Vector and method for making tissue factor pathway inhibitor (TFPI) analogues in yeast

A method for making TFPI analogues lacking part of the C-terminal end of the native TFPI molecule is described by cultivation of a yeast strain transformed with an expression vector containing a DNA sequence encoding such TFPI analogues. The TFPI analogues will at least contain the two first Kunitz domains and lack the third Kunitz domain.

FIELD OF THE INVENTION 
The present invention relates to a method of producing tissue factor 
pathway inhibitor (TFPI) analogues in yeast. 
BACKGROUND OF THE INVENTION 
Blood coagulation is a complex process involving many activating and 
inactivating coagulation factors. Anticoagulant proteins are known to be 
important for regulation of the coagulation process and anticoagulants are 
thus important in the treatment of a variety of diseases, e.g. thrombosis, 
myocardial infarction, disseminated intravascular coagulation etc. 
Thus heparin is used clinically to increase the activity of antithrombin 
III and heparin cofactor II. Antithrombin III is used for the inhibition 
of factor Xa and thrombin. Hirudin is used for the inhibition of thrombin 
and protein C may be used for the inhibition of factor V and factor VIII. 
Anticoagulant proteins may also be used in the treatment of cancer. 
Coagulation can be initiated through the extrinsic pathway by the exposure 
of tissue factor (TF) to the circulating blood (Y. Nemerson, Blood 71 
(1988) 1-8). Tissue factor is a protein cofactor for factor VII/VIIa and 
binding of tissue factor enhances the enzymatic activity of factor VIIa 
(FVIIa) towards its substrates factor IX and factor X. 
Recently a new anticoagulant protein, the tissue factor pathway inhibitor 
(TFPI) has been isolated (G. J. Broze et al., Proc. Natl. Acad. Sci. 84 
(1987) 1886-1890). 
On a molar basis TFPI has been shown to be a potent inhibitor of 
TF/FVIIa-induced coagulation (R. A. Gramzinski et al., Blood 73 (1989) 
983-989). TFPI binds and inhibits factor Xa (FXa) and the complex between 
TFPI and FXa inhibits TF/FVIIa (Rapaport, Blood 73 (1989) 359-365). TFPI 
is especially interesting as an anticoagulant/antimetastatic agent as many 
tumor cells express TF activity (T. Sakai et al., J. Biol. Chem. 264 
(1989), 9980-9988) and because TFPI shows anti-Xa activity like antistatin 
which has antimetastatic properties. 
TFPI has been recovered by Broze et al. (supra) from HepG2 hepatoma cells 
(Broze EP A 300988) and the gene for the protein has been cloned (Broze EP 
A 318451). A schematic diagram over the secondary structure of TFPI is 
shown in U.S. Pat. No. 5,312,736 (WO 91/01253). The amino acid sequence of 
TFPI with its natural 28 amino acid signal peptide (Sequence ID Number 1 
and 2) is shown in FIG. 1 where the N-terminal amino acid Asp is given the 
number 1. The protein consists of 276 amino acid residues and has in 
addition to three inhibitor domains of the Kunitz type, three potential 
glycosylation sites at position Asn117, Asn167 and Asn228. The molecular 
weight shows that some of these sites are glycosylated. Furthermore, it 
has been shown that the second Kunitz domain binds FXa while the first 
Kunitz domain binds FVIIa/TF (T. J. Girard et al., Nature 338 (1989) 
518-520). TFPI has also been isolated from HeLa cells (PCT/DK90/00016) and 
it was shown that HeLa TFPI binds heparin. 
In U.S. Pat. No. 5,312,736 certain TFPI analogues are described retaining 
the TFPI activity as well as anti Xa activity although parts of the 
molecule have been deleted. Furthermore, these analogues show a much lower 
affinity for heparin than full length TFPI, making them more useful as 
therapeutic agents than the native molecule. The TFPI analogues will 
furthermore have a longer half life as compared with native TFPI which 
will further reduce the amount of active ingredients for the medical 
treatment. 
These TFPI analogues are thus characterized in having TFPI activity but 
with no or low heparin binding capacity under physiological conditions 
(pH, ionic strength). 
In the present context the term "low heparin binding capacity" is intended 
to mean a binding capacity of about 50%, more preferably of about 25% and 
most preferably less than about 10% of that of native TFPI at 
physiological pH and ionic strength. 
It was thus shown that the heparin binding capacity is lost when the 
sequence from amino acid residue number 162 to amino acid residue number 
276 is deleted from the TFPI molecule. It was therefore concluded that the 
heparin binding domain is situated in this part of the TFPI molecule and 
it was assumed that the heparin binding domain comprises at least a region 
from Arg246 to Lys265 near the C-terminal end of the TFPI molecule being 
rich in positively charged amino acid residues. 
The present invention is based on the surprising fact that active TFPI 
analogues lacking C-terminal parts of the molecule are expressed in good 
yields in yeast. This is the more surprising because attempts to express 
native TFPI in yeast gave only neglible amounts. 
BRIEF DESCRIPTION OF THE INVENTION 
In its broadest aspect, the present invention is related to a method of 
making TFPI analogues in yeast, said TFPI analogues containing at least 
the first and second Kunitz domain and lacking part of the C-terminal end 
of the native TFPI molecule by cultivation of a yeast strain transformed 
with an expression vector containing a DNA-sequence encoding such TFPI 
analogues in a suitable nutrient medium under conditions which are 
conductive to the expression of the TFPI. 
In a more narrow aspect the present invention is related to a method of 
making TFPI analogues in yeast, said TFPI analogues containing at least 
the first and second Kunitz domain and lacking the third Kunitz domain 
from amino acid Cys189 to amino acid Cys239 as well as a substantial part 
of the amino acid sequence from Lys240 to Met276. With "a substantial 
part" is meant from about 70% to 100%. Preferably the TFPI analogues will 
lack the sequence from at least Cys189 to Lys265. The TFPI analogues may 
furthermore lack part of the amino acid sequence from amino acid Cys147 to 
Trp188 and part of the N-terminal sequence of native TFPI such as the 
sequence from amino acid residue 1 to 24. 
The TFPI analogues may also contain a Ser residue as the N-terminal residue 
for efficient cleavage of a signal peptide by a signal peptidase. Thus, 
the N-terminal in the TFPI molecule may be replaced by a Ser or an 
additional Ser may be inserted adjacent to the original N-terminal 
residue. 
The TFPI analogues may furthermore contain a modification of the potential 
N-glycosylation triade Asn117, Gln118, Thr119 to avoid glycosylation. Such 
modification will include a mutation and/or a deletion of one or more of 
the amino acid residues Asn117, Gln118, Thr119; see copending patent 
application Ser. No. 08/026,146. Asn117 may thus be replaced by Gln which 
cannot be glycosylated. 
The present invention is thus related to a method of expressing TFPI 
analogues in yeast, said TFPI analogues containing at least the amino acid 
sequence from Phe25 to Glu148 of the native TFPI molecule and lacking the 
third Kunitz domain from amino acid Cys189 to amino acid Cys239 and a 
substantial part of the amino acid sequence from Lys240 to Met276 of the 
native TFPI molecule. 
In a more preferred embodiment, the present invention is related to a 
method of expressing TFPI analogues in yeast, said TFPI analogues 
containing the amino acid sequence from Asp1 to Glu148 of the native TFPI 
molecule and lacking the third Kunitz domain from Cys189 to Cys239 and 
furthermore lacking a substantial part of the amino acid sequence from 
Lys240 to Met276 of the native TFPI molecule. 
In an even more preferred embodiment the present invention is related to a 
method of making TFPI analogues in yeast, said TFPI analogues lacking the 
amino acid sequence from Gln162 to Met276 of the native TFPI molecule in 
yeast. 
In a further aspect, the present invention is related to a recombinant 
yeast expression vector which comprises DNA sequences encoding functions 
facilitating gene expression, including a promoter and a terminator, 
linked functionally to a DNA sequence encoding the TFPI analogues and 
capable of expressing the TFPI analogues in yeast. 
In a still further aspect, the present invention is related to a yeast 
strain containing a recombinant yeast expression vector as defined above. 
DETAILED DESCRIPTION OF THE INVENTION 
The cDNA for the native TFPI has been cloned and sequenced (T. C. Wun et 
al., J. Biol. Chem. 263 (1988) 6001-6004). DNA sequences encoding the 
desired TFPI analogue may be constructed by altering TFPI cDNA by 
site-directed mutagenesis using synthetic oligonucleotides encoding the 
desired amino acid sequence in accordance with well-known procedures. 
The DNA sequence encoding the TFPI analogue of the invention may also be 
prepared synthetically by established standard methods. Thus 
oligonucleotides may be synthesized, by phosphoamidite chemistry in an 
automatic DNA synthesizer, purified, annealed, ligated and cloned in 
suitable vectors. 
The yeast expression vector according to the invention may be any vector 
which may conveniently be subjected to recombinant DNA procedures, and 
which is capable of expressing the gene for the TFPI analogues in yeast. 
Thus, the vector may be an autonomously replicating vector, i.e. a vector 
which exists as an extrachromosomal entity, e.g. a plasmid. Alternatively, 
the vector may be one which, when introduced into a host cell, is 
integrated into the host cell genome and replicated together with the 
chromosome(s) into which it has been integrated. 
In the expression vector, the DNA sequence encoding the TFPI analogue will 
be operably connected to a suitable promoter sequence. The promoter may be 
any DNA sequence which shows transcriptional activity in the yeast host 
cell of choice and may be derived from genes encoding proteins either 
homologous or heterologous to the yeast host cell. Suitable yeast 
promoters include promoters from yeast glycolytic genes (R. A. Hitzeman et 
al., J.Biol.Chem. 255 (1980) 12073-12080; T. Alber and G. Kawasaki, 
J.Mol.Appl.Gen. 1 (1982) 419-434) or alcohol dehydrogenase genes (T. Young 
et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender 
et al., eds.), Plenum Press, New York, 1982 pp. 335-361) or other highly 
expressed genes. Specific examples are the TPI1 (T. Alber and G. Kawasaki, 
op. cit., U.S. Pat. No. 4,599,311) or the ILV5 (J. G. L. Petersen and S. 
Holmberg, Nucl. Acids Res. 14 (1986) 9631-9651) promoter. 
The DNA sequence encoding the TFPI analogues should also be operably 
connected to a suitable terminator sequence which show transcription 
termination activity in yeast. Such terminator sequences may be derived 
from the 3' untranslated regions of yeast genes such as TPI1 (Alber and 
Kawasaki, op. cit.) and ILV5 (J. G. L. Petersen and S. Holmberg, op. 
cit.). The vector may further comprise elements such as polyadenylation 
signals, transcriptional enhancer sequences and translational enhancer 
sequences. 
Within the present invention it is preferred to express TFPI analogues in 
yeast host cells that can secrete the analogues into the culture media. To 
direct the TFPI analogues into the secretory pathway of the yeast host 
cell, a secretory signal sequence is operably linked to the TFPI analogue 
DNA sequence. The secretory signal should preferably be cleaved in vivo, 
e.g. by a signal peptidase or by the yeast KEX2 protease (D. Julius et 
al., Cell 37 (1984) 1075-1089) during export of the fusion protein to 
allow for secretion of the TFPI analogue having the correct N-terminal 
amino acid. Suitable secretory signals include the alpha factor signal 
sequence (J. Kurjan and I. Herskowitz, Cell 30 (1982) 933-943, U.S. Pat. 
No. 4,546,082 and EP 116,201), the PH05 signal peptide (WO 86/00637), 
secretory signal sequences derived from the BAR1 gene (U.S. Pat. No. 
4,613,572 and WO 87/002670), the SUC2 signal peptide (M. Carlson et al., 
Mol. Cell. Biol. 3 (1983) 439-447) and the human serum albumin 
prepropeptide (A. Dugaiczyk et al., Proc. Natl. Acad. Sci. USA, 79 (1982) 
71-75). Alternatively, a secretory signal sequence may be synthesized 
according to the rules established, for example, by G. von Heinje (Nucl. 
Acids Res. 14 (1986) 4683-4690). Examples of synthetic secretory signal 
sequences are described in WO 89/02463 and WO 92/13065. 
Suitable yeast vectors for use in the present invention include YRp7 (K. 
Struhl et al., Proc. Natl. Acad Sci. USA 76 (1987) 1045-1039), YEp13 (J. 
R. Broach et al., Gene 8 (1979) 121-133), POT vectors (U.S. Pat. No. 
4,931,373), pJDB249 and pJDB219 (J. Beggs, Nature 275 (1978) 104-109) and 
derivatives thereof. Such vectors will generally include a selectable 
marker, which may be one of any number of genes that exhibit a dominant 
phenotype for which a phenotypic assay exists to enable transformants to 
be selected. Preferred selectable markers are those that complement host 
cell auxotrophy, provide antibiotic resistance or enable a cell to utilize 
specific carbon sources, and include LEU2 (Broach et al., op.cit.), URA3 
(Botstein et al., Gene 8 (1979) 17), HIS3 (Struhl et al., op.cit.) or POT1 
(U.S. Pat. No. 4,931,373). 
Techniques for transforming yeast are well known in the literature, and 
have been described for instance by Beggs (op.cit.). The genotype of the 
host cell will generally contain a genetic defect that is complemented by 
the selectable marker present on the expression vector. Choice of a 
particular host and selectable marker is well within the level of ordinary 
skill in the art. To optimize production of heterologous proteins, it is 
preferred that the host strain carry a mutation, such as the yeast pep4 
mutation (E. W. Jones, Genetics 85 (1977) 23-33), which results in reduced 
proteolytic activity. 
The recombinant expression vector of the invention may further comprise a 
DNA sequence enabling the vector to replicate in the host cell in 
question. An example of such a sequence is the yeast 2-micron sequence. 
The procedures used to ligate the DNA sequences coding for the TFPI 
analogue of the invention, the promoter and the terminator, respectively, 
and to insert them into suitable vectors containing the information 
necessary for replication, are well known to persons skilled in the art. 
The yeast host cell may be any yeast species which is capable of producing 
the TFPI analogue. Examples of suitable yeast host cells include strains 
of Saccharomyces spp., Schizosaccharomyces spp. Kluyveromyces spp., Pichia 
spp. and Hansenula spp., in particular strains of Saccharomyces 
cerevisiae. 
The transformed yeast cells are grown according to standard methods in a 
growth medium containing nutrients required for growth of the particular 
yeast host cells. A variety of suitable media are known in the art and 
generally include a carbon source, a nitrogen source, essential amino 
acids, vitamins, minerals and growth factors. The growth medium will 
generally select for cells containing the DNA construct by, for example, 
drug selection or deficiency in an essential nutrient which is 
complemented by the selectable marker on the DNA construct or 
co-transfected with the DNA construct. 
Suitable growth conditions for yeast cells, for example, include culturing 
in a medium comprising a nitrogen source (e.g. yeast extract or 
nitrogen-containing salts), inorganic salts, vitamins and essential amino 
acid supplements as necessary at a temperature between 4.degree. C. and 
37.degree. C., with 30.degree. C. being particularly preferred. The pH of 
the medium is preferably maintained at a pH greater than 2 and less than 
8, more preferably pH 5-6. 
The TFPI analogue will preferably be secreted to the growth medium and may 
be recovered from the medium by conventional procedures including 
separating the host cells from the medium by centrifugation or filtration, 
precipitating the proteinaceous components of the supernatant or filtrate 
by means of a salt, e.g. ammonium sulphate, followed by purification by a 
variety of chromatographic procedures, e.g. ion exchange chromatography, 
affinity chromatography, or the like. 
The novel TFPI analogues may be used for the treatment of patients having 
coagulation disorders or cancer; see U.S. Pat. No. 5,312,736.

The invention is further described in the following examples which are not 
in any ways intended to limit the scope or spirit of the invention as 
claimed. 
Experimental Part 
Materials and Methods 
Standard recombinant DNA techniques were carried out as described (T. 
Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring 
Harbor Laboratory, 1982). 
Synthetic oligonucleotides were synthesized by the phosphoramidite method 
using an Applied Biosystems DNA Synthesizer Model 380B. 
Restriction endonucleases and T4 DNA ligase were obtained from New England 
Biolabs. Modified T7 DNA polymerase (Sequenase) was obtained from United 
States Biochemicals. Restriction endonucleases and other enzymes were used 
in accordance with the manufacturers recommendations. pBS+ (Stratagene) 
was used as cloning vector for construction of the synthetic TFPI gene by 
cloning of synthetic DNA fragments. 
E. coli strains XL-1 Blue (Stratagene) and MC1061 (M. J. Casadaban and S. 
N. Cohen, J. Mol. Biol. 138 (1980) 179-207) were used as bacterial 
recipients for plasmid transformations and as hosts for propagation and 
preparation of plasmid DNA. 
Strains of Saccharomyces cerevisiae used as hosts for expression of TFPI 
analogues were the two diploids E18 (MATa/MAT.alpha. 
.DELTA.tpi::LEU2/.DELTA.tpi::LEU2 leu2/leu2 +/his4 pep4-3/pep4-3) (U.S. 
Pat. No. 4,931,373) and YNG452 (MAT.alpha./MAT.alpha. ura3-52/ura3-52 
leu2-.DELTA.2/leu2-.DELTA.2 +/his4 pep4-.DELTA.1/pep4-.DELTA.1). The 
latter was derived from strain JC482 (J. F. Cannon and K. Tatchell, Mol. 
Cell. Biol. 7 (1987) 2653-2663). 
Yeast expression vectors used for expression of TFPI analogues in yeast 
were of the POT-type (US Patent No. 4,931,373) or the URA3-LEU2d-2.mu. 
plasmid pAB24 (P. J. Barr et al., in Proc. Alko Symp. on Industrial Yeast 
Genetics (Korkola and Nevalainen, eds.) Found. Biotech. Industr. Ferment. 
Res. 5 (1987) 139-148). 
DNA sequences were determined by the dideoxy chain termination method 
(Sanger et al., Proc. Natl. Acad. Sci. 74 (1977) 5463-5467) using double 
stranded plasmid DNA as template and .sup.32 P- or .sup.35 S labelled 
primers and Sequenase. 
Western blot analysis was carried out as described by J. Mikkelsen and J. 
Knudsen (Biochem. J. 248 (1987) 709-714). 
Affinity purification of the TFPI analogues was carried out from culture 
supernatants by affinity chromatography using polyclonal anti-TFPI 
immunoglobulin G coupled to Sepharose (O. Nordfang et al., Biochemistry 30 
(1991) 10371-10376). 
TFPI activity was measured in a chromogenic microplate assay, modified 
after the method of Sandset et al., (Thromb. Res. 47 (1987), 389-400). 
Heat treated plasma pool was used as a standard. This standard is defined 
as containing 1 U/ml of TFPI activity. Standards and samples were diluted 
in buffer A (0.05M Tris-HCl, 0.1M NaCl, 0.1M Na-citrate, 0.02% NAN.sub.3, 
pH 8.0) containing 2 .mu.g/ml polybrene and 0.2% bovine serum albumin. 
FVIIa/TF/FX/CaCl.sub.2 combination reagent was prepared in buffer A and 
contained 1.6 ng/ml FVIIa (Novo Nordisk A/S), human tissue factor diluted 
60 fold, 50 ng/ml FX (Sigma) and 18 mM CaCl.sub.2. The assay was performed 
in microplate strips at 37.degree. C. 50 .mu.l of samples and standards 
were pipetted into the strips and 100 .mu.l combination reagent was added 
to each well. After 10 minutes incubation, 25 .mu.l of FX (3.2 .mu.g/ml) 
was added to each well and after another 10 minutes 25 .mu.l of 
chromogenic substrate for FXa (S2222) was added 10 minutes after the 
addition of substrate. The reaction was stopped by addition of 50 .mu.l 
1.0M citric acid pH 3.0. The microplate was read at 405 nm. 
The inhibitory activity of TFPI analogues in the extrinsic pathway of 
coagulation was measured in PT clotting assay using human plasma and 
diluted human thromboplastin (O. Nordfang et al., op.cit.). 
EXAMPLE 1 
Construction of genes encoding TFPI analogues for secretion in yeast 
In U.S. Pat. No. 5,312,736, a synthetic gene for human TFPI with its 28 
amino acid signal peptide was described. The DNA sequence was derived from 
the published sequence of a cDNA coding for human TFPI (Wun et al., J. 
Biol. Chem. 263 (1988) 6001-6004). The synthetic gene was assembled by the 
step-wise cloning of synthetic restriction fragments into plasmid pBS(+). 
The resulting gene was contained on a 922 base pair (bp) SalI restriction 
fragment. The gene had 26 silent nucleotide substitutions in degenerate 
codons as compared to the cDNA resulting in fourteen unique restriction 
endonuclease sites in order to facilitate the introduction of mutations in 
TFPI as well as the in-frame insertion of new secretion signals at the 
N-terminal of mature TFPI. The DNA sequence of the 922 bp SalI fragment 
and the corresponding amino acid sequence of human TFPI (pre-form) is 
shown in FIG. 1. 
Using standard DNA manipulation technology, the coding sequences for TFPI 
analogues were constructed from the synthetic TFPI gene by replacing 
portions of the TFPI gene with appropriate synthetic DNA fragment. The DNA 
fragments were annealed oligodeoxynucleotides synthesized by 
phosphoramidite chemistry. Resulting plasmids were propagated in E. coli 
and the nucleotide sequences verified by DNA sequencing. The exemplary 
TFPI analogues constructed in this manner were all characterized by 
lacking different portions of the C-terminal one third of the TFPI 
polypeptide, and thus retaining at least the first two Kunitz Domains. 
Some derivatives had an Asn to Gln substitution at the potential 
N-glycosylation site at position 117 (Wun et al. op.cit., T. J. Girard et 
al., Thromb. Res. 55 (1989) 37-50) by changing the codon AAT (Asn) to CAA 
(Gln)) in order to avoid the addition of N-linked oligosaccharides at this 
site during expression and secretion in yeast. The following TFPI 
polypeptides were expressed in yeast: 
______________________________________ 
Name Characteristics 
Polypeptide 
______________________________________ 
TFPI full length Asp1--Met276 
TFPI-117Gln 
full length Asp1--Met276(117Gln) 
TFPI.sub.1-252 
lacking the 24 
Asp1--Leu252 
C-terminal amino 
acids 
TFPI.sub.1-161 
2-domain Asp1-Thr161 
TFPI.sub.1-161 -117Gln 
2-domain, lacking 
Asp1--Thr161(117Gln) 
N-glycosylation sites 
TFPI.DELTA.3-117Gln 
2-domain, internal 
Asp1--Gly150/ 
deletion of domain 
Phe243--Met276-- 
3 retaining the 
(117Gln) 
C-terminal, lacking 
N-glycosylation sites 
______________________________________ 
The TFPI polypeptides above were expressed in yeast in secretable forms by 
replacing the coding sequence for the 28 amino acid signal peptide of TFPI 
(FIG. 1A) with synthetic DNA fragments encoding two different heterologous 
secretion signals, namely the human serum albumin prepropeptide 
(pp.sub.HSA) (A. Dugaiczyk et al., Proc. Natl. Acad. Sci. USA, 79 (1982) 
71-75) and the synthetic secretion signal 212spx3 (WO 89/02463). The 
212spx3 signal consists of the signal peptide of mouse salivary gland 
.alpha.-amylase with residues Ala3Va14 changed to Phe, and Va17 to Leu, 
followed by a synthetic leader sequence. Both signals provided at their 
C-termini a pair of basic amino acids fused to the N-terminal Asp1 of 
mature TFPI to allow for secretion of correctly processed TFPI by cleavage 
in vivo by the yeast KEX2 protease (D. Julius et al., Cell 37 (1984) 
1075-1089). 
The DNA sequences and translated amino acid sequences for the two secretion 
signals fused to TFPI are shown in FIG. 2. 
EXAMPLE 2 
Construction of yeast expression plasmids 
In order to express the TFPI mutant forms in yeast, the genes were placed 
between a yeast promoter and a yeast terminator in yeast-E. coli vectors. 
In the present example, the strong constitutive promoters of the ILV5 (J. 
G. L. Petersen and S. Holmberg, Nucl. Acids Res. 14 (1986) 9631-9651) or 
TPI1 (T. Alber and G. Kawasaki, J. Mol. Appl. Genet. 1 (1982) 419-434) 
genes of S. cerevisiae were used. Transcription terminator sequences were 
derived from the same two genes. The ILV5 promoter fragment employed was 
from position -346 to -1 of the published sequence of ILV5 with a BamHI 
site added at the upstream end via a synthetic linker. The ATG translation 
start codon of the different signal-TFPI fusion genes were inserted at the 
putative start codon for the ILV5 gene. The ILV5 terminator fragment used 
was from position +1172 to +1823 of the published sequence with a SalI 
site at the upstream position and a poly-linker sequence downstream. The 
TPI promoter was from position -11 to -379 of the published sequence 
except that the SphI site in this fragment had been deleted. At the 
upstream end was added a SphI site, and at position -10 was added en EcoRI 
site, which could be conveniently used for the insertion of DNA fragments 
encoding 212spx3-TFPI fusions (see FIG. 2). The TPI1 terminator was from 
the XbaI site in the 3' end of the TPI1 coding region and 0.7 kb 
downstream where a BamHI site was inserted. 
The TFPI expression cassettes thus constructed were inserted in yeast 
vectors. In the present example, we used the POT-type (pRPOT, similar to 
CPOT, U.S. Pat. No. 4,931,373) characterized by carrying the 
Schizosaccharomyces pombe triosephosphate isomerase gene (POT) (P. R. 
Russell, Gene 40 (1985) 125-130) as selectable marker for the purpose of 
plasmid stabilization in tpi1 mutants of S. cerevisiae. The plasmids 
contained most of the yeast 2-micron plasmid for replication in yeast, and 
pUC13 sequences for selection and maintenance in E. coli. The POT vector 
contained in some cases also the defective LEU2d gene derived from plasmid 
PJDB219 (J. Beggs, Nature 275 (1978) 104-109). This marker was not used 
for selection in this example. A representative plasmid construct is shown 
in FIG. 3. 
Some TFPI analogues were also expressed from plasmid pAB24 (P. J. Barr et 
al., in Proc. Alko Symp. on Industr. Yeast Genet. Kornola and Nevalaiken, 
eds. Found. Biotech. Industr. Ferment. Res. 5 (1982 ) 139-148 ). 
EXAMPLE 3 
Production of TFPI analogues in yeast 
The TFPI expression plasmids with the POT gene as selectable marker were 
introduced into the diploid S. cerevisiae strain E18. The host strain 
grows poorly on media with glucose as the carbon source due to the 
chromosomal deletion of the gene for triose phosphate isomerase. For 
transformation, the strain was grown in medium with glycerol and lactate 
as the carbon sources and spheroblasts prepared according to J. Beggs 
(op.cit.). Transformants were selected in top-agar on minimal plates 
containing sorbitol and with glucose as the carbon source. In expression 
studies, reisolated transformants were grown in rich glucose medium (YPD) 
(F. Sherman, G. R. Fink and J. B. Hicks, Methods in Yeast Genetics, A 
Laboratory Manual. Cold Spring Harbor Laboratory, 1986) in shake flasks at 
30.degree. C. to stationary phase. Following centrifugation, cell pellets 
and media were stored at -20.degree. C. until further analysis. Expression 
plasmids based on vector pAB24 were transformed into strain YNG452 
selecting for uracil prototrophy on synthetic medium lacking uracil by the 
alkali cation transformation procedure (H. Ito et al., J. Bacteriol. 153 
(1983) 163- 168). Transformants of YNG452 were propagated in synthetic 
media without uracil. 
The activity of TFPI was measured in media supernatants by the chromogenic 
FXa/TF/FVIIa-dependent two-stage assay. The results are summarized in 
Table I. 
TABLE I 
______________________________________ 
Levels of secreted TFPI from yeast transformants of strain E18 
TFPI activity in the culture medium after three days of growth 
at 30.degree. C. was determined relative to absorbance of the culture at 
600 nm. Values are the mean of 2-10 independent experiments. 
Secreted TFPI 
Secretion TFPI/ activity 
Promoter 
signal TFPI analogue (U/ml .multidot. A.sub.600) 
______________________________________ 
ILV5 pp.sub.HSA 
TFPI 0.002 
TPI1 212spx3 TFPI 0.008 
TPI1 212spx3 TFPI.sub.1-252 
0.004 
ILV5 212spx3 TFPI-117Gln 0.007 
ILV5 pp.sub.HSA 
TFPI.sub.1-161 
0.45 
TPI1 212spx3 TFPI.sub.1-161 
4.9 
TPI1 212spx3 TFPI.sub.1-161 -117Gln 
4.0 
TPI1 212spx3 TFPI.DELTA.3-117Gln 
0.10 
-- -- placebo &lt;0.002 
______________________________________ 
As seen in Table I, plasmids for full length TFPI gave only low levels of 
activity in the culture medium with both secretion sequences. Full length 
TFPI with a substitution of one of the three potential sites for N-linked 
glycosylation (Asn117 to Gln) also showed very low levels of secretion. 
Expressing of a TFPI variant, TFPI.sub.1-252 lacking the C-terminal basic 
region of TFPI gave rise to about the same low levels of secreted activity 
as observed with full length TFPI. 
Expression of TFPI.DELTA.3-117Gln, which lacks the third Kunitz domain and 
potential N-glycosylation sites gave more than 10 times higher yields 
compared with full length TFPI. 
About a 200-fold increase in secreted activity relative to the full length 
protein was observed when TFPI.sub.1-161 was expressed in yeast using the 
ILV5 promoter and the prepropeptide of HSA. With the TPI1 promoter and 
signal sequence 212spx3, a further 10-fold increase was obtained. 
Substitution of the single potential N-glycosylation site in 
TFPI.sub.1-161 (resulting in TFPI.sub.1-161 -117Gln) did not affect the 
level of secreted activity. 
From these results it can be concluded that full length and near to full 
length TFPI containing the third Kunitz domain and a substantial part of 
the sequence from amino acid Lys240 to Met276 of native TFPI were 
expressed only poorly in yeast as measured by secreted activity, while 
surprisingly TFPI analogues lacking the third Kunitz domain and a 
substantial part of the sequence Lys240 to Met276 were secreted at 
significantly higher levels. 
EXAMPLE 4 
Anticoagulant properties of TFPI analogues from yeast 
In order to measure the anticoagulant activities of TFPI and TFPI.sub.1-161 
secreted from yeast, the polypeptides were partly purified by 
affinity-chromatography using a polyclonal antibody towards TFPI coupled 
to Sepharose and their anticoagulant activity determined in traditional 
prothrombin time (PT) coagulation assays. Anticoagulant units were 
normalized to chromogenic TFPI activity which was assumed to be similar to 
the different TFPI forms. In this assay, TFPI in human plasma used as 
standard was defined to have a relative anticoagulant activity of 1. The 
results are shown in Table II. A relative anticoagulant activity of 0.18 
was determined for full length TFPI produced in yeast. A comparable 
activity was found for TFPI with the Asn117 to Gln substitution. These 
activities are 5-9 times lower than for human, high-activity TFPI produced 
in BHK cells, but similar to C-terminally fragmented TFPI prepared as 
described by O. Nordfang et al. (Biochemistry 30 (1991) 10371-10376) and 
may be due to partial C-terminal fragmentation. In contrast, about 60-fold 
lower anticoagulant activities were observed for the two-domain 
polypeptides TFPI.sub.1-161 and TFPI.sub.1-161 -117Gln as compared to 
intact TFPI from BHK cells. 
TABLE II 
______________________________________ 
Anticoagulant activity of TFPI variants 
TFPI polypeptides secreted from yeast transformants were 
purified by affinity chromatography and their anticoagulant 
activity measured in a PT clotting assay relative to 
FXa/TF/FVIIa-dependent TFPI activity. 
TFPI Anticoagulant 
Relative 
activity activity anticoagulant 
TFPI molecule 
(U/ml) (units/ml) activity 
______________________________________ 
TFPI 8.3 1.5 0.18 
TFPI-117Gln 14.0 4.3 0.31 
TFPI.sub.1-161 
102.0 2.8 0.027 
TFPI.sub.1-161 -117Gln 
99.0 2.6 0.026 
From BHK cells: 
TFPI 10.0 15.8 1.6 
Heterogenous C-term. 
100.0 10.9 0.11 
______________________________________ 
EXAMPLE 5 
Western analysis of secreted and intracellular TFPI and TFPI.sub.1-161 
The analysis of secreted TFPI and TFPI analogues in Table I depended on 
activity measurements of the culture medium, and did not necessarily 
reflect the molar amounts of polypeptides being secreted. Thus, the low 
levels of full length TFPI could be a result of mainly inactive product 
being secreted, e.g. as a result of proteolytic degradation or improper 
folding. Therefore, the expression of TFPI and TFPI.sub.1-161 was analyzed 
by Western blotting using different antibodies towards TFPI (FIG. 4). The 
samples of supernatant media or cell extracts were partially purified by 
anti-TFPI affinity chromatography before SDS polyacrylamide gel 
electrophoresis and Western blot analysis. Expression of full length TFPI 
(lanes 2 and 5) gave rise to a predominant protein band with an apparent 
molecular mass of 50 kDa. The polypeptide reacted both with antibodies 
against the N- and C-terminal regions suggesting that the polypeptide was 
full length. The lower mobility as compared to TFPI expressed in 
transfected BHK cells (lane 3) suggested that more extensively 
glycosylation takes place in yeast. TFPI.sub.1-161 reacted as expected 
only with the anti-N antibody (lanes 4 and 6). The major immunoreactive 
species appeared as a broad band of about 25 kDa. 
Comparable staining intensities were obtained in Westerns with TFPI from 
yeast or BHK cells, and with TFPI.sub.1-161 from yeast (FIG. 4). Since the 
same amounts of activity were analyzed (0.7 U per lane) it appears as the 
three preparations have similar specific activities. 
A similar analysis by Western blotting was carried out with cell extracts 
of transformants expressing TFPI and TFPI.sub.1-161 fused to the 
prepropeptide of HSA. The cell extract of the full length TFPI 
transformant upon partial immuno-affinity purification showed 2-3 bands of 
34-39 kDa which could represent full length, underglycosylated TFPI, while 
the TFPI.sub.1-161 transformant showed a major band of 25 kDa and a minor 
band of 22 kDa, probably representing glycosylated and unglycosylated 
TFPI.sub.1-161, respectively. 
Pilot-scale purification of TFPI.sub.1-161 
In order to obtain a large quantity of homogeneously pure TFPI.sub.1-161, a 
yeast transformant expressing the analogue fused to the prepropeptide of 
HSA was propagated in a pilot-scale fermentor to high cell density. The 
resulting fermentation medium after removal of the cells by centrifugation 
contained about 650 U/ml of TFPI activity. The purification scheme for 
TFPI.sub.1-161 from this batch is summarized in Table III. At each step, 
fractions with high TFPI activity and low protein content were pooled for 
further purification. 
TABLE III 
______________________________________ 
Purification of TFPI.sub.1-161 
Yeast transformants of strain YNG452 expressing TFP.sub.1-161 
fused to the prepropeptide of HSA was grown in a 2000 l 
fermentor and secreted TFPI.sub.1-161 purified from the 
cleared supernatant medium. 
Total 
Volume TFPI activity 
Yield 
Purification step 
(1) (10.sup.6 U) 
% 
______________________________________ 
Fermentation medium 
1050 680 100 
Cation exchange (pH 3.0) 
73 92 13 
FF-Q anion exchange 
7.8 73 11 
(pH 9.3) 
Freeze-drying and gel 
1.2 78 11 
filtration on Superdex 75 
(pH 3.4) 
S-Sepharose cation exchange 
1.4 57 8 
(pH 3.4) 
Precipitation by -- 49 7 -isopropanol, dissolution 
in H.sub.2 O, freeze-drying 
______________________________________ 
About 5.times.10.sup.7 U were isolated corresponding to a purification 
yield of about 7%. The rather low yield was mainly caused by loss of 
material during the first step of purification involving cation exchange 
chromatography. 
SDS-PAGE analysis of the purified TFPI.sub.1-161 showed a single protein 
band of 25 kDa after staining with Coomasie Brilliant Blue (FIG. 5, lanes 
3 and 4). After the initial cation and anion exchange chromatography 
several additional polypeptides were present (lane 2). Among these, the 
major 25 kDa polypeptide and the 14 kDa polypeptide reacted with 
antibodies against the N-terminal region of TFPI (data not shown). This 
result suggested that the 14 kDa polypeptide is a proteolytic break-down 
product of TFPI.sub.1-161, possibly consisting of the first Kunitz domain. 
As was the case for TFPI.sub.1-161 produced in COS-7 cells (copending 
patent application Ser. No. 07/828,90), TFPI.sub.1-161 produced in yeast 
differed from full length TFPI in its binding characteristics towards 
heparin. Whereas full length TFPI binds strongly to heparin-Sepharose with 
about 1M NaCl required for effective elution, TFPI.sub.1-161 did not bind 
to heparin under physiological conditions (0.1M NaCl, 50 mM Tris-HCl, 0.1% 
BSA, pH 7.4). 
Based on weighing of the freeze-dried preparation of purified 
TFPI.sub.1-161 from yeast, a specific activity of 23,000 U/mg in the 
chromogenic FXa/TF/FVIIa-dependent assay was determined. The activity 
agrees well with the estimated 30,000 and 10,000 U/mg reported for TFPI 
isolated from transfected BHK cells (A. H. Pedersen et al., J. Biol. Chem. 
265 (1990) 16786-16793) and from the HepG2 hepatoma cell line (Broze, G. 
J., Jr. et al., Thromb. Res 48 (1987) 253-259). 
The anticoagulant activity of purified TFPI.sub.1-161 was further analyzed 
by its ability to inhibit the activity of tissue factor (TF) in direct PT 
clotting assay where TFPI.sub.1-161 was added to the plasma without 
preincubation with TF. It was found that a concentration of 65 .mu.g/ml of 
TFPI.sub.1-161 inhibited 90% of the TF activity. This inhibition was 
obtained for both high and low concentrations of the TF preparation 
(thromboplastin). With full length TFPI from transfected BHK cells only 
0.5 .mu.g/ml was required for 90% inhibition of TF activity. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 928 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 8..919 
(ix) FEATURE: 
(A) NAME/KEY: sigpeptide 
(B) LOCATION: 8..91 
(ix) FEATURE: 
(A) NAME/KEY: matpeptide 
(B) LOCATION: 92..919 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GTCGACCATGATT TACACAATGAAGAAAGTACATGCACTTTGGGCTAGC49 
MetIleTyrThrMetLysLysValHisAlaLeuTrpAlaSer 
28-25-20-15 
GTATGCC TGCTGCTTAATCTTGCCCCTGCCCCTCTTAATGCTGATTCT97 
ValCysLeuLeuLeuAsnLeuAlaProAlaProLeuAsnAlaAspSer 
10-51 
GAGGAA GATGAAGAACACACAATTATCACAGATACGGAGCTCCCACCA145 
GluGluAspGluGluHisThrIleIleThrAspThrGluLeuProPro 
51015 
CTGAAACTTAT GCATTCATTTTGTGCATTCAAGGCGGATGATGGGCCC193 
LeuLysLeuMetHisSerPheCysAlaPheLysAlaAspAspGlyPro 
202530 
TGTAAAGCAATCATGAAAA GATTTTTCTTCAATATTTTCACTCGACAG241 
CysLysAlaIleMetLysArgPhePhePheAsnIlePheThrArgGln 
35404550 
TGCGAAGAATTTATA TATGGGGGATGTGAAGGAAATCAGAATCGATTT289 
CysGluGluPheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPhe 
556065 
GAAAGTCTGGAAGAG TGCAAAAAAATGTGTACAAGAGATAATGCAAAC337 
GluSerLeuGluGluCysLysLysMetCysThrArgAspAsnAlaAsn 
707580 
AGGATTATAAAGACAAC ACTGCAGCAAGAAAAGCCAGATTTCTGCTTT385 
ArgIleIleLysThrThrLeuGlnGlnGluLysProAspPheCysPhe 
859095 
TTGGAAGAGGATCCTGGAATAT GTCGAGGTTATATTACCAGGTATTTT433 
LeuGluGluAspProGlyIleCysArgGlyTyrIleThrArgTyrPhe 
100105110 
TATAACAATCAGACAAAACAGTGTGAAAGG TTCAAGTATGGTGGATGC481 
TyrAsnAsnGlnThrLysGlnCysGluArgPheLysTyrGlyGlyCys 
115120125130 
CTGGGCAATATGAACAATTTTGAGACA CTCGAGGAATGCAAGAACATT529 
LeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLysAsnIle 
135140145 
TGTGAAGATGGTCCGAATGGTTTCCA GGTGGATAATTATGGTACCCAG577 
CysGluAspGlyProAsnGlyPheGlnValAspAsnTyrGlyThrGln 
150155160 
CTCAATGCTGTTAACAACTCCCTGACTC CGCAATCAACCAAGGTTCCC625 
LeuAsnAlaValAsnAsnSerLeuThrProGlnSerThrLysValPro 
165170175 
AGCCTTTTTGAATTCCACGGTCCCTCATGGTGT CTCACTCCAGCAGAT673 
SerLeuPheGluPheHisGlyProSerTrpCysLeuThrProAlaAsp 
180185190 
AGAGGATTGTGTCGTGCCAATGAGAACAGATTCTACTACAAT TCAGTC721 
ArgGlyLeuCysArgAlaAsnGluAsnArgPheTyrTyrAsnSerVal 
195200205210 
ATTGGGAAATGCCGCCCATTTAAGTACTCCGGATGTGG GGGAAATGAA769 
IleGlyLysCysArgProPheLysTyrSerGlyCysGlyGlyAsnGlu 
215220225 
AACAATTTTACTAGTAAACAAGAATGTCTGAGGGCAT GCAAAAAAGGT817 
AsnAsnPheThrSerLysGlnGluCysLeuArgAlaCysLysLysGly 
230235240 
TTCATCCAAAGAATATCAAAAGGAGGCCTAATTAAAACC AAAAGAAAA865 
PheIleGlnArgIleSerLysGlyGlyLeuIleLysThrLysArgLys 
245250255 
AGAAAGAAGCAGAGAGTGAAAATAGCATATGAAGAAATTTTTGTT AAA913 
ArgLysLysGlnArgValLysIleAlaTyrGluGluIlePheValLys 
260265270 
AATATGTGAGTCGAC9 28 
AsnMet 
275 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 304 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetIleTyrThrMetLysLysValHisAlaLeuTrpAlaSerValCys 
28 -25-20- 15 
LeuLeuLeuAsnLeuAlaProAlaProLeuAsnAlaAspSerGluGlu 
10-51 
AspGluGluHisThrIleIleT hrAspThrGluLeuProProLeuLys 
5101520 
LeuMetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLys 
25 3035 
AlaIleMetLysArgPhePhePheAsnIlePheThrArgGlnCysGlu 
404550 
GluPheIleTyrGlyGlyCysGluGlyAsnGlnAsn ArgPheGluSer 
556065 
LeuGluGluCysLysLysMetCysThrArgAspAsnAlaAsnArgIle 
707580 
IleLysThrTh rLeuGlnGlnGluLysProAspPheCysPheLeuGlu 
859095100 
GluAspProGlyIleCysArgGlyTyrIleThrArgTyrPheTyrAsn 
10 5110115 
AsnGlnThrLysGlnCysGluArgPheLysTyrGlyGlyCysLeuGly 
120125130 
AsnMetAsnAsnPheGluThrLeuG luGluCysLysAsnIleCysGlu 
135140145 
AspGlyProAsnGlyPheGlnValAspAsnTyrGlyThrGlnLeuAsn 
150155160 
AlaValAsnAsnSerLeuThrProGlnSerThrLysValProSerLeu 
165170175180 
PheGluPheHisGlyProSerTrpCysLeuThrProAlaAspArgGly 
185190195 
LeuCysArgAlaAsnGluAsnArgPheTyrTyrAsnSerValIleGly 
200205210 
LysCysArgProP heLysTyrSerGlyCysGlyGlyAsnGluAsnAsn 
215220225 
PheThrSerLysGlnGluCysLeuArgAlaCysLysLysGlyPheIle 
230235 240 
GlnArgIleSerLysGlyGlyLeuIleLysThrLysArgLysArgLys 
245250255260 
LysGlnArgValLysIleAlaTyrGluGluIlePheVal LysAsnMet 
265270275 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 81 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..81 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..54 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 55..72 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ATGA AGTGGGTTACTTTCATCTCTTTGTTGTTCTTGTTCTCTTCTGCT48 
MetLysTrpValThrPheIleSerLeuLeuPheLeuPheSerSerAla 
151015 
TAC TCTAGAGGTGTTTTCAGGAGGGATTCTGAG81 
TyrSerArgGlyValPheArgArgAspSerGlu 
2025 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetLysTrpValThrPheIleSerLeuLeuPheLeuPheSerSerAla 
151015 
TyrSerArg GlyValPheArgArgAspSerGlu 
2025 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 231 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(iii) HYPOTHETICAL: NO 
( iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 76..231 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 76..222 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GAATTCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTC ATACACAATA60 
TAAACGATTAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATCGGA111 
MetLysAlaValPheLeuValLeuSerLeuIleGly 
15 10 
TTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAGATT159 
PheCysTrpAlaGlnProValThrGlyAspGluSerSerValGluIle 
1520 25 
CCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAACGTC207 
ProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsnVal 
303540 
GCCATGGCTAAGAGAGATTCTGAG231 
AlaMetAlaLysArgAspSerGlu 
4550 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 52 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla 
151015 
GlnProValThrGlyAspGlu SerSerValGluIleProGluGluSer 
202530 
LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaLys 
3540 45 
ArgAspSerGlu 
50