
Purified recombinant FIBAC can be refolded and remains
soluble with 100% recovery of protein by following the protocol
described below:
(1) Dilute, purified FIBAC (less than 300 ug/ml) in
6 Id urea, 50 MES, pH 6.0, 14 mM 2-mercaptoethanol,
is incubated in the presence of 70 mM oxidized
glutathione.
(2) After a 10-minute incubation period at room tem-
perature, the sample is diluted ten-fold with
50 mM Tris, pH 9Ø
(3) The sample is then incubated overnight at 4°C.
SDS-PAG1: analysis of this reactivated material indi-
cates the presence of both intact and degraded FIBAC. The de-
graded FIBAC is carried through from the purification procedure
and does not appear to degrate further during the reactivation
procedure.
This sol.ubilized FIBAC has been demonstrated to have
both collagenase binding activity (ELISA) and collagenase inhib-
itory activity. The amount of activity relative to the amount of
protein appears to be greater than 90% as determined by the col-
lagenase inhibition assay. This number was derived from calcula-
tions based on the estimated amount of collagenase in the assay.
1340971
_,8_
Although this is an estimate, it is not believed it to be off by
more than 50%. l3inding activity as measured against the FIBAC
standard has enabled us to monitor the relative reactivation of
different sample:.
The re:Eolding process has also been shown to work on
50% pure FIHAC preparations. The nature and amount of
contaminating protein that can be tolerated is still uncertain,
however, some act=ive FIBAC has been detected in total cell
lysates without purification demonstrating that at least some
yields are obtainable at less than 5% purity.
It will. be apparent to those skilled in the art that
various modifications and variations can be made in the processes
and products of t:he present invention. Thus, it is intended that
the present invention cover the modifications and variations of
this invention provided they come within the scope of the
appended claims and their equivalence.
._s
,.
