Note: Descriptions are shown in the official language in which they were submitted.
~.-:. 2043953
Title: Method for the isolation and expression of a gene
which codes for streptokinase, nucleotide sequence obtained,
recombinant DNA and transformed microorganisms.
The present invention relates to the field of
biotechnology and genetic engineering techniques and in
particular a method for the isolation and cloning of a novel
nucleotide sequence which codes for a streptokinase, as well
as the recombinant DNA obtained therefrom which is used for
the transformation of different host organisms.
Streptokinases are plasminogen activators from
prokaryotic. cells, which are usually secreted into the culture
medium by a large number of haemolytic Strepto oc ~ of
different serotypes. Being proteins of bacterial origin, some
antigenic responses to them have been detected
(Dykewies, M.S. et al., 1985 and McGroth, K.G. et al., 1985).
Their molecular weight is approximately 97,000 dalton.
The function in the pathogenicity of tr p~~oCGi is not
known exactly, although potentially it must contribute to
elimination or avoidance of the formation of fibrin barriers
around the infection.
During interaction of streptokinase with the plasminogen
which is in human plasma, it was found that the protein is
capable of converting the latter to plasmin, which displays
proteolytic activity and can degrade fibrin clots into soluble
products.
Streptokinase, urokinase and tissue-type plasminogen
activator are at present used~as thrombolytic agents in the
treatment of disorders which collectively represent one of the
greatest causes of death in the world, such as myocardial
infarct, pulmonary, arterial or venous thromboembolism,
surgical complications and other cases of thrombosis.
There are physicochemical and immunological differences
anal differences in rESpect. of .~~ubstrate specificity which beat
witness to molecular heterogeneity of streptokinases of
A
. . . _ . .. .. . .. .. ~04~~~~ ..
2
different origins, although they are all closely related in
respect of function.
Combined with the low yields obtained in production of
the protein and the pathogenicity of its natural host, the
,tre~tQcoccus strains used for the industrial production of
streptokinase secrete other products into the culture medium,
such as deoxyribonucleases, streptolysin br hyaluronidase and
proteases, which makes the process of purifying the desired
protein difficult. On the other hand, it has not yet been
possible to obtain genetically improved strains from these
hosts due to the lack of a developed methodology for the gene
transfer.
It is due to these drawbacks that the cloning of
different isolated genes which code for these proteins has
been attempted, using prokaryotic and eukaryotic hosts.
The German Democratic Republic patent no. 249493,
IPC: C12 N 15/00, describes the cloning and expression of a
gene which codes for streptokinase from strain H46A of
Streptococcus ~cuisimilis, belonging to Streptococcus type C,
using ~. coli bacteria as the host, wherein levels of
excretion into the medium from 0.1 to 1.8 mg/1 are obtained,
depending on the age of the culture.
The coding sequence for this gene, which was called SKC,
was subsequently determined, including its signal peptide, as
well as the identification of the primary structure of
adjacent regions involved in the control of transcription and
translation of said gene (Malke, H. et al., 1985).
Other nucleotide sequences of genes which code for
streptokinases from Streptococcus types G and A have been
characterised (Water, F. et al., 1989), In particular, the
gene which determines streptokinase type A (gene SKA) from
strain 49 type M of Streptococcus pio,genes was cloned and
expressed in strain JM109 of ~, coli and in Streptococcus
sanauis Challis 57E (Huan , T.T, et al., 1989). In both cases,",;
.. . .,. ..~.. ... ... . , .. _. , , , ... ..,~.,.,,:»~.~~~ . .., .... ... ,.
.. ,, .. , . . . .. . . . . .
protein levels of 0.64 mg/1 and 40 ~.g/1 respectively were
produced. In the case of ~. coii, 94$ of the'protein recovered
,,, . .
.204393 _
3
was in the periplasmic space and 6~ in the cytosol, whereas in
pan uis all the enzyme was found extracellularly. Moreover,
many clones producing streptokinase in ~. coli were very
unstable, as in some cases the SKA gene was deleted or the
host cells died owing to some lethal activity of the gene
product in question.
In the case of ~. s~"uis, the protein molecule obtained
was approximately 3,000 dalton less than native streptokinase
and the absence of 32 amino acids from the C-terminal end was
detected; however, biological activity was not affected
(Huang, T.T. et al., 1989).
Subsequently, conclusive results were obtained with
respect to the difficulty of cloning and expressing the
isolated SKC gene in ~. coli, it being found that the gene
product interferes with the normal physiology of the host,
which is shown by the mucosity of the cells which carry this
gene, by the incomplete export of streptokinase into the
periplasmic space, by the structural instability of the
.plasmids which carry the SKC gene and which are designated for
the expression of high levels of the protein, as well as by
the drawbacks encountered in cloning streptokinase genes from
additional serotypes of Streptococcus in plasmids of ~. _roli
and unsuccessful attempts to express heterologous genes under
the control of expression-excretion signals of the SKC gene
itself (Muller, J. et al., 1989).
More recently the company Phillips Petroleum (patent
DD257646 IPC: C 12 N 15/00, and Ha
, ggenson, M.J. et al., 1989)
has obtained the expression of~~streptokinase in the
methylotrophic yeast Pichia pastoris, under the control of
gene regulatory sequences of alcohol oxidase, wherein yields
of the desired product using continuous fermentation of the
order of 77-250 mg/1 culture medium were obtained, with an
. intermediary cell density of 46 g/1. This system uses the
~KG~ge~ne,- contained i~n the vpla'si~~ti ~~MFS "Which is'''lice~rised' to
this company by Dr. J.J Ferretti of Oklahoma University, USA,
in the expression vectors.
2043953
9
The controllability of the system makes it attractive,
taking into account that it can easily be repressed by using
glucose or glycerol and induced with methanol; nevertheless,
its application is limited to this host.
It is the object of the present invention to obtain high
levels of streptokinase yield in different host systems, by
the use of expression vectors which carry a novel nucleotide
sequence which represents a genetic variant not described
before and which codes for a bioactive streptokinase, which
contains the active portion corresponding to streptokinase
from ~t~rP.~gt-ococcus ~gllssims>>s of type C, strain ATCC-9592.
The novel isolated gene is called SKC-2, and codes for a
protein of the same molecular weight as the one encoded by
SKC; it has the fundamental characteristic of stability in
vectors of E_. soli and yeasts, adverse effects on cells growth
or viability not being found in a single case, which makes it
possible to obtain yields greater than those reported up to
now in both hosts, in respect of the product obtained, which
indicates a streptokinase different to those known up to nova,
with the desired biological activity.
A
s ,
2443953
4a
Drawings: The invention will now be described with reference
to the following drawings:
Figure 1. Plasmid pEKG-3, which carries the sequence of the
SKC-2 gene under the tryptophan promoter of E. coli
and displays at the 3~ end of the gene the signal
for termination of bacteriophage T4 to give greater
stability to expression.
Figure 2. Comparison between the amino acid sequences derived
from the base sequences of the genes SKC-2, SKC,
SKG and SKA.
Figure 3. Plasmids pPESKC-4 and pPISKC-6, for extracellular
and intracellular expression respectively in P.
pastoris.
Figure 4. Relative positions of DNA fragments SK1, SK2 and
SK3.
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2043953
The present invention relates to a method for the
isolation and expression of a novel nucleotide sequence
corresponding to the SKC-2 gene, the product of which is a
protein of approximately 97,000 dalton. Said protein belongs
to the streptokinases, which are distinguished by their
fibrinolytic activity.
The present invention also relates to this gene, l.he
sequence of which corresponds to seq. id. No. 1 in the
Sequence T~isting.
It was obtained from the genome of the ~ re~tocsccus
eauis~.rnilis type C strain (ATCC-9592) by gene amplificat.ian
using the polymerase chain reaction (PCR) from three synthetic
oligonucleot.ides called SK1, SK2 and SK3 having the seyuer~c
S..K1 5' . ... TGG1AATTCAT.G11AA1~~'~'1~~',TTj~T,CT . . . 3_' sect . id . Ne .
2
SK2 5'...'TGGATCCTTATTTGTCGTTAGGGTTATC...3' seq. id. Nm. i
SK3 5'...GGnATTCATGATTGCTGGACCTGAGTGGCTG...3' seq. id. No. 4
JJ:lcd
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2043953
and constitute a novel aspect for this method. The relative
positions of the DNA fragments SK1, SK2 and SK3 are depicted
in Fig. 4. These primers further carry restriction sites
which are not found in the gene and which allow direct cloning
5 in an expression vector. On the other hand, the SK3 which
hybridises at the 5~ end carries an ATG which acts as a site
for initiating translation and removes the signal peptide of
SKC-2. The three oligonucleotides were synthesised from the
SKC sequences published by Malke et al., 1985; and with them
were marked the boundaries of the exact fragment of the gene
which codes for the mature protein or the complete gene
including the signal peptide.
Another novel aspect of this method is the
possibility of expressing the isolated gene in both bacteria
and yeasts, high levels of expression being achieved in both
cases.
The present invention also relates to recombinant
DNA which includes the SKC-2 gene, such as vectors for the
expression of this gene, in bacteria pEKG3 (Fig. 1), and in
yeasts pPESKC-4 and pPISKC-6 (Fig. 3). In particular for
expression in ~. coli, the SKC-2 gene with or without its
signal peptide is cloned under the tryptophan promoter and
with the transcription termination signal of phage T4. For
yeasts there was used an expression vector in which the SKC-2
gene is located behind the signal peptide of sucrose invertase
(SUC2) controlled by the alcohol oxidase gene promoter (AOX1)
of ich' a pastoris and which carries the termination signal of
the glyceraldehyde-3-phosphate dehydrogenase (GAPt) gene of
Saccharomyces cerevisiae for the extracellular expression
variant, this vector being called pPESKC-4. For the
intracellular expression of SKC-2, the vector pPISKC-6 is used
which does not contain the signal peptide of SUC-2 behind the
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2043953 _
5a
AOX1 promoter, and this is obtained by inserting the SKC-2
gene in the expression vector pNAO (kindly provided by Muzio,
OIGB, Havana, Cuba) (Fig. 3). The HISS gene of S. cerevisiae
is used as a selection marker in both vectors, and the
expression cassette referred to above
JJ:lcd
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6 2043953
is flanked by 5' and 3' sequences of the AOX gene of ~ hia
gast-oris for integration.
The present invention also relates to the microorganisms
resulting from transformation of ~.. ,~oli strain 4V3110 with
vector pEKG3, and to mutant MP-36 of P.ichia ~asroris
with expression
vectors pPESKC-4 and pPISKC-6, which axe characterised as
expressing high levels of streptokinase, and having good
viability (the product has no adverse effects on cell growth)
and high stability of the strains transformed.
The transformed ~.. coli clone was called HSK-M and
exhibits levels of expression of the product of the SKC-2 gene
greater than 350 mg/1 culture medium..
The transformed Pichia has oris strains MSK-M9 and MSK-M6
produce streptokinase levels intracellularly and
extracellularly respectively which vary between 1.0 and
1.2 g/1 culture medium.
The method described in the present invention, given the
levels of expression never before reported for this product,
makes it possible to reach optimum purity thereof for its
administration to human beings and animals, without the need
to develop a complex and costly process for purification.
Examples
The following examples are intended to illustrate but not
limit the invention. ~. ~oli and ~i~hia ~~storis are used as
host systems in these examples; nevertheless, other eukaryot.i.c
and prokaryotic cells can be~used for the method described in
the present invention.
Fxamg,~e 1
For the isolation of genomic DNA of ~trPp~~occus
~,L~imilis of type C, strain ATCC-9542 was used as a source
thereof for cloning the SK gene. The cells of ~~rPnrococcus
aauis.;mills were =grown -in Br..aHin Heart .Infusion .Medi..um .(GLBCO)
at 240 r.p.m. for 12 hours at 37°C in 5-ml pre-cultures acting
as an inoculum which was grown for 12 hours in a 300-ml
*Trade-mark
,~ 7 203953
Erlenmeyer flask. The cells were collected by centrifugation
at 3,000 r.p.m. and resuspended in 8 ml lyse (4.5 g glucose,
1.$6 g EDTn, 1.51 g tris-HC1, in 500 ml sterile water, pH=8),
80 ~1 of lysozyme were added at a concentration of 10 mg/ml,
and the suspension was incubated for 30 minutes at 37°C. Next,
to obtain efficient cell rupture, 500 ~1 pronase (Boehringer),
1 ml SDS at 10~ and 200 ail EDTA, 0,5 M, pH=8, were added and
the suspension was incubated for 2 hours at 50°C with smooth
agitation. Successive treatments with phenol, phenol-
chloroform and chloroform were carried out, and the genomic
DNA was precipitated with absolute ethanol and NHqAc" 7.5 M.
The yield obtained was 100 Hg per 300-ml Erlenmeyer
culture flask. The presence of the gene which codes for
streptokinase was verified by the Southern blot technique
(Maniatis et al., 1982).
For subcloning in bacteria, 1 ~g genomic DNA of the
~trPntococcus Pau~s~m>>;s type C strain (ATCC-9592) was taken
and the gene which codes for SKC-2 was amplified by a PCR
(Randall et al., 1988) using the oligonucleotides SK1 and SK2
for cloning the gene with its signal peptide and SK2-SK3 for
cloning without it.
In each reaction 100 pmol of each oligonucleotide,
2 units of Taq polymerase (Perkin Elmer, USA) and 200 )unol of
each dNTP were used and the reactions were performed in
10 mM MgCl~, 100 mM dTT, 10 mM NaCl and 100 ~g/ml gelatine.
Thirty amplification cyc~.es were performed, wherein in
each one the reaction was incubated at 95°C for 1 minute for
denaturisation, at 52°C for 95 seconds for hybridisation of
the oligonucleotides and at 70°C for 80 seconds for extension.
An efficiency greater than 5~ of amplification was obtained.
For cloning in bacteria (F. co i), a new genetic
construct was used, wherein the tryptophan promoter of E_. coli
and the termination signal of bacteriophage T4 are used.
A
8 2043953
The fragments amplified in the PCR were digested with BamHI
and ligated with the vector ptrip-N_coI-S1-CHI
(Estrada et al., 1988). This construct was transformed into a
preparation of competent cells prepared according to
Dagert et al. (1979) and Hanahan et al. (1983), of ~. coli
strain HB101 ((rg-mg-), sunEq4, ara-19, aalK-2, cYl, proA2,
rnsL20, (StrR) , ~cyl-5, ~1-5, ~1-1, recAl3 } , which had a
frequency greater than 10~ transformants per g DNA.
The colonies obtained were applied to plates of LB medium
(10 g/1 trypton, 5 g/1 yeast extract, 10 g/1 sodium chloride)
and 50 ~tg/ml ampicillin, and hybridised according to
Maniatis et al. (1982), using as a probe the fragment
resulting from the amplification in the PCR, which was marked
using dATP32 (Amersham, UK) and the Klenow fragment of DNA-
polymerase I of ~. ~oli (Maniatis et al., 1982) in whatman 541
filters for 30 minutes at 37°C, the reaction being terminated
by EDTA and heat. 4~ of the colonies were positive clones,
which were examined by restriction analysis and had the same
pattern of digestion with more than 10 restriction enzymes;
moreover the positive clones were checked by double chain DNA
sequencing (Sanger et al., 1977), using therefor an oligo-
nucleotide of 17 bases (5'...ATCATCGAACTAGTTAA...3', seq. id.
No. 5) which hybridises at the 3' end of the promoter, with
which it was corroborated that joining of the latter to the
SKC-2 gene was as desired.
The selected clone was called pEKG-3 (E'ig. 1), which was
subjected to fermentation to realise the characterisation of
the product.
The plasmid of clone pEKG3 was purified using a CsCl
gradient and the sequences of the SKC-2 gene were established,
each time using 2 g of plasmid, and using the oligonucleotides
which appear below as primers:
SSK-O1 5'...GAATCAAGACATTAGTC...3' seq. id. No. 6
SSK-02 5'...GTGGCGCGATGCCAC..,3' seq. ld. NO. 7
SSK-03 5'...GCAACCATTACTGATCG...3' seq. id. No. 8
SSK-04 5'...CCAGTACAAAATCAAGC...3' seq> id. No. 9
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,~~ 204395
9
SSK-05 5'...CTAGCTATCGGTGACAC...3' seq. id. No. 10
SSK-06 5'...CAGAGATCAGGTCAG...3' seq.~id. No. ll
SSK-07 5'...GTTAAGAGCTGCTCGC...3' seq. id. No. 12
SSK-08 5'...CCAGTTAAGGTATAGTC...3' seq. id. No. 13
SSK-09 5'...TCTCGTTCTTCTTCGG...3' seq. id. No. 14
The protocol followed was basically according to
Sanger et al. (1977), and dATP32 and S35dATP (Amersham, UK)
were used.
Fig. 2 shows a comparison between the amino acid sequence
derived from the base sequence of SKC-2, and those of the
genes SKC (Malke et al., 1986), SKA and SKG (Water, F. et al.,
1989) .
The plasmid pEKG3 was transformed in several E,, coli
strains such as W-3110, JM-101, LE392 and MC-1061 and the
expression of streptokinase was compared between them. The
best results were obtained with strain W3110 (F- supF suoE
~R g~",],K ~gR metB ~Y tonA), owing to which it was selected
to be subjected to fermentation, wherein stable expression
levels greater than 20~ of the total protein content of the
cells were obtained, and 350-400 mg streptokinase per litre of
culture medium were obtained.
ExamBle 3
For subcloning of the SKC-2 gene in yeast, strain MP36 of
Piehia nastori~ was used as the host, and variants were made
for intracellular and extracellular expression from the
plasmids pNAO and pPS7 (Fig. 3), using the signal peptide of
sucrose invertase for the extY~acellular construct, and in both
cases subcloning the gene under the control of the alcohol
oxidase (AOX) promoter, using as the terminator at the 3' end
the termination signal of the glyceraldehyde-3-phosphate
'. dehydrogenase gene of ~. e-erevisiae and a non-coding 3' region
of the AOX gene for integration by homology in the genome of
the ~'e.ast,.... fuxt,~e~;, ,~e.~'~,n,~ ~ ~ ;k~~", ~e ~nco, i
... 35~....,..which w 1~ ' .."~, ..k~ ,9.~ . .." ,.. ~
,~.9.:~~;~,~~~~~.~?~,"~~... ..,....",... , .
as used for selection in strain MP36 his'.
204395"3
The vector pPESKC4 (plasmid for extracellular
expression of the protein) was obtained from the vector
construct pPS7-NcoI-S1 nuclease-phosphatase ligated with the
SKC-2 gene amplified by PCR from pEKG3 with the
5 oligonucleotide SK2 and a new primer which hybridises with the
5' end of the gene and eliminates the ATG which had been
inserted for expression in bacteria. This new primer is a
fragment of SK-3 which does not contain the ATG. In the case
of intracellular expression, pPISKC6 was obtained (plasmid for
10 intracellular expression of the protein) from the vector pNAO-
NcoI-EcoRI-S1 nuclease-phosphatase ligated with the band of
SKC-2 amplified by PCR, with the primers SK2 and SK3 with
which is obtained the exact gene which codes for streptokinase
with an ATG at its 5' end (Fig. 3). Both plasmids were
transformed in strain MP36 his-, using the protocol according
to Cregg, J. et al. (1985).
The positive clones were studies by the Southern
blot method (Maniatis et al., 1982), and out of those which
had the correct integration were selected the most productive
in each case for characterisation of the product.
The expression of recombinant streptokinase obtained
in P_. pastoris extracellularly was 1-1.2 g/1 culture
supernatant, and in case of the intracellular construct more
than 1.0 g/1 culture.
In the construct for extracellular expression, the
glycosylated protein was obtained with a molecular weight
greater than 67,000 dalton, it being corroborated by Western
blotting that it decreases to the molecular weight of native
streptokinase when it is digested with endoglycosidase H.
This was carried out by taking a portion having a
concentration equal to 1 mg/ml in a sodium citrate solution,
0.05 molar, pH=5.5, and denaturing it by the addition of SDS
JJ:1 cd
2043953
10a
(final concentration 0.02%) and heating at 100°C for 10
minutes, then leaving it to cool to ambient temperature and
adding 20 milli-units (mU) of endoglycosidase H (endo H) and
leaving it for 16 hours at 37°C, at the end of which it is
subjected to subsequent heating at 100°C for 5 minutes and 10
mU of endo H are added, followed by 12 hours incubation at
37°C, and it is
JJ:Icd
°',
~,'"'""""
. . . . . . . _ . . . 204~:9..~~ ...
11 '
applied to a 12.5% polyacrylamide ge,l and compared with an
undeglycosylated sample.
The streptokinase produced in .fichia gastoris in both
constructs maintains biological activity, not varying its
affinity for plasminogen and being in fact another variant for
the use of this protein in clinical medicine.
Example 4
To verify the biological activity of the product of the
SKC-2 gene, the pure recombinant streptokinase obtained from
both bacteria and yeast was used for acute and subacute
toxicology tests on rats, wherein satisfactory and acceptable
results were obtained to allow its use in human and animal
therapeutics. Its in vivo fibrinolytic activity was verified
in clinical tests on animals, wherein there was success in
dissolving clots in the coronary and femoral arteries of dogs,
blood parameters being maintained similar to those reported in
the literature with this type of product.
The product of the SKC-2 gene showed a specific activity
of 50,000-100,000 IU/mg, which was measured on plates of
agarose-fibrin (Astrup et al.., 1952), chromogenic substrate
(Friberger et al., 1982) and in vitro clot lysis according to
Westlund et al. (1985).
Exam 1~ a 5
To verify the amino acid sequence derived from the base
sequence of the SKC-2 gene, an analysis was made of the pure
product by high-performance Liquid chromatography in reverse
phase (HPLC-RP), using therefor a C8 4.6 x 250 mm column
(Baker, USA), wherein there was used the gradient 5 minutes at
0% buffer B and up to 90% B in 55 minutes, with buffer A
(trifluoroacetic acid (TFA, Pierce, USA) at 0.1% in distilled
water) and buffer 8 (TFA at 0,.,5% ~.n acetonitrile .
,, , ~ ,(~L.ichrosolv, ,Merck,T, FRG) ),,..,. ~a~"~,~air)in~ :a, flow ,
r,ate",off ,, M , "" , ,. , ""
0.8 ml/min.
."
. _ . _ _ .. 12 ~0 ~3~ j~ .
With the protein with a high degree of purity, the amino
acid sequence derived from the base sequence obtained from the
SKC-2 gene was verified by sequencing it by mass spectrometry.
For this the protein was digested with different enzymes
and with combinations of them. The enzymes used were
chymotrypsin, endoproteinase Glu-C, endoproteinase Lys-C and
trypsin .
From the analysis of the mass spectra of the°peptides
obtained in each of the digestions with the different enzymes,
the map of the amino acid sequence of the protein was
constructed by superposition, which made it possible to verify
that there is in this case 100 correspondence between the
sequence of the SKC-2 gene and the amino acid sequence of the
protein obtained.
Strain deposits
The ~. coli HSK-M [pEKG3] strain, based on the ~. coli
strain W3110 and containing the plasmid pEKG3, was deposited
on June 11, 1990, with the Centraalbureau voor
Schimmelcultures (CBS), Baarn, The Netherlands, and obtained
deposit number CBS 243.90.
Likewise, the Pichia pas oris MSK-M4 [pPESKC-4] strain,
based on the Pir,~.zia gastoris strain MP-36 and containing the
plasmid pPESKC-4, was deposited on June 11, 1990, with the
Centraalbureau voor Schimmelcultures (CBS), Baarn, The ,
Netherlands, and obtained deposit number CBS 244.90.
_ .:. W . . ...~ . , . , ,. ,. . , . . . . . . t ~E~~ ~ : . . .. .., . .. . .
. . . . .. . . .. . .
' . ,.
',: '
13
2043953
R1~ FERENCES
Astrup, 'T., and Mullertz, S., 1952, Arch. Biochem. Biophys.
40: 346-351
Burnett, N.N., 1981, nnal. Biochemistry 112: 195-203
Cregg, J.M., Barringer, K.J., Iiessler, A.Y., and Madden, K.R.,
1985, Mol. Cell. Biol. 5: 3376-3385
Dagert, M., and Ehrlich, S.D., 1974, Gene 6: 23-28
Dykewicz, M.S., McGrath, K.G., Harris, K.E., and Patterson,
R., 1985, Int. Arch. Allergy Appl. Immun. 78: 386-390
Estrada, M.P., Hernandez, O., and de la Fuente, J., Interferon
y Biotecnolog~.a 5: 152-156
Fiberger, P., 1982, J. Clin. Lab. Invest. 92, Suppl. 162:
9 9-59
Hanaha~~, D., 1983, J. Mol. Biol. 166: 557-580
Huang, T.T., Malke, Ii., and Ferrett_i, J.J., 1989, Molec.
Microb . 3 (2 ) : 197-205
Malke, ti., and Ferretti, J.J., 1984, Proc. Natl. Acad. Sci.
USA, 81: 3557-3561
Malke, H., Roe, B., and Ferretti, J.J., 1985, Gene 34: 357-362
Maniatis, T., Frisch, E.F., and Sambrook, J., 1982, Cold
Spring Harbor Laboratory, USA
McGrath, K.G., Zeffren, B., Alexander, J., Kaplan, K., and
Patterson, R.J., 1985, Allergy Clin. Immunol. 76: 453-457
Muller, J . , Reinert, fi . , and Malke, H . , 1989, Journal of
Bacteriology, Apr.: 2202-2208
Randall, K., Gelfond, D.H., Stoffel, S., Scharf, S., t~iguchi,
R., Horn, G.T., Mullis, K.B., and Erlich, H.A., 1988, Science
239: 987-491
Sanger, F., Nickler, S., and Coulson, A.K., 1977, Proc. Natl.
Acad. Sci. USA 74: 5463-5467
Tombin, H., Stahelin, T., and Gordon, J., 1979, Proc. Natl.
Acad. Sci. USA 76: 4350-4354
Walter, F., Siegel, M., and Malke, H., 1989, Nucl. Acids Res.
17 (3) : 1262
Westtund, L.E., and Anderson, L.O., 1985, Thrombosis Research
37: 213-223
Haggenson M.J. et al., Enzyme Microb. and Technol. 11,
650-656 (1989)
~4 20,4,3953
SEQUENCE LISTING
SEQ ID NO:1
SEQUENCE TYPE: Nucleotide with corresponding protein
SEQUENCE LENGTH: 1245 base pairs
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: genomic DNA
ORIGINAL SOURCE ORGANISM: Streptococcus equisimilis from group C
of Lanfield definition
IMMEDIATE EXPERIMENTAL SOURCE: ATCC-9542 strain
FEATURES: from 1 to 1245 by mature peptide
PROPERTIES: Streptokinase gene
The gene product binds to human plasminogen
The gene product is an activator of human plasminogen
ATT CTG 48
GCT CTA
GGA GAC 96
CCT CGT
GAG CCA
TGG TCT
Ile GTC
Ala AAC
Gly AAC
Pro AGC
Glu Leu
Trp Leu
1 Asp
Arg
CAA Pro
TTA Ser
GTT Val
GTT Asn
AGC Asn
GTT Ser
Gln 10
Leu 15
Val GCT
Val GGT
Ser ACT
Val GTT
20 GAG
GGG
ACG
AAT
CAA
GAC
Ala
Gly
Thr
Val
Glu
Gly
Thr
Asn
Gln
Asp
25
30
ATTAGT CTT AAA TTTTTT GAAATT GAC CTAACA TCA CGA CCTGCT CAT 144
IleSer Leu Lys PhePhe GluIle Asp LeuThr Ser Arg ProAla His
35 40 45
GGAGGA AAG ACA GAGCAA GGCTTA AGT CCAAAA TCA AAA CCATTT GCT 192
GlyGly Lys Thr GluGln GlyLeu Ser ProLys Ser Lys ProPhe Ala
50 55 60
ACTGAT AGT GGC GCGATG CCACAT AAA CTTGAA AAA GCT GACTTA CTA 240
ThrAsp,Ser Gly AlaMet ProHis Lys LeuGlu Lys Ala AspLeu Leu
65 70 75 80
AAGGCT ATT CAA GAACAA TTGATC GCT AACGTC CAC AGT AACGAC GAC 288
LysAla Ile Gln GluGln LeuIle Ala AsnVal His Ser AsnAsp Asp
85 90 95
TACTTT GAG GTC ATTGAT TTTGCA AGC GATGCA ACC ATT ACTGAT CGA 336
TyrPhe Glu Val IleAsp PheAla Ser AspAla Thr Ile ThrAsp Arg
100
105 110
AAC AAG GTC TAC GAC AAA GATGGT TCG GTA ACCTTG CCG 384
GGC Lys Val TTT Asp Lys AspGly Ser Val ThrLeu Pro
Asn 115 GCT
Gly Tyr 120 125
Phe
Ala
t~.
~5 2043953
ACCCAACCT GTC CAA GAATTT TTGCTA AGC GGA CATGTG CGCGTT AGA 432
ThrGlnPro Val Gln GluPhe LeuLeu Ser Gly HisVal ArgVal Arg
130 135 ~ 140
CCATATAAA GAA AAA CCAATA CAAAAT CAA GCG AAATCT GTTGAT GTG 480
ProTyrLys Glu Lys ProIle GlnAsn Gln Ala LysSer ValAsp Val
145 150 155 160
GAATATACT GTA CAG TTTACT CCCTTA AAC CCT GATGAC GATTTC AGA 528
GluTyrThr Val Gln PheThr ProLeu Asn Pro AspAsp AspPhe Arg
165 170 175
CCAGGTCTC AAA GAT ACTAAG CTATTG AAA ACA CTAGCT ATCGGT GAC 576
ProGlyLeu Lys Asp ThrLys LeuLeu Lys Thr LeuAla IleGly Asp
180 185 190
ACCATCACA TCT CAA GAATTA CTAGCT CAA GCA CAAAGC ATTETA AAC 624
ThrIleThr Ser Gln GluLeu LeuAla Gln Ala GlnSer IleLeu Asn
195 200 205
AAAACC,CAC CCA GGC TATACG ATTTAT GAA CGT GACTCC TCAATC GTC 672
LysThrIsisPro Gly TyrThr IleTyr Glu Arg AspSer SerIle Val
210-~ 215 220
ACTCATGAC AAT GAC ATTTTC CGTACG ATT TTA CCAATG GATCAA GAG 720
ThrHisAsp Asn Asp IlePhe ArgThr Ile Leu ProMet AspGln Glu
225 230 235 240
TTTACTTAC CAT GTC AAAAAT CGGGAA CAA GCT TATGAG ATCAAT AAA 768
PheThrTyr His Val LysAsn ArgGlu Gln Ala TyrGlu IleAsn Lys
245 250 255
AAATCTGGT CTG AAT GAAGAA ATAAAC AAC ACT GACCTG ATCTCT GAG 816
LysSerGly Leu Asn GluGlu IleAsn Asn Thr AspLeu IleSer Glu
260 265 270
AAATAT,TAC GTC CTT AAAAAA GGGGAA AAG CCG TATGAT CCCTTT GAT 864
LysTyrTyr Val Leu LysLys'GlyGlu Lys Pro TyrAsp ProPhe Asp
275 28Q 285
CGCAGTCAC TTG AAA CTGTTC ACCATC AAA TAC GTTGAT GTCAAC ACC 912
ArgSerHis Leu Lys LeuPhe ThrIle Lys Tyr ValAsp ValAsn Thr
290 295 300
AACGAATTG CTA AAA AGCGAG CAGCTC TTA ACA GCTAGC GAACGT AAC 960
AsnGluLeu Leu Lys SerGlu GlnLeu Leu Thr AlaSer GluArg Asn
305 310 315 320
TTAGACTTC AGA GAT TTATAC GAT,~ CGT GAT AAGGCT AAACTA CTC 1008
CT
LeuAspPhe Arg Asp "LeuTyr Asp'1 Arg Asp LysAla LysLeu Leu
~~ro
325 330 335
TACAACAAT CTC GAT GCTTTT GGTATT ATG GAC TATACC TTAACT GGA 1056
TyrAsnAsn Leu Asp AlaPhe GlyIle Met Asp TyrThr LeuThr Gly
340 345 350
a
2p~3953
AAAGTA GAGGAT AAT CACGAT GACACC AAC CGTATC ATA ACC GTTTAT 1104
LysVal GluAsp Asn HisAsp AspThr Asn ArgIle Ile Thr ValTyr
355 360 365
ATGGGC AAGCGA CCC GAAGGA GAGAAT GCT AGCTAT CAT TTA GCCTAT 1152
MetGly LysArg Pro GluGly GluAsn Ala SerTyr.His Leu AlaTyr
370 375 380
GATAAA GATCGT TAT ACCGAA GAAGAA CGA GAAGTT TAC AGC TACCTG 1200
AspLys AspArg Tyr ThrGlu GluGlu Arg GluVal Tyr Ser TyrLeu
385
390 395 400
CGTTAT ACAGGG ACA CCTATA CCTGAT AAC CCTAAC GAC AAA TAA 1245
ArgTyr ThrGly Thr ProIle ProAsp Asn ProAsn Asp .Lys
405 410
A
17
2043953 _.
SEQ ID N0:2
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 26 bases
TGGAATTCAT GAAAAATTAC TTATCT
SEQ ID N0:3
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 28 bases
TGGATCCTTA TTTGTCGTTA GGGTTATC
SEQ ID N0:9
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 31 bases
GGAATTCATG ATTGCTGGAC CTGAGTGGCT G
SEQ ID N0:5
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 17 bases
ATCATCGAAC TAGTTAA
SEQ ID N0:6
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 17 bases
GAATCAAGAC ATTAGTC
SEQ ID N0:7
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 15 bases
GTGGCGCGAT GCCAC
SEQ ID N0:8
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 17 bases
GCAACCATTA CTGATCG
.,, 18 204r395~3
SEQ ID N0:9
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 17 bases
CCAGTACAA71 ATCAAGC
SEQ ID NO:10
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 17 bases
CTAGCTATCG GTGACAC
SEQ ID NO:11
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 15 bases
CAGAGATCAG GTCAG
SEQ ID N0:12
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 16 bases
GTTAAGAGCT GCTCGC
SEQ ID N0:13
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 17 bases
CCAGTTAAGG TATAGTC
SEQ ID N0:14
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 16 bases
TCTCGTTCTT CTTCGG
A
