Note: Descriptions are shown in the official language in which they were submitted.
- WO91/17250 2 0 ~ 3 ~ 7 ~ PCT/EP91/00~3
-- 1 --
METHOD FOR THE RECOMBINANT PRODUCTION OF HIRIDIUS AND HIRUDIN-LIKE POLYPEPTIDES
The present invention relates to the preparation of
hirudin, which was originally isolated from the leech ~irudo
5 medicinalis, as well as to the preparation of derivatives
thereof.
The most popular anticoagulant peptides are
probably those belonging to the family of hirudins.
Hirudin, originally isolated from the medicinal leech,
10 Hirudo medicinalis, is a well known and well characterized
polypeptidic inhibitor of thrombinl~2. More particularly,
it binds thrombin by ionic interactions thus preventing the
cleavage of fibrinogen to fibrin and the subsequent fibrin-
clot formation. In animal studies hirudin has demonstrated
- 15 efficacy in preventing venous thrombosis, vascular shunt
occlusion and thrombin-induced disseminated intravascular
coagulation. In addition, hirudin exhibits low toxicity,
little or no antigenicity and a very short clearance time
from circulation3.
Three natural variants of hirudin are known. The
sequence of a first variant designated HVl was determined by
Dodt et al, FEBS 165 (1984) 180-184. The sequence of HVl
is, according to the three letter code (Eur. J. ~iochem.
138, 9-37, 1984):
25 Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-
Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-
Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-
~- Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
Glu-Glu-Tyr-Leu-Gln.
SO3H
A second variant designated HV2 has been described
by Dodt et al, Biol. Chem. Hoppe-Seyler 367 (1986) 803-811.
HV2 differs from HVl in the following respects: Ile at
position 1 instead of Val, Thr at position 2 instead of Val,
35 Lys at position 24 instead of Gln, Asn at position 33
;'
.:
: ' - .
- , ,: - ,,,
~091/17250 2 ~ ~ 3 5 7 5 PCT/EP91/00~
instead of Asp, Lys at position 35 instead of Glu, Gly at
position 36 instead of Lys, Asn at position 47 instead of
Lys, Glu at position 49 instead of Gln and Asn at position
53 instead of Asp.
A third variant designated HV3 has been described
by Harvey et al, Proc. Natl. Acad. Sci. USA (1986) 1084-
1088. HV3 is identical to HV2 from positions 1 to 32 and
then differs from HV1 in the following respects: Gln at
position 33 instead of Asp, Lys at position 35 instead of
10 Glu, Asp at position 36 instead of Lys, Gln at position 53
instead of Asp, Pro at position 58 instead of Glu, Asp at
position 62 instead of Glu, Ala at position 63 instead of
Tyr (S03H), Asp at position 64 instead of Leu and Glu at
position 65 instead of Gln.
A new approach to the preparation of hirudins and
hirudin-like polypeptide has now been devised. This
approach is based on chemical synthesis of a nucleotide
sequence encoding a hirudin or a hirudin-like polypeptide,
and expression of the hirudin or hirudin-like polypeptide in
20 recombinant organisms. The cultivation of the genetically
modified organisms leads to the production of the desired
product displaying full biological activity.
Accordingly, the present invention provides an
expression vector comprising a DNA sequence encoding a
25 hirudin or hirudin-like polypeptide. The invention further
provides a host transformed with a compatible expression
vector according to the invention, and also provides a
synthetic DNA encoding a hirudin or hirudin-like
polypeptide. A DNA fragment encoding a hirudin or hirudin-
30 like polypeptide may be single or double stranded.
A host in which a hirudin or hirudin-like
polypeptide is able to be expressed is prepared by
transforming a host with a compatible expression vector of
the invention. The expression vector is generally prepared
35 by:
(a) chemically synthesising DNA encoding a hirudin
: . , : .
, - , -: ~ . :
, ;. . . . ..
,. . .
- ' , : :~ ,' " ,:.
-, . - ~
~ - WO91/17250 2 0 6 ~ ~ ~ 5 PCT/EP91/00~3
or hirudin-like pol~peptide; and
(b) inserting the said DNA into an expression
vector.
A hirudin or hirudin-like polypeptide is
5 consequently prepared by providing a transformed host
according to the invention under such conditions that a
hirudin or hirudin-like polypeptide is expressed therein.
The hirudin or hirudin-like polypeptide can then be
isolated. In this way, a hirudin or hirudin-like
lO polypeptide may be obtained in pure form.
The term "a hirudin or hirudin-like polypeptide" as
used here n refers to hirudin in its natural HVl, XV2 and
XV3 forms as well as derivatives thereof, e.g. by way of
amino acid substitutions, deletions, insertions, extensions,
15 functionalisations and chemical modifications. The
invention can therefore also be applied to derivatives of
HVl, HV2 or HV3 having anti-thrombin activity.
The amino acid sequence HVl, HV2 or HV3 may be
modified by one or more amino acid substitutions, insertions
20 and/or deletions and/or by an extension at either or each
end. A derivative composed of such a modified sequence must
of course still exhibit anti-thrombin activity. Typically
` there is a degree of homology of 75% or more between the
- amino acid sequence of HVl, HV2 or HV3 and the amino acid
25 sequence of a derivative thereof. The degree of homology
may be 85% or more or 95~ or more.
For example, one or more amino acid residues of the
sequence of HYl, HV2 or HV3 may be substituted or deleted or
one or more additional amino acid residues may be inserted.
, 30 The physicochemical character of the original sequence can
be preserved, i.e. in terms of charge density,
hydrophobicity/hydrophilicity, size and configuration.
Candidate substitutions are, based on the one-letter code
(Eur. J. Biochem. 138, 9-37, 1984):
35 A for G and vice versa,
V by A, L or G;
.
.,:
- . - .. . . . ~ . ,~ .. ..
WO91/17250 2 ~ 6 ~ ~ 7 ~ PCT/EPgl/006 r
-- 4
K by R;
S by T and vice versa;
E for D and vlce versa; and
Q by N and vice versa.
As far as extensions are concerned, a short
sequence of up to 50 amino acid residues may be provided at
either or each ~erminal. ~he sequence may have up to 30,
for example up to 20 or up to 10, amino acid residues.
The hirudin or hirudin-like polypeptide may be
10 subjected to one or more post-translational modification
such as glycosylation, sulphation, COOH-amidation, acylation
or chemical alterations of the polypeptide chain. The Tyr
residue at position 63, for example, may be sulphated. A
recombinant hirudin or hirudin-like polypeptide obtained
15 according to the invention would not normally be sulphated
at this position, unlike the natural HV1, HV2 and HV3.
`~ Further, the invention may be applied to the production of
; lower molecular weight derivatives which do not have the N-
terminal or C-terminal portions of HVl, HV2 or HV3.
The invention is particularly applicable to the
production of HVl and of a XVl derivative composed of the
first 46 residues of HVl followed by the amino acid sequence
from residue 47 to 65 of the HV2 variant:
HVl:
25 Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-
Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-
Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-
Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
Glu-Glu-Tyr-Leu-Gln
30 Hybrid HVl/HV2 (designated HV12):
Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-
~ Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-
: Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-
Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-GlY-Asp-phe-Glu-Glu-Ile-pr
35 Glu-Glu-TYr-Leu-Gln
.
~.
', .' ' , '
.
. '', ' ' ~ '~ ' ' ~ ' '
` '`' ` ' ' ' ' ` `
2~3~7~
WO91/172~0 PCT/EP91/00~3
where the underlined sequence is the HV2 portion. The
polypeptide HV12 as depicted above and derivatives thareof
form another aspect of the invention.
The hirudins and hirudin-like polypeptides are
5 prepared by recombinant DNA technology. A synthetic gene
encoding a hirudin or hirudin-like polypeptide is prepared.
The DNA coding sequence typically does not include introns.
The synthetic gene is inserted in an expression vector able
to drive production of the recombinant product. The
lO synthetic gene is typically prepared by chemically
synthesising oligonucleotides which, in total, correspond to
the desired gene. The oligonucleotides are then assembled
to obtain the gene.
A gene may therefore be constructed from four
15 chemically synthesised oligonucleotides, each
oligonucleotide representing about half of one strand of a
double-stranded ~NA gene. The oligonucleotides are ligated
and annealed ~o obtain the desired gene. If desired, the
; gene sequence may be modified by site-directed mutagenesis
20 to introduce one or more codon changes. Typically, a gene
is constructed with restriction sites at each end to
facilitate its subsequent manipulation.
A preferred DNA sequence encoding HVl is shown in
Figure l of the accompanying drawings. A preferred DNA
25 sequence encoding HV12 is shown in Figure 3. Either
sequence may be modified to code for a derivative.
A DNA sequence may be provided which further
` encodes a leader peptide. The leader peptide is capable of
directing secretion of the hirudin or hirudin-like
30 polypeptide from cells in which the hirudin or hirudin-like
polypeptide is to be expressed. The sequence encoding the
leader peptide is typically fused to the 5'-end of the DNA
sequence encoding the hirudin or hirudin-like polypeptide.
The leader peptide is prefarably the OmpA leader
35 peptide when expression in a bacterial host, such as E.
coli, is required. The leader peptide is preferably the
, ~ . ~. - ., . - . : -
: . .: -, . . - . . . . . . .
- ' ,' , - : - ' ' .
WO91/17250 ~ 3 ~ ~ ~ PCT~EP91/00~ `
-- 6 --
leader peptide of vesicular stomatitis virus G protein
; (VSV G protein) where expression is to be in insect cells.
Appropriate DNA sequences encoding the OmpA and VSV G
protein leader sequences are shown in Figures 5 and 8
5 respectively.
A DNA sequence may be provided which encodes a
fusion protein which is cleavable to release a hirudin or
hirudin-like polypeptide. A DNA sequence may be used which
encodes a carrier polypeptide sequence fused via a cleavable
l0 linkage to the N-terminus of a hirudin or hirudin-like
polypeptide. The cleavable linkage may be one cleavable by
cyanogen bromide.
For expression of a hirudin or hirudin-like
polypeptide, an expression vector is constructed which
15 comprises a DNA sequence encoding a hirudin or hirudin-like
polypeptide and which is capable of expressing the hirudin
or hirudin-like polypeptide when provided in a suitable
host. Appropriate transcriptional and translational control
elements are provided, including a promoter for the DNA
20 sequence, a transcriptional termination site, and
translational start and stop codons. The DNA sequence is
provided in the correct frame such as to enable expression
of the polypeptide to occur in a host compatible with the
vector.
The expression vector typically comprises an origin
of replication and, if desired, a selectable marker gene
such as an antibiotic resistance gene. A promoter is
operably linked to the DNA sequence encoding a hirudin or
hirudin-like polypeptide. The expression vector may be a
30 plasmid. In that case, preferably a promoter selected from
~he Ptrp and Plcc/lac promoters is operably linked to the
DNA sequence. Alternatively, the expression vector may be a
virus. The virus may be a recombinant baculovirus in which
the polyhedrin promoter is operably linked to the DNA
35 sequence encoding a hirudin or hirudin-like polypeptide.
An expression vector capable of expressing a
.. ..
- : - : : .
. . . :
~ WO91/172~0 2 0 6 3 ~ 7 ~ PCT/EP91/00~3
hirudin or hirudin-like polypeptide may be prepared in any
convenient fashion. A DNA fragment encoding hirudin or
hirudin-like polypeptide may be inserted into an appropriate
restriction site of an expression vector, for example a
5 plasmid vector. A recombinant baculovirus ~ay be prepared
by:
(i) cloning a gene encoding a hirudin or hirudin-
like polypeptide into a baculovirus transfer vector at a
restriction site downstream of the polyhedrin promoter; and
: 10 (ii) co-transfecting ins~ct cells susceptible to
baculGtirus infection with the recombinant transfer vector
from step (i) and intact wild-type baculovirus DNA.
~ omologous recombination occurs, resulting in a
recombi,ant baculovirus harbouring the hirudin gene or gene
15 encoding a hirudin-like polypeptide downstream of the
polyhedrin promoter. The baculovirus transfer vector may be
: one having a unique cloning site downstream of the
polyhedrin ATG start codon. The product that is then
expressed by the resulting recombinant baculovirus will be a
; 20 fusion protein in which a N-terminal portion of the
polyhedrin protein is fused to the N-terminus of a hirudin
or hirudin-like polypeptide. As indicated above, a
cleavable linkage may be provided at the fusion junction.
The insect cells employed in step (ii) are
25 typically Spodoptera fruai~erda cells. The wild-type
baculovirus is typically Autoarapha californica nuclear
polyhedrosis virus ~AcNPV).
; An expression vector encoding a hirudin or hirudin-
like polypeptide is provided in an appropriate host. Cells
30 are transformed with the gene for a hirudin or hirudin-like
polypeptide. A transformed host is provided under such
conditions that the ~ ldin or hirudin-like polypeptide is
expressed therein. I ansformed cells, for example, are
cultivated so as to enable expression to occur. Any
35 compatible host-vector system may be employed.
The transformed host may be a prokaryotic or
.
- ., . , . ~ .
. . ~
WO91/17250 2 ~ ~ 3 5 7 5 PCr/EP91/00~ -`
- 8 -
eukaryotic host. A bacterial or yeast host may be employed,
for example E. coli or S. cerevisiae. Gram positive
bacteria may be employed. A preferred bacterial host is a
strain of E. coli type B. Insect cells can alternatively be
5 used, in which case a baculovirus expression system is
appropriate. The insect cells are typically S~odoptera
fruqiperda cells. As a further alternative, cells of a
mammalian cell line may be transformed. A transgenic
animal, for example a non-human mammal, may be provided in
lO which a hirudin or hirudin-like polypeptide is produced.
- The hirudin or hirudin-like polypeptide that is expressed
may be isolated and purified.
The hirudin or hirudin-like polypeptide prepared
according to the invention may be used in a pharmaceutical
15 formulation, together with a pharmaceuticall~ acceptable
carrier or excipient therefor. Such a formulation is
typically for intravenous administration (in which case the
` carrier is generally sterile saline or water of acceptable
. purity). The hirudin or hirudin-like polypeptide prepared
20 according to the invention is an anti-thrombin and is
~- suitable for treatment of thromboembolic events, such as the
~ coagulation of blood, typically in a human patient. In one
; embodiment of the invention, the hirudin or hirudin-like
polypeptide is coadministered with a plasminogen activator,
: 25 such as tissue plasminogen activator. The hirudin or
hirudin-like polypeptide prepared according to the invention
has been found to be compatible with the latter.
The following Examples illustrate the invention.
In the accompanying drawings:
Figure l shows the nucleotide sequence of the four
oligonucleotides coding for most of the hirudin HVl chain.
- 5 The sequence underlined indicates the BalI site which has
been used for further constructions. The lower part of the
Figure shows the mode of assembling of the four oligos.
, .
.
- .: .
'
WO91/17250 2 0 6 3 ~ 7 ~ PCT/EP9t/00~3
_ g _
HindIII and PstI sites were i~cluded to allow subsequent
manipulations.
Figure 2 shows the scheme of the construction of
the intermediate plasmid Ml3-HVl, which is the source of a
5 BalI-BamHI DNA fragment for all further bufrudin
~ constructions.
; Figure 3 shows the nucleotide sequence of the four
oligonucleotides coding for most of the hirudin HV12 chain.
The sequence underlined indicates the BalI site which has
l0 beer used for further constructions. The lower part of the
, figure shows the mode of assembling of the four oligos.
;~ HindIII and PstI sites were included to allow subsequent
manipulations.
Figure 4 shows the scheme of the construction of
- 15 the intermeidate plasmid Ml3-HV12, which is the source of a
BalI-BamHI DNA fragment for all further hirudin
constructions.
Figure 5 shows schematically the construction of
new recombinant Ml3s, named OMP-HVl and OMP-HVl2, which
20 carry respectively the complete HVl gene and the complete
HVl2 gene linked to the OmpA leader peptide. The leader
peptide sequence is underlined twice while the BalI blunt
end and the HindIII sticky end are underlined once.
Figure 6 shows schematically the construction of
25 pFC-HVl and pFC-HVl2 which are the plasmids used for the
production of HVl and HVl2 in E. coli.
Figure 7 shows the general structure of the plasmid
pOMP-HVl used for the production of hirudin in E. coli. We
; employed traditional gene manipulation techniques to prepare
30 this new plasmid where the hirudin gene is under
transcriptional control of the hybrid promoter Plpp/lac~
Even in this case, the OmpA leader peptide drives secretion
~ .
,
'
:
, . . , : :: , :'. ., ' . '~
WO 91/17250 2 0 6 3 ~ 7 ~ PCT/EP91/00~
- 10
- of bufrudin to the periplasm of E. coli.
Figure 8 shows the nucleotide sequence and
assembling of the synthetic oligos used for the secretion of
hirudin from insect cells. The sequence underlined
5 indicates the VSV G protein leader peptide.
Figure 9 i5 a schematic representation of the
construction of a new recombinant M13, named VSV-HV12, where
the complete bufrudin gene is linked to the VSV G protein
leader peptide.
Figure 10 shows schematically the construction of
pAc-HV12 which has been used as a transfer vector to the
baculovirus genome. pAcYM1 is the starting plasmid widely
used as an acceptor of heterologous sequences to be
transferred to the virus.
Figure 11 shows the nucleotide sequence and
assembling of the synthetic oligos coding for the beginning
of the hirudin chain. The ATG codon coding for the
additional methionine residue is underlined.
Figure 12 shows schematically the construction of
20 pAcFT1 which has been used for intracellular hirudin
expression.
Figure 13 is a schematic representation of the new
transfer plasmids, named pAcFTl-HVl and pAcFTl-HV12, which
carry respectively the complete HVl and HV12 sequences
25 linked to the first 18 amino acids of polyhedrin. These
plasmids have been used to transfer the heterologous
sequence to the baculovirus genome.
Example 1: Chemical synthesis of the HVl and HV12 qenes
; The nucleotide coding sequences were designed on
30 the basis of the Escherichia coli preferred codons4.
Moreover, a BalI restriction site was engineered very close
to the 5' end of the synthetic genes to allow insertion of
the coding sequences in different expression vectors.
Indeed, the same synthetic genes were used for expression in
3S bacterial and insect cells. In the case of insect cells
, "
.
~- WO9l/17250 2 0 6 3 ~ 7 ~ PCT/EP9l/0~3
methods were developed which yielded secreted or cytoplasmic
products.
All plasmid DNA manipulations were carried out as
described by Maniatis et al5.
A HVl gene was synthesised and assembled as
follows. Four synthetic complementary oligonucleotides were
prepared using an automated DNA synthesiser (Applied
Biosystems) and their sequence is shown in Figure 1.
Following enzymatic phosphorylation the four oligos were
10 assembled using DNA ligase and the resulting double-strand
sequence was inserted in the M13 phage vector mpl8,
obtaining the recombinant plasmid M13-HV1 which is shown in
Figure 2. In order to enable insertion of the hirudin gene
in the M13 vector, HindIII and PstI sites were also added in
15 the synthetic oligos. The correct nucleotide sequence has
been verified by the Sanger method carried out on the single
strand phage DNA6.
The recombinant plasmid M13-HV1 was used as the
source of the HVl gene for all the expression vectors used
20 in the Examples.
A HV12 gene was synthesised and assembled in the
same way. The oligos used to assemble the gene are shown in
Figure 3. Oligos 3 and 4 code for different amino acids
than oligos 3 and 4 of Figure 1. A recombinant plasmid M13-
25 HV12, shown in Figure 3, was obtained.
Example 2: Expression and secretion of hirudin from E. colicells
In order to obtain secretion to the periplasm of
the recombinant product, it is necessary to synthesize the
30 HV1 and XV12 molecules each in the form of a pre-protein.
More particularly, an amino acid sequence named "leader
peptide", responsible for an efficient secretion must be
present at the NH2 end of HV1 or HV127~. This extra
sequence is then cleaved off, ln vivo, during secretion, by
35 a specific E. coli leader peptidase, yielding the correct
'''
" . . . . . . . . . - , . ..
,. . . . .
:' - ,: , ~ ' ' '
' ' ' '' .' ': .' '~, ', : , , '
- ,, ': , ' ' . .' ':. ' . ~ ..
': . : ' .
WO91/l7250 2 ~ 5 PCT/EP9t/00
- 12 -
mature sequence9.
Many examples o~ secretion systems have been
described in the literature10~11. Among them, we have
selected the system based on the secretion signal of the
5 Outer Membrane Protein of E. coli (Omp A) previously
publishedl2. We therefore designed two additional
complementary oligonucleotides coding for the OmpA leader
peptide preceded by the OmpA Shine-Dalgarno sequence known
to be responsible for an efficient translation of the
10 messenger RNA13.
Their sequence, shown in Fig. 5, includes also the
beginning of the HV1 gene coding for the first 10 amino
acids. The presence of the BalI site allowed the joining of
this synthetic piece to the rest of the HV1 coding sequence
15 while the presence of the upstream HindIII site allowed the
joining to the M13 vector. Thus, the synthetic HindIII-BalI
fragment was ligated to a BalI-BamHI piece from M13-HVl and
inserted in M13mpl8, obtaining a new plasmid named OMP-HVl.
The schematic representation of this new plasmid
20 construction is also shown in Figure 5. Equivalent
manipulations starting from M13-HV12 gave OMP-HV12 (Figure
5).
From OMP-HVl the hirudin gene can be excised as a
HindIII-BamHI fragment which codes for the OmpA Shine-
-25 Dalgarno and leader peptide followed by the hirudin coding
sequence. This restriction fragment is now ready to be
;inserted in an appropriate expression vector. Several
--expression systems could, theoretically, bP employed to
obtain high level production of heterologous proteins in
30 bacteria. The system based on the promoter Ptrp has been
used with success in our laboratory in the past13. Again,
even in the case of the selected promoter, the levels of
expression of a given polypeptide cannot be predicted. The
use of the promoter Ptrp for the expression of hirudin has
35 neve been reported to date.
Plasmid pFC33, shown in Figure 6, has already been
'
"~ . . .
~ ~ ' ', :
.
~$~3~
WO91/17250 PCTJEP91/00~3
: -
13 -
described in the literature13. It carries the resistance to
the antibiotic ampicillin and the bacterial promoter Ptrp
which drives expression of proapolipoprotein Al. Following
; digestion of pFC33 with ~indIII and BamHI, the large
5 HindIII-BamHI fragment, carrying the antibiotic resistance
gene and the promoter, was isolated and joined to the
HindIII-BamHI fragment from O~P-HV1 or from OMP-HV12 coding
for the hirudin HVl or the hirudin derivative ~V12. The
details of this construction are shown in Figure 6. We
10 isolated new plasmids, named pFC-HV1 and pFC-HV12, which are
the final plasmids for the production of HV1 and HV12 in E.
col i .
A main object of the present invention is the use
of E. ~li strains of the type B for the expression and
15 secre 1 to the periplasm of hirudin and its derivatives.
Indeec ~e have found that insertion of plasmid pFC-HVl in
type ~ rains of the bacterium E. coli brings high level
produc; jn of hirudin. Interestingly, different strain
types ef E. coli do not work as efficiently and it seems,
20 therefore, that the host strain type is crucial for the -
successful production of hirudins.
Several type ~ strains of E. coli are available and
can be used for the production of a hirudin or hirudin-like
polypeptide. Preferred strains are ATCC 12407, ATCC 11303,
25 NCT~ 10537. Below is an example of transformation of strain
NCTC 10537 with plasmid pFC-HVl and subsequent cultivation
;:~ of the transformant.
Competent cells of strain NCTC 10S37 were-prepared
using the calcium chloride procedure of Mandel and Higal~.
30 Approximately 200~1 of a preparation of these cells at
1 x 109 cells per milliliter were transformed with 2~1 of
- plasmid DNA (approximate concentration 5~g/ml).
Transformants were selected on plates of L-agar containing
100~g/ml ampicillin. Two small colonies were streaked with
35 wooden tooth picks (each as three streaks about 1 cm long)
onto L-agar containing the same antibiotic. After 12 hours
.. . . -
WO91/17250 2 0 ~ 3 5 7 5 P~T/EP91/00~ `
- 14 -
incubation at 37C, portions of the streaks were tested for
hirudin production by inoculation onto 10 ml of LB medium
(containing ampicillin at a concentration of 150~g/ml) and
incubated overnight at 37C. The following day the cultures
5 were diluted 1:100 in M9 medium, containing the same
concentration sf ampicillin, and incubated for 6 hours at
37C.
-20 ml of such culture were centrifuged at 12000xg,
4C, for 10 minutes. The bacterial pellet was resuspended
10 in 2 ml of 33 mM HCl Tris pH 8; an equal volume of a second
solution 33 mM EDTA, 40% sucrose was then added and the
total mixture was incubated under mild shaking conditions at
37c for 10 minutes. Following centrifugation, the
permeabilized cells were resuspended in 2 ml of cold water
15 and left for 10 minutes in ice. The resulting supernatant
was isolated by centrifugation and represents the
periplasmic fraction of the bacterial cell.
Using a chromogenic assay that is based on the
inhibition of the thrombin ability to cleave a synthetic
20 substrate S-223815, we have measured the presence of anti-
; thrombin activity in the periplasmic fraction of hirudin-
producing cells. In these samples, hirudin activity was
present at the level of about 50~g/ml. This activity was
absent in control periplasmic fractions. In the case of
~25 pFC-HV1~ the productivity of the hirudin variant HV12 was
: equivalent to 80 ~g/ml.
With the similar approach we have also constructed
a new expression/secretion plasmid for hirudin where the
promoter Plpp/lacl6 is present instead of the promoter Ptrp.
30 This different plasmid, named pOMP-HV1, is shown in Figure
7. Following insertion of this plasmid in E. coli strains
of the type B, high levels of active hirudin were also
obtained (40-80 ~g/ml). As starting plasmid for the
construction of pOMP-HV1 we used the plasmid pIN-III-ompA3
35 described by Ghrayb et all6. Conditions for cultivation and
-induction of expression with isopropyl-~-D-
.
.~:
- WO91/17250 2 0 6 3 ~ 7 ~ PCT/EP91/00~3
- 15 -
thiogalactopyranoside (IPTG) were as previously describedl6.
Example 3: Anticoaqulant activity of recombinant HVl
obtained from E. coli
The anticoagulant activity of the hirudin variant
5 HVl was also tested in an activated partial thromboplastin
time (aPTT) test and in a thrombin time (T.T.) test. Both
tests were performed using an automatic coagulometer (ACL300
Research, Instrumentation Laboratory, Milan, Italy).
To normal citrated human plasma, were added
l0 increasing concentrations of the recombinantly produced
hirudin HVl. The samples were then assayed with the
automatic coagulometer which permits determination of aPTT
and T.T. by adding automatically to the plasma the
; appropriate reagents and recording the rate of formation of
15 the clot. aPTT was determined using cephaline and calcium
chloride (automated APTT reagent, General Diagnostic, USA)
and T.T. was determined using human thrombin (Fibrindex,
Ortho Diagnostic, Milan, Italy) at a concentration of
5IU/ml. The reagents were prepared, stored and used
20 according to the manufacturer's instructions.
The clotting times obtained in the aPTT and T.T. -
tests were plotted against the concentrations of the
recombinant protein. In each test, the concentration which
doubled clotting times relative to a normal plasma was
; .......................................................................... . .
25 calculated. The value obtained for the aPTT test was 210
ng/ml, whereas for the T.T. test the value was 90 ng/ml.
~ :.
Exam~le 4: Expression and secretion of HV12 from insect
cells
To obtain secretion of HV12 from recombinant insect
30 cells we had to join the HVl2 coding sequence to a leader
peptide that is efficiently recognized by these cells. We
have used the leader peptide of the Vescicular Stomatitis
Virus (VSV) G proteinl7. The use of such sequence for the
production of hirudin or its derivatives in insect cells has
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WOgl~17~50 2 ~ ~ 3 ~ ~ ~ PCT/EP91/00~ -`
- 16 -
never been reported to date. Similarly to what is described
above, a synthetic DNA sequence coding for the VSY G protein
leader peptide followed by the beginning of the HV12 gene
has been prepared and the nucleotide sequence is given in
5 Figure 8. Also in this case we provided convenient
restriction sites (HindIII, BamHI and BalI) to allow joining
to the rest of the HV12 gene and to the expression vector.
The synthetic HindIII-BalI fragment was joined to a
purified BalI-BamHI fragment from M13-HV12 carrying the HV12
10 gene and inserted in M13mpl8 previously cut with HindIII and
BamHI. This construction which yielded a new plasmid named
VSV-HV12 is schematically shown in Figure 9. From VSV-HV12
we have excised a BamHI-BamHI DNA fragment carrying the HV12
gene fused to the VSV leader peptide which was then inserted
lS into the vector pAcYM118, as shown in Figure 10. The
resulting plasmid was named pAc-HV12.
To obtain expression in insect cells, the VSV-HV12
coding sequence must be transferred to the baculovirus
genome under the transcriptional control of the polyhedrin
20 promoter. For this purpose, we co-transfected insect cells
with a wild-type baculovirus DNA and with the transfer
;~ vector pAc-HV12. As insect cells, S~odoptera fru~iperda
cells were chosen as host cells. Experimental details are
as follows:
S. fruqiperda cells were transfected with a mixture
of infectious AcNPV DNA and plasmid DNA representing the
individual recombinant transfer vectors by a modification of
the procedure described by Summers et al19. One microgram
of viral DNA was mixed with 25-100 ~g of plasmid DNA and
30 precipitated with (final concentrations) 0.125 M calcium
chloride in the presence of 20 mM HEPES buffer, pH 7.5, 1 mM
disodium hydrogen orthophosphate, 5mM potassium chloride,
140 mM sodium chloride and 10 mM glucose (total volume lml).
The DNA suspension was inoculated onto a monolayer
35 of 106 S. frugiperda cells in a 35-mm tissue culture dish,
allowed to adsorb to the cells for 1 h at room temperature,
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WOgl/17250 PCT/EP91/00~3
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- 17 -
then replaced with 1 ml of medium. After incubation at 28C
f~r 3 days the supernatant fluids were harvested and used to
produce plaques in S. fruaiperda cell monolayers. Plaques
containing recombinant virus were identified by their lack
5 of polyhedra when examined by light microscopy. Virus from
such plaques was recovered and after further plaque
purification was used to produce polyhedrin-negative virus
stoc~s.
The above procedure allowed us to isolate a
10 recombinant baculovirus whose genome carried the HV12 gene
under control of the polyhedrin promoter and of the VSV G
protein leader peptide. We used this virus to infect S.
fruqi~erda cells accordinq to well-established procedures19,
at a multiplicity of infection of 10. Infected cells were
15 then cultivated in spinner culture or in monolayers in the
presence of 10% foetal calf serum according to published
methodsl9. At different times post-infection, anti-thrombin
activity (ATU) was measured in the supernatant using the S-
2238 chromogenic assay. The following Table summarizes the -
20 results:
Table: HIRUDIN ACTIVITY
ATU/106 cells
.
time post-infection
0 hours 24 hrs 48 hrs 72 hrs
25 Spinner culture 0.8 0.9 1.4 1.8
Monolayers 0.8 0.8 2.0 5.1
Example 5: Expression of HVl and HV12 in the cvtoplasm of
insect cells
Hirudin and its derivatives could also be produced
30 and accumulated in the cytoplasm of S. fruqiPerda cells.
This approach generally gives a better yield of heterologous
proteins since it utilizes the expression signals of
polyhedrin which is a non-secreted viral protein.
Our approach to obtain large quantities of
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WO~1/17250 2 0 6 3 ~ 7 ~ PCT/EP91/00~
- 18 -
recombinant HV1 and HV12 is based on the expression of a
fusion polypeptide where the first 18 amino acids of
polyhedrin are joined in frame to the 65 amino acids of HVl
or HV12. The presence of the NH2 end sequence of polyhedrin
; 5 allows high level expression20. In addition, between the
polyhedrin portion and the HV1 or HV12 sequence we put a
methionine residue which allows the release of the HV1 or
HV12 moiety by treatment of the hybrid protein with CNBr.
Similarly to the previous approaches, we prepared a
10 synthetic DNA fragment which could allow the joining of the
BalI-BamHI fragment from M13-HVl or M13-HV12 to an
appropriate transfer vector. The new synthetic piece, shown
in Figure 11, includes also samHI and BalI sites for
subsequent manipulations.
A different transfer vector, pAcFTl, carrying the
nucleotide sequence coding for the first 18 amino acids of
polyhedrin has been obtained (Figure 12). Briefly, the
EcoRV-BamHI fragment of pAcYM118 has been replaced by a
synthetic oligonucleotide containing the polyhedrin gene
- 20 sequence from nucleotide -92 to nucleotide +55. A
convenient BamHI site is present after this sequence and it
has been used for insertion of the complete HVl or HV12
coding sequence according to a scheme illustrated in Figure
13. Through this construction, we obtained two new
25 plasmids, named pAcFTl-HVl and pAcFT1-HV12, which have been
used to transfer the hybrid genes to the baculovirus genome.
The recombinant baculoviruses were obtained as
described in Example 4. Infection of S. fruaiperda cells
was carried out according to standard proceduresl9.
30 Cultivation of infected insect cells lead to the cytoplasmic
accumulation of the fusion protein. This hybrid protein was
the source of recombinant HVl or HV12. Several methods are
available from the literature which can be used to cleave
the hybrid with CNBr21~22. The application of the method of
35 Olson et al22, has allowed us to obtain the HVl and HV12 of
the correct polypeptide sequences. These two molecules
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displayed anti-thrombin acti ~y.
References
1) Markwardt, F. 1970, Methods in Enzymology, 19, p. 924
2) Markwardt, F. 1985, Biomed. Biochim. Acta. 44, p. 1007
5 3) Markwardt, F. Hauptmann, J., Nowak, G., Xlessen, C., and
Walsmann, P. 1982. Thromb. Haemostasis 47, p. 226.
4) Grosjeans H. and Fiers W. 1982. Gene, 18, p. 199
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35 D.H.L. 1987. J. Gen. Virol. 68, p. 1233
'
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WO91/17250 2 0 6 3 5 7 5 PCT/EP91/00~
- 20 -
19) Summers, M.D., and Smith, G.E. 1987, Texas Agricultural
Experiment Station Bulletin No. 1555
- 20) Luckow, V.A. and Summers, M.D. 1988, Virology, 167, p.56
- 21) Gross E. 1967. Methods in Enzymology, 11, p. 238
S 22) Olson H., Lind P., Pohl G., Henrichson C., Mutt V~,
- Jornvall H., Josephson S., Uhlen M. and Lake M. 1987,
~ Peptides, 9, p. 301
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