Source: http://www.google.com/patents/US5373090?dq=6,460,050
Timestamp: 2014-11-29 07:54:54
Document Index: 550049556

Matched Legal Cases: ['application No. 238', 'application No. 4501', 'application No. 4501', 'application No. 0214826', 'application No. 195691', 'application No. 163529']

Patent US5373090 - Aprotinin analogues and a process for the production thereof - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThis invention relates to novel aprotinin analogues having a selected inhibition profile against serine proteases....http://www.google.com/patents/US5373090?utm_source=gb-gplus-sharePatent US5373090 - Aprotinin analogues and a process for the production thereofAdvanced Patent SearchPublication numberUS5373090 APublication typeGrantApplication numberUS 07/598,737PCT numberPCT/DK1989/000096Publication dateDec 13, 1994Filing dateApr 25, 1989Priority dateApr 26, 1988Fee statusPaidAlso published asDE68914022D1, DE68914022T2, EP0339942A2, EP0339942A3, EP0339942B1, WO1989010374A1Publication number07598737, 598737, PCT/1989/96, PCT/DK/1989/000096, PCT/DK/1989/00096, PCT/DK/89/000096, PCT/DK/89/00096, PCT/DK1989/000096, PCT/DK1989/00096, PCT/DK1989000096, PCT/DK198900096, PCT/DK89/000096, PCT/DK89/00096, PCT/DK89000096, PCT/DK8900096, US 5373090 A, US 5373090A, US-A-5373090, US5373090 A, US5373090AInventorsKjeld Norris, Lars C. PetersenOriginal AssigneeNovo Nordisk A/SExport CitationBiBTeX, EndNote, RefManPatent Citations (5), Non-Patent Citations (4), Referenced by (18), Classifications (13), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetAprotinin analogues and a process for the production thereofUS 5373090 AAbstract This invention relates to novel aprotinin analogues having a selected inhibition profile against serine proteases.
We claim: 1. An aprotinin analogue having an inhibitory effect against serine protease of the following formula:X1 -Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-X2 -X3 -X4 -X5 -X6 -X7 -X8 -X9 -Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-X10 -Arg-Ala-X11 -X12 -Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala (SEQ ID NO: 2)wherein X1 is Arg-Pro,Pro or hydrogen; X2 is Gly; X3 is Pro; X4 and X10 are both Cys; X5 is Lys or Arg X6 is Ala or Gly; 2. The aprotinin analogue having the following sequence:Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-Ala-Ile-Glu-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala (SEQ ID NO: 22). 3. The aprotinin analogue having the following sequence:Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-Ala-Ile-Glu-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala (SEQ ID NO: 24). 4. The aprotinin analogue having the following sequence:Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala (SEQ ID NO: 26). 5. The aprotinin analogue having the following sequence:Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala (SEQ ID NO: 29). 6. The aprotinin analogue having the following sequence:Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Arg-Ala-Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala (SEQ ID NO: 31). Description
FIELD OF THE INVENTION The present invention relates to novel aprotinin analogues and to a process for their production.
BACKGROUND OF THE INVENTION Throughout the present specification the term naturally occuring amino acid (or amino acid residue) refers to one of the α-amino acids which are listed below together with the symbols used to designate the individual amino acid residues:
______________________________________Asp      Aspartic acid Ile      IsoleucineThr      Threonine     Leu      LeucineSer      Serine        Tyr      TyrosineGlu      Glutamine acid                  Phe      PhenylalaninePro      Proline       His      HistidineGly      Glycine       Lys      LysineAla      Alanine       Arg      ArginineCys      Cysteine      Trp      TryptophaneVal      Valine        Gln      GlutamineMet      Methionine    Asn      Asparagine______________________________________
All amino acids (or amino acid residues) mentioned in the present specification have the L-configuration, except glycine which has no chiral center.
Aprotinin (bovine pancreatic trypsin inhibitor, BPTI) is a polypeptide present in several bovine organs and tissues, such as lymph nodes, pancreas, lung, parotid gland, spleen and liver. It is a single chain polypeptide consisting of 58 amino acid residues cross-linked by three disulphide bridges in the following sequence: ##STR1##
The three disulphide bridges are situated between Cys(5)-Cys(55), Cys(14)-Cys(38) and Cys(30)-Cys(51), respectively.
Aprotinin inhibits various serine proteases, such as trypsin, chymotrypsin, plasmin and kallikrein and is used for the treatment of acute pancreatitis, various states of shock syndroms, hyperfibrinolytic haemorrhage, and myocardial infarction. Administration of aprotinin in high doses significantly reduces blood loss in connection with cardiac surgery.
Aprotinin is extracted from various bovine organs or tissues, such as lung, pancreas and parotid glands. Extraction from animal tissues is a cumbersome process and requires large amounts of the bovine organ or tissue. Aprotinin may also be produced by recombinant DNA-technology by insertion of a gene coding for aprotinin in a suitable microorganism which when cultured in a suitable nutrient medium produces the desired product.
Production of aprotinin analogues in E. coli is described in EP published patent application No. 238,993 and production of aprotinin in yeast is described in Danish patent application No. 4501/87.
Certain aprotinin analogues and derivatives have been described, see for instance Jering H. and Tschesche H., Eur.J.Biochem. 61 (1976), 453-463 describing replacement of Lys(15) with Arg, Phe or Trp or U.S. Pat. No. 4,595,674 describing aprotinin analogues in which the lysine residue in position 15 in the active center of the aprotinin has been replaced by Gly, Ala, Val, Leu, Ile, Met, Arg, L-α-amino butyric acid, L-norvaline, L-norleucine, dehydroalanine or L-homoserine. Also, the above mentioned EP No. 238,993 describes aprotinin analogues having Lys(15) substituted by Arg, Val, Ile, Leu, Phe, Gly, Ser, Trp, Tyr or Ala and/or Met(52) substituted by Glu, Leu, Val, Thr or Ser.
The known aprotinin analogues are claimed to have modified effects and efficacies towards different proteinases. For instance aprotinin(15 Val) has a relatively high selectivity for granulocyte elastase and an inhibition effect on collagenase, aprotinin(15Ala) has only a weak inhibitory effect on elastase and aprotinin(15Gly) has an outstanding antitrypsin activity and surprisingly inhibits kallikrein.
SUMMARY OF THE INVENTION It is the purpose of the present invention to provide novel aprotinin analogues having a more specific inhibitory effect towards certain serine proteases, such as elastase, kallikrein, t-PA, urokinase and coagulation factors, such as thrombin.
The present invention is based on the surprising fact that replacement of Arg in position 17 of aprotinin(3-58;42Ser) with Ala or of Arg in position 17 with Ala and of Ile in position 19 of aprotinin (3-58;42Ser) with Glu gives rise to a substantial increase in the inhibition of human plasma kallikrein. This is even more pronounced on replacement of Lys in position 15 with Arg and of Arg in position 17 with Ala in aprotinin (3-58; 42Ser).
Accordingly, the present invention is related to aprotinin analogues in which at least one of the amino acid residues 16 to 19 have been replaced by another naturally occuring amino acid residue.
In the above mentioned Danish patent application No. 4501/87 aprotinin analogues with certain amino acid residue substitutions and/or deletions are described. The aim of these amino acid residue substitutions and/or deletions is to avoid proteolytical cleavage at certain basic amino acid residues during the production of the aprotinin analogue in yeast. In particular amino acid residues 1 and 2 may be deleted and one of the basic amino acid residues at the dibasic sequence 41- 42 may be replaced by another amino acid residue. It has been shown that such aprotinin analogues are produced in high yields in yeast and exhibit the same characteristics as native aprotinin.
To ensure high production in yeast the present aprotinin analogues may further be modified in a similar way. Accordingly the present aprotinin analogues besides being modified in the sequence from amino acid 16 to 19 may also be modified at sequence 1-2 and 41-42, respectively provided that such further modification does not have an adverse effect on the goal of the present invention.
The present aprotinin analogues may furthermore be modified in position 15 by insertion at this position of another naturally occuring amino acid residue including the known substitutions with naturally occuring amino acid residues.
Native aprotinin contains a disulphide bridge between Cys(14) and Cys(38) which is strongly involved in the tertiary structuring around the active site at Lys (15) . This disulphide bond may be split by reducing agents. A more convenient way to avoid having a disulphide bridge at this position in the molecule would be to substitute the cysteine residues with other residues or simply to delete these residues. Accordingly, the present aprotinin analogues may furthermore be modified so that they do not contain a disulphide bridge between Cys(14) and Cys(38). This might impart further interesting characteristics to the molecule.
Finally modifications at positions 12 and 13 might be considered.
Accordingly in its broadest aspect the present invention relates to aprotinin analogues being modified in the sequence from amino acid residue 12 to 19 provided that if Lys(15) has been replaced by another amino acid then at least one further amino acid residue in said sequence has been substituted or deleted. The present aprotinin analogues may furthermore as described above be modified in the known way at the sequences 1-2 and 41-42.
According to a further aspect of the present invention there is provided a method for producing the above aprotinin analogues by cultivation of a yeast strain containing a replicable expression vector containing a gene encoding the aprotinin analogues in a suitable nutrient medium followed by recovery of the desired product from the culture medium.
BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further illustrated by reference to the accompanying drawings in which:
FIG. 1 shows a synthetic gene encoding aprotinin (3-58; 42Ser);
FIG. 2 illustrates the construction of the plasmid pKFN 306;
FIG. 3 illustrates the construction of the plasmid pKFN 504;
FIG. 4 illustrates the construction of the plasmid pMT 636;
FIG. 5A illustrates the inhibition of plasma kallikrein by native aprotinin and by the aprotinin analogues KFN 396 and KFN 399; and
FIG. 5B illustrates the inhibition of plasma kallikrein by native aprotinin and by the aprotinin analogues KFN 396, KFN 772 and KFN 773.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS The present aprotinin analogues may be represented by the following formula (I): ##STR2## in which X1 is Arg-Pro, Pro or hydrogen, preferably Arg-Pro or hydrogen, most preferred hydrogen; X2 is any naturally occuring amino acid residue, preferably Gly; X3 is any naturally accuring amino acid residue, preferably Pro; X4 and X10 are each any naturally ocurring amino acid residue, preferably they are both Cys or both Ala, most preferred they are both Cys;X5 is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala, preferably Lys or Arg, with the proviso that when X5 is different from Lys, then at least one of X2 to X4 and X6 to X9 are different from the amino acid residue at the corresponding position in native aprotinin; X6 is Ala or Gly, preferably Ala; X7 is any naturally occuring amino acid residue, preferably Ala or Gly; X8 is Ile, Leu, Met, Val or Phe, preferably Ile; X9 is any naturally occuring amino acid residue, preferably Ile or Glu; X11 is any naturally Occuring amino acid residue, preferably Lys or Arg; X12 is Lys, Arg or Set at least one of the amino acid residues X2 to X9, preferably X5 to X9, more preferred X6 to X9 being different from the corresponding amino acid residue in native aprotinin.
According to a more narrow aspect the present aprotinin analogues may be represented by the following formula (II): ##STR3## in which X1, X5, X6, X7, X8, X9, X11 and X12 are as defined above for formula (I), at least one of the amino acid residues X5 to X9, preferably X6 to X9 being different from the corresponding amino acid residue in native aprotinin.
According to an even narrower aspect the aprotinin analogues may be represented by the following formula (III): ##STR4## in which X1, X6, X7, X8, X9, X11 and X12 are as defined above for formula (I) , at least one of the amino acid residues X6 to X9 being different from the corresponding amino acid residue in native aprotinin.
Examples of preferred aprotinin analogues according to the present invention are aprotinin(3-58; 17Ala+42Ser ) , which lacks the first two amino acid residues of native aprotinin and has Ala substituted for Arg in position 17 and Ser substituted for Arg in position 42; aprotinin (3-58; 17Ala+19Glu+42Ser), which lacks the first two amino acid residues of native aprotinin and has Ala substituted for Arg in position 17, Glu substituted for Ile in position 19 and Ser substituted for Arg in position 42; and aprotinin (3-58; 15Arg+17Ala+42Ser) which lacks the first two amino acid residues of native aprotinin and has Arg substituted for Lys in position 15, Ala substituted for Arg in position 17 and Ser substituted for Arg in position 42, respectively.
Further examples of aprotinin analogues according to the present invention are:
Aprotinin(3-58; 17Ala)
Aprotinin(3-58; 17Ala+19Glu)
Aprotinin(3-58; 15Arg+17Ala)
Aprotinin(17Ala+42Ser)
Aprotinin(15Arg+17Ala+42Ser)
Aprotinin(17Ala)
Aprotinin(17Ala+19Glu)
Aprotinin(15Arg+17Ala)
For secretion purposes the DNA-sequence encoding the desired aprotinin-analogue is fused to a DNA-sequence encoding a signal and leader peptide sequence. The signal and leader peptides are cleaved off by the transformed microorganism during the secretion of the expressed protein product from the cells ensuring a more simple isolation procedure of the desired product. A well suited leader peptide system for yeast is the yeast MFαl leader sequence of a part thereof (Kurjan, J. and Herskowitz, I., Cell 30 (1982) 933-943). However, any signal- or leader-sequence which provides for secretion in yeast may be employed and the present invention is not contemplated to be restricted to a specific secretion system.
For expression purposes a promoter sequence is positioned upstream to the DNA-sequence for the desired protein product. Preferably a promoter from a gene indigenous to the yeast host organism is used, e.g. the promoter of the TPI-(triose phosphate isomerase) gene. The DNA-sequence for the desired product will be followed by a transcription terminator sequence, preferably a terminator sequence from a gene indigenous to the host yeast organism, e.g. the terminator of the TPI-gene or the MFαl gene.
The DNA-sequence encoding the desired aprotinin analogue fused to appropriate promoter, signal, leader and terminator sequences is inserted into an expression vector for expression of the aprotinin analogue in yeast.
The expression vector may be a plasmid capable of independent replication in yeast or capable of integration into the yeast chromosome. The plasmid may preferably be stabilized against plasmid loss by the host microorganism by incorporation of a gene essential for the viability or normal growth of the host cells, e.g. a gene coding for cell division, cell wall biosynthesis, protein synthesis, etc.
EXAMPLE 1 Aprotinin(3-58; 17Ala+42Ser) (KFN 396) A sequence encoding aprotinin(3-58; 42Ser) was constructed from a number of oligonucleotides by ligation.
The oligonucleotides were synthesized on an automatic DNA synthesizer using phosphoramidite chemistry on a controlled pore glass support (S. L. Beaucage and M. H. Caruthers (1981) Tetrahedron Letters 22, 1859-1869).
The following 10 oligonucleotides were synthesized: ##STR5##
5 duplexes A-E were formed from the above 10 oligonucleotides as shown in FIG. 1 and 2.
20 pmole of each of the duplexes A-E was formed from the corresponding pairs of 5'-phosphorylated oligonucleotides I-X by heating for 5 min. at 90� C. followed by cooling to room temperature over a period of 75 minutes. The five duplexes were mixed and treated with T4 ligase. The synthetic sequence was isolated as a 176 bp band after electrophoresis of the ligation mixture on a 2% agarose gel.
The synthetic sequence was ligated to a 330 bp EcoRI-HgaI fragment from plasmid pKFN9 coding for MFαl signal and leader sequence(l-85) and to the large EcoRI-XbaI fragment from pUC19. The construction of pKFN9 containing a HgaI site immediately after the MFαl leader sequence is described in European patent application No. 0214826.
The ligation mixture was used to transform a competent E. coli strain (r-, m30) selecting for ampicillin resistance. Sequencing of a 32 p-XbaI-EcoRI fragment (Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) showed that plasmids from the resulting colonies contained the correct DNA-sequence for aprotinin(3-58; 42Ser) .
One plasmid pKFN306 was selected for further use. The construction of plasmid pKFN306 is illustrated in FIG. 2.
To introduce Ala in position 17 the following oligonucleotides were synthesized as described above: ##STR6##
The oligonucleotides were 5'-O-phosphorylated by treatment with ATP and T4 -kinase.
A duplex formed by annealing 5'-phosphorylated oligonucleotides Ia and IIa was ligated to the 352 bp EcoRI-PflMI fragment and the 3 kbp EcoRI-StyI fragment, both from pKFN306. pKFN306 encodes the S.cerevisiae mating factor αl signal-leader (1-85) fused to the synthetic aprotinin (3-58; 42Ser) gene.
The ligation mixture was used to transform a competent E. coli strain (r-, m+) selecting for ampicillin resistance. Sequencing of a 32 P-XbaI-EcoRI fragment (Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) showed that plasmids from the resulting colonies contained the correct DNA-sequence for aprotinin(3-58; 17Ala+42Ser).
One plasmid, pKFN501 was selected for further use. The construction of plasmid pKFN501 is illustrated in FIG. 3.
pKFN501 was cut with EcoRI and XbaI and the 0.5 kb fragment was ligated to the 9.5 kb NcoI-XbaI fragment from pMT636 and the 1.4 kb NcoI-EcoRI fragment from pMT636, resulting in plasmid pKFN504, see FIG. 3. Plasmid pMT636 was constructed from pMT608 after deletion of the LEU-2 gene and from pMT479, see FIG. 4. pMT608 is described in European patent application No. 195691. pMT479 is described in European patent application No. 163529. pMT479 contains the Schizo. pombe TPI gene (POT), the S. cerevisiae triosephosphate isomerase promoter and terminator, TPIp and TPIT (Alber, T. and Kawasaki, G. (1982) J.Mol.Appl.Gen. 1, 419-434). Plasmid pKFN504 contains the following sequence:
TPIP -MFαl-signal-leader(1-85)-aprotinin(3-58;17Ala+42Ser)-TPIT where MFαl is the S. cerevisiae mating factor alpha 1 coding sequence (Kurjan, J. and Herskowitz, I. (1982) Cell 30, 933-943), signal-leader(1-85) means that the sequence contains the first 85 amino acid residues of the MFαl signal-leader sequence and aprotinin(3-58; 17Ala+42Ser) is the synthetic sequence encoding an aprotinin derivative lacking the first two amino acid residues at the N-terminus and having amino acid residues 17 and 42 replaced by an Ala and a Ser residue, respectively.
S. cerevisiae strain MT663 (E2-7B XE11-36 a/α, ΔtpiΔtpi, pep 4-3/pep 4-3) was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an optical density at 600 nm of 0.6.
100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of (1.2M sorbitol, 25 mM Na2 EDTA pH=8.0, 6.7 mg/ml dithiotreitol). The suspension was incubated at 30� C. for 15 minutes, centrifuged and the cells resuspended in 10 ml of (1.2M sorbitol, 10 mM Na2 EDTA, 0.1M sodium citrate pH=5.8, 2 mg Novozym� 234). The suspension was incubated at 30� C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2M sorbitol and 10 ml of CAS (1.2M sorbitol, 10 mM CaCl2, 10 mM Tris HCl (Tris=Tris(hydroxymethyl)-aminomethan) pH=7.5) and resuspended in 2 ml of CAS. For transformation 0.1 ml of CAS-resuspended cells were mixed with approximately 1 μg of plasmid pKFN504 and left at room temperature for 15 minutes. 1 ml of (20% polyethylenglycol 4000, 10 mM CaCl2, 10 mM Tris HCl, pH=7.5) was added and the mixture left for further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2M sorbitol, 33% v/v YPD, 6.7 mM CaCl2, 14 μg/ml leucine) and incubated at 30� C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2M sorbitol. 6 ml of top agar (the SC medium of Sherman et al. , (Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) containing 1.2M sorbitol plus 2.5% agar) at 52� C. was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium. Transformant colonies were picked after 3 days at 30� C., reisolated and used to start liquid cultures. One such transformant KFN396 was chosen for further characterization.
Yeast strain KFN396 was grown on YPD medium (1% yeast extract, 2% peptone (from Difco Laboratories), and 2% glucose). A 1 liter culture of the strain was shaken at 30� C. to an optical density of 600 nm of 13. After centrifugation the supernatant was purified by FPLC ion exchange chromatography. The yeast supernatant was filtered through a 0.22 μm Millex� GV filter unit and 1 ml was applied on a MonoS cation exchange column (0.5�5 cm) equilibrated with 20 mM Bicine, pH 8.7. After wash with equilibration buffer the column was eluated with a linear NaCl gradient (0-1M) in equilibration buffer. Trypsin inhibitor activity was quantified in the eluated fractions by spectrophotometric assay and furthermore by integration
______________________________________E 280 1%  (aprotinin) = 8.3______________________________________
The yield was about 4.3 mg/liter of aprotinin(3-58; 17Ala+42Ser).
For amino acid analysis the yeast supernatant (7 ml) was adjusted to pH 8.7 with 0.1M NaOH and filtered (0.22 μm). The effluent from a Q-Sepharose anion exchange column (1�4 cm) equilibrated with 20 mM Bicine, pH 8.7 was applied to a MonoS cation exchange column (0.5�5 cm). The cation exchange chromatography was carried out as described above. Concentration of the gradient eluated aprotinin (3-58) was made by rechromatography on MonoS and eluation with steep NaCl-gradient. The collected fractions were further concentrated by vacuum centrifugation to about 100 μl and applied to a RP-HPLC column (Vydac C4, 4.6�250 mm). Eluation was carried out with CH3 CN gradient in 0.1% TFA. The collected fractions were concentrated to about 100 μl by vacuum centrifugation and samples were taken for amino acid analysis.
The amino acid analysis appears from the following Table 1. From this table it appears that the product has the expected amino acid composition.
TABLE 1______________________________________                Aprotinin                (3-58;17 Ala + 42 Ser)Amino acid Theoretical                (found)______________________________________Asx        5         4.90Thr        3         2.95Ser        2         2.10Glx        3         3.01Pro        3         3.14Gly        6         5.93Ala        7         6.69Cys        6         5.91Val        1         1.02Met        1         0.99Ile        2         2.00Leu        2         1.98Tyr        4         3.73Phe        4         3.75Lys        4         4.29Arg        3         3.21Total      56        55.60______________________________________
EXAMPLE 2 Aprotinin(3-58;17Ala+19Glu+42Ser) (KFN399) A synthetic gene encoding aprotinin(3-58; 17Ala +19Glu+42Ser) was constructed as described in Example 1. The following oligonucleotides Ib and IIb were used instead of Ia and IIa: ##STR7##
The pUC19 derived plasmid pKFN503 was constructed in a similar way as pKFN501.
By following the Procedure of Example 1 a plasmid pKFN507 was obtained containing the following construction
TPIP -MF&#945;l-signal-leader (1-85)-aprotinin(3-58;17Ala+19Glu +42Ser)-TPIT where aprotinin(3-58; 17Ala+19Glu+42Ser) is the synthetic gene encoding an aprotinin derivative lacking the first two amino acid residues at the N-terminal and having the residues 17, 19 and 42 of native aprotinin replaced by an alanine, a glutamic acid and a serine residue respectively.
Plasmid pKFN507 was transformed in yeast strain MT663 as described above and culturing of the transformed strain KFN399 gave about 10 mg/liter of aprotinin(3-58; 17Ala+19Glu+42Ser).
The amino acid analysis appears from the following Table 2 and confirms the expected amino acid composition.
TABLE 2______________________________________               Aprotinin               (3-58;17 Ala + 19 Glu + 42 Ser)Amino acid    Theoretical               (found)______________________________________Asx      5          4.96Thr      3          2.83Ser      2          1.90Glx      4          4.08Pro      3          2.98Gly      6          5.98Ala      7          6.92Cys      6          5.06Val      1          0.99Met      1          0.86Ile      1          0.99Leu      2          1.99Tyr      4          3.77Phe      4          3.89Lys      4          4.07Arg      3          3.06Total    56         54.36______________________________________
EXAMPLE 3 Aprotinin(3-58;15Arg+17Ala+42Ser) (KFN773). A synthetic gene encoding aprotinin(3-58; 15Arg +17Ala+42Ser) was constructed as described in Example 1. The following oligonucleotides Ic and IIc were used instead of Ia and IIa: ##STR8##
The pUC19 derived plasmid pKFN777 was constructed in a similar way as pKFN501.
By following the procedure of Example 1 a plasmid pKFN807 was obtained containing the following construction
TPIP -MF&#945;l-signal-leader(1-85)-aprotinin(3-58;15Arg+17Ala +42Ser)-TPIT where aprotinin(3-58; 15Arg+17Ala+42Ser) is the synthetic gene encoding an aprotinin derivative lacking the first two amino acid residues at the N-terminal and having the residues 15, 17 and 42 of native aprotinin replaced by an arginine, an alanine and a serine residue, respectively.
Plasmid pKFN807 was transformed in yeast strain MT663 as described above and culturing of the transformed strain KFN773 gave about 8.5 mg/liter of aprotinin(3-58; 15Arg+17Ala+42Ser).
The amino acid analysis is shown in Table 3 and confirms the expected amino acid composition.
TABLE 3______________________________________               Aprotinin               (3-58;15 Arg + 17 Ala + 42 Ser)Amino acid    Theoretical               (found)______________________________________Asx      5          4.95Thr      3          2.85Ser      2          1.81Glx      3          3.01Pro      3          3.05Gly      6          5.92Ala      7          6.91Cys      6          5.31Val      1          1.02Met      1          0.73Ile      2          1.41Leu      2          1.99Tyr      4          3.80Phe      4          3.94Lys      3          2.97Arg      4          4.24Total    56         53.91______________________________________
The slightly lowered content of Ile compared with the theoretical value can most probably be ascribed to incomplete hydrolysis of Ile(18)-Ile(19). This is well known in the art.
EXAMPLE 4 Inhibition of Serine Proteases from Plasma by Aprotinin(3-58; 17Ala+42Ser) (KFN396) and Aprotinin(3-58; 17Ala+19Glu+42Ser) (KFN399), Aprotinin(3-58;15Arg+42Ser) (KFN772) and Aprotinin(3-58; 15Arg+17Ala+42Ser) (KFN773).
Aprotinin(3-58; 17Ala+42Ser) (KFN396), aprotinin(3-58; 17Ala+19Glu+42Ser) (KFN399) and aprotinin (3-58; 15Arg+17Ala+42Ser) (KFN773) were purified as described above. As native, bovine pancreatic aprotinin(1-58) batch B 5029-65 (67000 KIU/mg) from NOVO (Bagsvaerd, Denmark) was used. The concentration was calculated using E280 nm=8.3 and Mr =6500. Human plasma kallikrein was obtained from Sigma (St. Louis, Mo.), bovine factor Xa was purified according to (H. Nobukazu et al. J.Biochem. 97 (1985)1347-1355), human factor IIa (thrombin) was a gift from Dr. W. Lawson (New York State Dept. of Health, Albany, N.Y.), recombinant human factor VIIa was from NOVO (Bagsvaerd, Denmark) and recombinant human protein Ca was from ZymoGenetics, Inc. (Seattle, Wash.). Substrate S 2302 (H-D-Pro-Phe-Arg-p-nitroanilide) substrate S2238 (H-D-Phe-Pip-Arg-p-nitroanilide) and substrate S2366 (Glu-Pro-Arg-p-nitroanilide) were from Kabi (Stockholm, Sweden). Substrate FXa-1 (methoxycarbonyl DCH-Gly-Arg-p-nitroanilide) was from NycoMed (Oslo, Norway). The experiments were performed in 100 mM NaCl, 50 mM Tris-HCl 0.01% Tween80, pH 7.4 at 25� C.
Human plasma kallikrein (3 nM) was incubated with aprotinin (0-20 nM) for 30 minutes in a micro-titer well. Substrate S 2302 (0.6 mM) was added to a final volume of 300 μand the rate of nitroaniline generation was measured at 405 nm by means of a Micro ELISA� Autoreader MR 580 from Dynatech Laboratories. The rate is proportional to the concentration of free enzyme. The inhibition of plasma kallikrein by native aprotinin and the four analogues KFN 396, KFN 399, KFN 772 and KFN 773 is shown in FIGS. 5A and 5B. With native aprotinin a moderate inhibition was observed. The inhibition was strongly increased by analogues KFN 396 and KFN 399 containing Ala in position 17 (FIG. 5A).
A further increase of the inhibition was obtained with Arg in position 15 (KFN 772); and the strongest inhibition was observed with the analogue (KFN 773) with substitution of both position 17 (Ala) and position 15 (Arg) (FIG. 5B).
The analogues were also tested for inhibition of the amidolytic activity of the serine proteases: bovine factor Xa, human factor IIa, human recombinant factor VIIa and human recombinant protein Ca. The experiments were performed essentially as described for plasma kallikrein only appropriate substrates were used. Finally the analogues were analysed for an effect on the coagulation factors of human plasma by means of two clotting tests. These tests, the prothrombin time (PTT) and the activated thromboplastin time (APTT) were performed with General Diagnostics� reagents from Organon (Durham, N.C.) according to the directions given by the manufacturer. The results of the inhibition experiments are summarized in Table 4 which describes the inhibition profile of the four aprotinin analogues. KFN 773 is characterized by an extraordinaryly strong inhibition of human plasma kallikrein, which is ten fold stronger than that of the Arg 15 analogue (KFN 772). A reverse effect is observed with activated protein C. In this case the relatively strong inhibition obtained by substitution of Lys 15 to Arg is weakened by further substitution of Arg 17 to Ala.
TABLE 4______________________________________Inhibition profile of aprotinin analoguesKi *.sup.) (nM);Amidolytic activityof serin proteases � ClotPlasma                    Prot.   assaysProduct  kallikrein           FIIa   FVIIa FXa  Ca    PTT  APTT______________________________________Native 180      -      -     -    400   -    -AprotininKFN 396  12       -      -     -          -    -KFN 399  12KFN 772  1        -      -     1800  10   -    +KFN 773  0.1      -      -      150 100   -    +______________________________________ - No inhibition at 1.0 &#956;M aprotinin analogue + Prolonged clotting time at 1.0 &#956;M aprotinin analogue *.sup.) Inhibition constants estimated according to the graphical Dixon method (M. Dixon, Biochem. J. 129 (1972)197-202) � Substrates: Plasma kallikrein: S2302; FIIa: S2238; FVIIa: Substrat FXa1: Substrate FXa1; Prot. Ca: S2366.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 34(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 58 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: bovine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla151015ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCy sGlnThr202530PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla354045 GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:XaaAspPheCysLeuGluProProTyrThrXaaXaaXaaXaaXaaXaa151015XaaXaaArgTyrPheTyrAsnAlaLysAla GlyLeuCysGlnThrPhe202530ValTyrGlyGlyXaaArgAlaXaaXaaAsnAsnPheLysSerAlaGlu3540 45AspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:XaaAspPheCysLeuGluProProTyrThrGlyProCysXaaXaaXaa151015XaaXaaArgTyrPheTyrAsn AlaLysAlaGlyLeuCysGlnThrPhe202530ValTyrGlyGlyCysArgAlaXaaXaaAsnAsnPheLysSerAlaGlu35 4045AspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:XaaAspPheCysLeuGluProProTyrThrGlyProCysLysXaaXaa151015XaaXaaArgTyrP heTyrAsnAlaLysAlaGlyLeuCysGlnThrPhe202530ValTyrGlyGlyCysArgAlaXaaXaaAsnAsnPheLysSerAlaGlu35 4045AspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 37 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:AAAGAGATTTCTGTTTGGAACCTCCATACACTGGTCC37(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B ) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TTACATGGACCAGTGTATGGAGGTTCCAAACAGAAACT38(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:ATGTAAAGCTAGAATCATCAGATACTTCTACAACG 35(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CTTGGCGTTGTAGAAGTATCTGATGATTCTAGCT 34(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CCAAGG CTGGTTTGTGTCAAACTTTCGTTTACGGTGGCT39(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 40 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:CTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC40(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GCAGAGCTAAGTCCAACAACTTCAAGT27(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:AGCAGACTTGAAGTTGTTGGACTTAG26(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CTGCTGAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT39(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:CTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTC 38(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:CTGGTCCATGTAAAGCTGC TATCATCAGATACTTCTACAACGC43(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic( xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:CTTGGCGTTGTAGAAGTATCTGATGATAGCAGCTTTACATGGACCAGTGT50(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:ThrGlyProCysLysAlaAlaIleIleArgTyrPheTyrAsnAla151015(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:CTGGTCCATGTAAAGCTGCTATCGAAAGATACTTCTACAACGC43(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:CTTGGCGTTGTAGAAGTATCTTTCGATAGCAGCTTTACATGGACCAGTGT 50(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:CTGGTCCATGTAGAGCTGCTATC ATCAGATACTTCTACAACGC43(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:CTTGGCGTTGTAGAAGTATCTGATGATAGCAGCTCTACATGGACCAGTGT50(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 58 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla151015AlaIleGluArgTyrPheTyrAsnAlaLys AlaGlyLeuCysGlnThr202530PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla3540 45GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle151015GluArgTyrPheTyrAsn AlaLysAlaGlyLeuCysGlnThrPheVal202530TyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAlaGluAsp35 4045CysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 58 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla151015AlaIleGluArgT yrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr202530PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla35 4045GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle151015Gl uArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal202530TyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAlaGluAsp 354045CysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 58 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla151015 AlaIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr202530PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla354045GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle1510 15IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal202530TyrGlyGlyCysArgAlaLysArgAsnAsnPheLys SerAlaGluAsp354045CysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle1510 15IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal202530TyrGlyGlyCysArgAlaLysSerAsnAsnP heLysSerAlaGluAsp354045CysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 58 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla15 1015AlaIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr202530PheValTyrGlyGlyCysArgAlaLy sSerAsnAsnPheLysSerAla354045GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:AspPheCysLeuGluProProTyrThrGlyProCysArgAlaAlaIle15 1015IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal202530TyrGlyGlyCysArg AlaLysSerAsnAsnPheLysSerAlaGluAsp354045CysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 58 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE:(A) ORGANISM: synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:ArgProAspPheCysLeuGluProProTyrThrGlyProCysArgAla1 51015AlaIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr202530PheValTyr GlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla354045GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 177 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(vi) ORIGINAL SOURCE:(A) ORGANISM: Synthetic(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 6..173(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:AAAGAG ATTTCTGTTTGGAACCTCCATACACTGGTCCATGTAAAGCT47AspPheCysLeuGluProProTyrThrGlyProCysLysAla1510AGAATCATCAGATACT TCTACAACGCCAAGGCTGGTTTGTGTCAAACT95ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr15202530TTCGTTTACGGT GGCTGCAGAGCTAAGTCCAACAACTTCAAGTCTGCT143PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla354045GAAGACTGCATG AGAACTTGTGGTGGTGCCTAAT177GluAspCysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:AspPheCysLeuGluProProTyrThrGlyProCysLysAlaArgIle151015IleArgTyrPheTyrAsnAl aLysAlaGlyLeuCysGlnThrPheVal202530TyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAlaGluAsp3540 45CysMetArgThrCysGlyGlyAla5055(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 176 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:CTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTCAGCAGACTTGAAGTTGTTGGAC60TTAGCTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGCCTTGGCGTTGTAGAAG120TATCTGATGATTCT AGCTTTACATGGACCAGTGTATGGAGGTTCCAAACAGAAATC176
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4595674 *Mar 13, 1985Jun 17, 1986Bayer AktiengesellschaftPharmaceuticalsUS5118668 *Jul 20, 1988Jun 2, 1992Bayer AktiengesellschaftPeptide having sequence of appotinin where in one or more amino acids are replaced at position 15,16,17,18 34-39 and 52. by naturally occuring amino acid useful for hyperfibrinolytic or traumatic hemorrhageEP0297362A2 *Jun 16, 1988Jan 4, 1989Bayer AgHuman aprotinine whose Lys residue in position 15 is substituted by another protogene redisue of amino-acidGB2188322A * Title not availableGB2188933A * Title not available* Cited by examinerNon-Patent CitationsReference1Fiovett: et al. "Primary Structure and Antiproteolytic Activity of a Kunits-type Inhibitor from Bovine Spleam." JBC, 260 (21) 11451-11455. 1985.2 *Fiovett: et al. Primary Structure and Antiproteolytic Activity of a Kunits type Inhibitor from Bovine Spleam. JBC, 260 (21) 11451 11455. 1985.3 *Schnabel et al., Biol. Chem. Hoppe Seyler, vol. 367, pp. 1167 1176 (1986).4Schnabel et al., Biol. Chem. Hoppe-Seyler, vol. 367, pp. 1167-1176 (1986).* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS5747449 *Jul 1, 1993May 5, 1998Corvas International, Inc.Bovine pancreatic trypsin inhibitor derived inhibitors of factor XAUS5780265 *Jun 5, 1995Jul 14, 1998Genentech, Inc.Kunitz type plasma kallikrein inhibitorsUS5786328 *Jun 5, 1995Jul 28, 1998Genentech, Inc.Use of kunitz type plasma kallikrein inhibitorsUS5962266 *Apr 2, 1997Oct 5, 1999Scios, Inc.Protease inhibitor peptidesUS6376648Jan 21, 1999Apr 23, 2002Scios Nova, Inc.PolypeptidesUS6613890Jan 21, 1999Sep 2, 2003Scios, Inc.Analogues of Kunitz protease inhibitor domain of amyloid precursor protein bind to and inhibit activity of serine proteases including kallikrein, plasmin, and coagulation factors VIIa, IXa, Xa, XIa, and XIIa; for treating blood lossUS6906033Feb 19, 2002Jun 14, 2005Scios Nova, Inc.Isolated DNA encoding amino acid sequences, serine protease inhibitor, host cell transformed with a DNA molecule, Kunitz type Inhibitor, blood coagulation factorsUS6953674Jun 11, 2002Oct 11, 2005Dyax Corp.Inhibitors of human plasmin derived from the kunitz domainsUS6989369Feb 7, 2003Jan 24, 2006Dyax Corp.Kunitz domain peptidesUS7550427Jul 20, 2006Jun 23, 2009Dyax Corp.Poly-pegylated protease inhibitorsUS7919462Oct 30, 2007Apr 5, 2011Dyax Corp.Inhibitors of human plasmin derived from the Kunitz domainsUS8431359Mar 18, 2005Apr 30, 2013Dyax Corp.Nucleotide sequences coding tissue factor pathway inhibitor for use in treatment and prevention of blood disordersUS8637454Jan 6, 2010Jan 28, 2014Dyax Corp.Treatment of mucositis with kallikrein inhibitorsUS8663629Aug 9, 2012Mar 4, 2014Dyax Corp.Kallikrein-binding �kunitz domain� proteins and analogues thereofUS8710007Nov 22, 2010Apr 29, 2014Dyax Corp.Prevention and reduction of blood lossUS8715672Dec 21, 2012May 6, 2014Csl Behring GmbhTreatment of diseases linked to pathological kinin formationUS8716225Apr 27, 2012May 6, 2014Dyax Corp.Kallikrein inhibitors and anti-thrombolytic agents and uses thereofUS8822653Jan 6, 2011Sep 2, 2014Dyax Corp.Plasma kallikrein binding proteins* Cited by examinerClassifications U.S. Classification530/324, 435/69.2International ClassificationA61K38/00, C07K1/20, C12N9/99, C07K14/81, C12P21/02, C12N15/09, C12R1/865, C12N15/12Cooperative ClassificationA61K38/00, C07K14/8117European ClassificationC07K14/81B1A1Legal EventsDateCodeEventDescriptionMay 3, 2006FPAYFee paymentYear of fee payment: 12Jun 4, 2002FPAYFee paymentYear of fee payment: 8May 11, 1998FPAYFee paymentYear of fee payment: 4Nov 28, 1995CCCertificate of correctionDec 10, 1990ASAssignmentOwner name: NOVO NORDISK A/S, NOVO ALLE, DK-2880, BAGSVAERD, DFree format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:PETERSEN, LARS C.;NORRIS, KJELD;REEL/FRAME:005539/0275;SIGNING DATES FROM 19901122 TO 19901123RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google