# Patent

## Title
Protease inhibition

## Abstract

Proteins including engineered sequences which inhibit proteases are disclosed, including proteins having two or more engineered Kunitz domains, and uses of such proteins.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of prior U.S. provisional application 60/754,903, filed Dec. 29, 2005.


BACKGROUND
Aspects of the pathology of many diseases include the excessive activity of proteases. Pharmaceutical agents that reduce such protease activity can be used to ameliorate disease conditions.
SUMMARY
Kunitz domains are robust protease inhibitors. Examples of Kunitz domains include non-naturally occurring Kunitz domains that inhibit human plasma kallikrein, human plasmin, human hepsin, human matriptase, human endotheliase 1 (hET-1) human endotheliase 2 (hET-2), and human neutrophil elastase (also known as human leukocyte elastase). Kunitz domains can be engineered to have very high affinity and specificity for most serine proteases.
Sometimes in a single disease state there is more than one protease whose activity is detrimental. In different patients, a given symptom may result from the aberrant activity of two different proteases, one in one patient, another in another patient. In cases such as these, it would be useful to have a single active pharmacologic agent that inhibits two or more proteases. Often, low-specificity inhibitors (e.g., small molecule inhibitors) cause serious side effects by inhibiting proteases other than the intended targets. A molecule that contains multiple domains, each having high affinity and specificity for a different protease can be used to provide inhibition of different target proteases without causing side effects due to cross reaction of the inhibitor with protease(s) other than the target(s). This allows the development of a single agent for the treatment of patients with multiple or various dysregulated proteases.
In one aspect, the disclosure features an isolated protein that includes one or more engineered protease inhibitory sequences, wherein the protein inhibits two or more human proteases. For example, the protein inhibits the two or more human proteases, each with a Ki of less than 100 nM, 10 nM, 1 nM, or 0.1 nM.
In one embodiment, the protein of the invention comprises two or more Kunitz domains, each of which inhibit the same protease and the protein has longer serum residence than would a single Kunitz domain due to increased molecular mass. The protein may or may not be glycosylated.
In one embodiment, the protein contains a single engineered Kunitz domain that inhibits two or more human proteases as the engineered protease inhibitory sequence.
The protease inhibitory sequences can be, for example, Kunitz domains, peptides (e.g., cyclic peptides or linear peptides of length less than 24 amino acids), and variable domains of antigen binding fragments. For example, the protease inhibitory sequences can include the amino acid sequence of a heavy chain variable domain that binds and inhibits a human protease in conjunction with a light chain variable domain.
In one embodiment, the protein includes at least two, three, four, five, six, seven, or eight Kunitz domains, as the engineered proteases inhibitory sequences. For example, the protein contains two, three, four, five, or six Kunitz domains. In some cases, at least one of the Kunitz domains comprises the framework of a naturally occurring human Kunitz domain (e.g., TFPI or other human protein mentioned herein), but that differs from the naturally occurring human Kunitz domain at no less than one residue in the protease binding loops.
In one embodiment, the protein has a molecular weight of at least 15 kiloDaltons and/or a beta-phase half life of at least 30 minutes in mice. Molecular weight of proteins and/or beta-phase half life may be increased by, for example, modifying the protein to include one or more PEG moieties or by covalently linking the protein to a carrier such as human serum albumin (HSA). Such covalent linkage may be accomplished by covalent cross-linking of the protein and the carrier following production of the protein or by expression of the protein as a fusion protein with the carrier.
In one embodiment, at least one of the Kunitz domains contains only two disulfide bonds. For example, the protein does not include cysteines at positions corresponding to amino-acid positions 14 and 38 in bovine pancreatic trypsin inhibitor (BPTI).
Exemplary proteins can inhibit:

    
    
        two or more proteases whose excessive activity contributes to rheumatoid arthritis, multiple sclerosis, Crohn's disease, cancer, and chronic obstructive pulmonary disorder;
        two or more of (e.g., all of) neutrophil elastase, proteinase 3, and cathepsin G;
        two or more of (e.g., all of) human neutrophil elastase, proteinase 3, cathepsin G, and tryptase.
        human plasma kallikrein and human neutrophil elastase;
        two or more of (e.g., all of) human kallikrein 6 (hK6), human kallikrein 8 (hK8), and human kallikrein 10 (hK10);
        hK6 and human neutrophil elastase;
        human tissue kallikrein (h.K1) and human plasma kallikrein
        two or more of human plasmin, human hepsin, human matriptase, human endotheliase 1, and human endotheliase 2.
    
    


For example, the protein includes at least two Kunitz domains, a first Kunitz domain specific for neutrophil elastase and a second Kunitz domain specific for proteinase 3. The first Kunitz domain can be N-terminal or C-terminal to the second Kunitz domain.
DX-88 is a Kunitz-domain inhibitor of human plasma kallikrein (hpKall) and DX-890 is a Kunitz-domain inhibitor of human neutrophil elastase (hNE). In one embodiment, the protein includes (i) DX-88, a domain at least 85, 90, 92, 95, 96, 97, 98 or 99% identical to DX-88, or a Kunitz domain that includes at least 70, 80, 85, 90, 95, or 100% of the amino acid residues in the protease binding loops of DX-88 and (ii) DX-890, a domain at least 85, 90, 92, 95, 96, 97, 98 or 99% identical to DX-890, or a Kunitz domain that includes at least 70, 80, 85, 90, 95, or 100% of the amino acid residues in the protease binding loops of DX-890.
In one embodiment, the protein includes at least two Kunitz domains, and at least two of which are identical or at least 90 or 95% identical. In one example, the protein includes at least two Kunitz domains, each of which is DX-88, a domain at least 85, 90, 92, 95, 96, 97, 98 or 99% identical to DX-88, or a Kunitz domain that includes at least 70, 80, 85, 90, 95, or 100% of the amino acid residues in the protease binding loops of DX-88. In another example, the protein includes at least two Kunitz domains, each of which is DX-890, a domain at least 85, 90, 92, 95, 96, 97, 98 or 99% identical to DX-890, or a Kunitz domain that includes at least 70, 80, 85, 90, 95, or 100% of the amino acid residues in the protease binding loops of DX-890.
In one embodiment, the protein includes at least two Kunitz domains, and the Kunitz domains differ from each other.
In one embodiment, the at least two Kunitz domains are connected by a linker, e.g., a flexible hydrophilic linker. For example, the linker is less than 35, 22, 15, 13, 11, or 9 amino acids in length. The linker can include at least two, three, or four glycine residues. In one embodiment, the at least two Kunitz domains are separated by a serum albumin moiety. In another embodiment, the at least two Kunitz domains are not separated by a serum albumin moiety. In one embodiment, the at least two Kunitz domains are connected by a linker that does not include T cell epitopes. For example, the at least two Kunitz domains are connected by a linker that lacks medium to large hydrophobic residues and charged residues. The linker may or may not include a glycosylation site. For example, the linker has fewer than three, two, or one glycosylation sites, i.e., no glycosylation sites. The linker can be devoid of N-linked glycosylation sites.
In one embodiment, the protein inhibits the human proteases in the presence of neutrophil defensins. The protein can be resistant to degradation by resident proteases, e.g., metalloproteases, e.g., MT6-MMP (Leukolysin). For example, the protein does not include cleavage sites recognized by MMP-8 or MMP-12. In one embodiment, the protein does not inhibit both of factor VIIa and factor Xa. The protein may differ from human bikunin by at least two amino acids.
In another aspect, the disclosure features a method of treating a subject. The method includes administering, to the subject, a protein described herein, e.g., a protein that includes one or more engineered protease inhibitory sequences, wherein the protein inhibits two or more human proteases. For example, the subject is a subject in need of a reduction in the protease activity of at least two different proteases. The subject can have a disorder to which first and second human proteases contribute and the protein administered inhibits the first and second human proteases. For example, the protein includes a Kunitz domain that specifically inhibits the first human protease and a Kunitz domain that specifically inhibits the second human protease.
In another aspect, the disclosure features an isolated protein that includes (i) a Kunitz domain that inhibits a human serine protease, and (ii) a polypeptide sequence that contributes to binding and inhibition of a human protease, e.g., a metalloprotease. In one example, the protein consists of a single polypeptide chain. In another example the protein includes at least two polypeptide chains. In one embodiment, one polypeptide chain includes a heavy chain variable domain of an antigen-binding fragment and another polypeptide chain includes a light chain variable domain of an antigen-binding fragment, such that the antigen binding binds and inhibits a human protease, e.g., a metalloprotease.
In one embodiment, the Kunitz domain is a component of the same polypeptide chain as the heavy chain variable domain of the antigen-binding fragment. In another embodiment, the Kunitz domain is a component of the same polypeptide chain as the light chain variable domain of the antigen-binding fragment. For example, the Kunitz domain may be C-terminal to a constant domain of an antibody or an antigen-binding fragment thereof or N-terminal to a variable domain. In another embodiment, one Kunitz domain is N-terminal to a variable antibody domain and a second Kunitz domain is N-terminal to the final constant domain of the same chain. The two Kunitz domains may have the same inhibitory activity or different inhibitory activities.
In another aspect, the disclosure features a method of providing a protein that inhibits two or more human proteases. The method includes: providing amino acid sequences of a first Kunitz domain that inhibits a first human protease and a second Kunitz domain that inhibits a second human protease; providing the amino acid sequence of a recipient protein that includes a plurality of Kunitz domains; and preparing a polynucleotide encoding a modified amino acid sequence of the recipient protein, wherein the recipient protein is modified such that (a) amino acids of one of the Kunitz domains in the recipient protein is substituted with amino acid residues that contribute to binding or specificity of the first Kunitz domain and (b) amino acids of another of the Kunitz domains in the recipient protein is substituted with amino acid residues that contribute to binding or specificity of the second Kunitz domain.
In another aspect, the disclosure features a method of providing a protein that inhibits two or more human proteases. The method includes: providing amino acid sequences of a first Kunitz domain that inhibits a first human protease and a second Kunitz domain that inhibits a second human protease; and preparing a polynucleotide encoding a polypeptide in which sequences encoding the first and second Kunitz domains are in frame. The domains can be separated by a sequence encoding a linker sequence. In certain embodiments, (a) the linker does not include medium or large hydrophobic residues, nor charged residues, or (b) MHC I epitopes are not formed by amino acids from the first and second Kunitz domain and the linker. In one embodiment, the first and second human proteases contribute to a single human disease.
These methods can further include expressing the prepared polynucleotide in a cell, e.g., a cell in a transgenic animal (e.g., a mammary cell), or a cultured host cell. The method can further include formulating a protein encoded by the prepared polynucleotide as a pharmaceutical composition.
In another aspect, the invention features a method of treating rheumatoid arthritis. The method includes: administering, to the subject having or suspected of having rheumatoid arthritis, an effective amount of a protein that comprises one or more engineered protease inhibitory sequences. In some embodiments, the protein inhibits both human plasma kallikrein and human tissue kallikrein.
In another aspect, the disclosure features a method of treating lung inflammation. The method includes: administering, to a subject having or suspected of having lung inflammation, an effective amount of a protein that comprises one or more engineered protease inhibitory sequences. In some embodiments, the protein inhibits two or more of human neutrophil elastase, proteinase 3, cathepsin G, and tryptase.
In another aspect, the disclosure features a method of treating multiple sclerosis. The method includes: administering, to a subject having or suspected of having multiple sclerosis, an effective amount of a protein that comprises one or more engineered protease inhibitory sequences. In some embodiments, the protein inhibits two or more of human kallikrein 6 (hK6), human kallikrein 8 (hK8), and human kallikrein 10 (hK10).
In another aspect, the disclosure features a method of treating spinal chord injury. The method includes: administering, to a subject having or suspected of having spinal chord injury, an effective amount of a protein that comprises one or more engineered protease inhibitory sequences. In some embodiments, the protein inhibits both of hK6 and human neutrophil elastase.
In another aspect, the disclosure features a method of treating chronic obstructive pulmonary disorder. The method includes: administering, to a subject having or suspected of having chronic obstructive pulmonary disorder, an effective amount of a protein that comprises one or more engineered protease inhibitory sequences. In some embodiments, the protein inhibits two or more of human neutrophil elastase, PR3, and cathepsin G.
In another aspect, the disclosure features a method of treating an inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis). The method includes: administering, to a subject having or suspected of having Crohn's disease, an effective amount of a protein that comprises one or more engineered protease inhibitory sequences. In some embodiments, the protein inhibits human tissue kallikrein (hK1) and human plasma kallikrein.
In another aspect, the disclosure features a protein comprising an engineered Kunitz domain that inhibits a human protease, wherein the Kunitz domain lacks the 14-38 disulfide found in most naturally occurring Kunitz domains. In some embodiments, the protease is selected from the group consisting of plasma kallikrein, human neutrophil elastase, plasmin, or other protease described herein. In one embodiment, the binding loops of the Kunitz domain are identical to the binding loops of DX-88, DX-890, or DX-1000, or at least 80, 85, 90, or 95% identical. In one embodiment, the Kunitz domain is at least 85, 90, 95, or 100% identical to DX-88, DX-890, or DX-1000, but does not include cysteines at the positions corresponding to cysteine 14 and cysteine 38 of BPTI.
In another aspect, the disclosure features a library of polynucleotides. The library includes a plurality of polynucleotides, each of which comprises a sequence encoding a Kunitz domain that lacks cysteines at positions corresponding to position 14 and 38 of BPTI. The polynucleotides of the plurality vary such that one or more residues in the first and/or second binding loops of the Kunitz domains differs among the polynucleotides of the plurality. In one embodiment, codons encoding positions 13, 15, 16, 17, 18, 19, 31, 32, 34, 39, and 40 (numbered with respect to the sequence of BPTI) are varied.
In another aspect, the invention features a polynucleotide encoding a polypeptide that includes at least two Kunitz domains that differ by no more than 80, 85, 90, 95, 98%. The polynucleotide sequences encoding the Kunitz domains use different codons at positions corresponding to one or more residues that are identical between the at least two Kunitz domains. In one embodiment, the amino-acid sequences of the Kunitz domains are identical. For example, the amino-acid sequences of the Kunitz domains are identical, yet the polynucleotide sequences encoding the identical Kunitz domains differ at least ten or twenty codons. For example, the nucleic acid sequences encoding the Kunitz domain are less than 90, 80, 70, 60, or 50% identical.
It is possible to engineer proteins that contain multiple Kunitz domains, e.g., at least two, three, four, five, or six Kunitz domains. Each Kunitz domain is engineered to have high affinity and specificity for one or more serine proteases such that the resulting protein can inhibit one to several proteases with high affinity and specificity. In addition, the size of the engineered protein can be such that the lifetime in serum is enhanced (e.g., by including amino acid residues outside of the Kunitz domains to increase the molecular weight of the protein). Any method can be used to engineer a protein with multiple Kunitz domains. Examples including fusing different domains and selecting one or more domains from a library of varied Kunitz domains.
In one aspect of the invention, a library of Kunitz domains lacking the 14-38 disulfide is constructed and high-affinity inhibitor are selected. For example, the library includes variations at one or more positions in the first and/or second binding loops of the Kunitz domains. For example, positions 13, 14, 15, 16, 17, 18, 19, 31, 32, 34, 38, 39, and 40 with respect to the sequence of BPTI are varied. In other aspects, the invention comprises the construction of genetic diversity that encodes constant or nearly constant amino acid sequences to avoid genetic instability, e.g., recombination between nucleic acid sequences encoding different segments of the protein.
All patents, patent applications, and other references cited herein are incorporated by reference in their entireties.


DETAILED DESCRIPTION
A number of disorders involve multiple proteases (Churg and Wright, 2005, Curr Opin Pulm Med, 11:153-9), and thus proteins that inhibit at least two human proteases would be useful pharmacological agents for treating (e.g., improving and/or preventing) such disorders. Such proteins can include one or more Kunitz domains, or other sequences that inhibit proteases.
Some of these disorders are associated with the excessive protease activity of dissimilar serine proteases. In such cases, the catalytic sites of any pair of serine proteases may differ enough that no single Kunitz domain can bind both proteases with useful affinity. Accordingly, provided herein are compositions which include two or more Kunitz domains, wherein the composition includes at least two different engineered Kunitz domains (or Kunitz domains with different specificities) bound via a covalent linkage. The Kunitz domains are typically linked via a peptide bond (although it should be understood that the Kunitz domains need not be directly linked by a peptide bond in the sense that the carboxy terminus of one Kunitz domain directly linked via a peptide bond to the amino terminus of another Kunitz domain); a linker of one or more amino acids may separate the Kunitz domains). A peptide linker may be any desired length, but in some embodiments the linker is limited in size, for example, less than 35, 22, 15, 13, 11, or 9 amino acids in length. The linker can include at least two, three, or four glycine residues.
In one embodiment, the at least two Kunitz domains are not separated by a serum albumin moiety. In one embodiment, the at least two Kunitz domains are connected by a linker that does not include T cell epitopes. For example, the at least two Kunitz domains are connected by a linker that lacks medium to large hydrophobic residues and charged residues. The linker may or may not include a glycosylation site. For example, the linker has fewer than three, two, or one glycosylation sites, i.e., no glycosylation sites. The linker can be devoid of N-linked glycosylation sites.
In some embodiments, proteins comprising multiple different engineered Kunitz domains are produced by transferring the binding determinants of an engineered Kunitz domain to a recipient protein that naturally includes a plurality of Kunitz domains (e.g., modifying the naturally occurring sequences by reference to an engineered sequence so as to produce a protein comprising at least two engineered Kunitz domains (or Kunitz domains with different specificities).
If two or more serine proteases are similar, one can select for a Kunitz domain that inhibits all of the target serine proteases, e.g., inhibits each target protease with a Ki of less than 10 nM. As used herein, two serine proteases are “similar” if the approximately 20 residues that correspond to the trypsin residues that touch BPTI in the trypsin-BPTI complex are 80% identical. As will be understood, the likelihood of obtaining a single Kunitz domain that inhibits two or more target serine proteases increases with increasing identity between the target proteases. In some embodiments, proteins comprising Kunitz domains with specificity for more than one protease have a K, of less than 100 nM, 10 nM, 1 nM, or 0.1 nM for each of the target proteases.
Additionally, in some embodiments, protein comprises a Kunitz domain linked to a antibody (or antigen binding fragment or derivative thereof, such as Fab's, (Fab)2, scFv, Fd, Fv) or non-Kunitz domain peptide which binds and inhibits a serine protease.
Some disorders involve not only serine proteases, but other types of proteases as well (e.g., metalloproteases, such as the matrix metalloproteases, or MMPs, or cysteine proteases). Accordingly the invention also provides proteins which comprise at least one Kunitz domain linked to at least one peptide or protein which inhibits a metalloprotease or a cysteine protease. Exemplary proteins which inhibit metalloproteases are antibodies and antigen-binding fragments or derivatives thereof (e.g., Fab's, (Fab)2, scFv, Fd, Fv) which bind to and inhibit metalloproteases, particularly those that bind to and inhibit members of the matrix metalloprotease (MMP) family. Exemplary proteins which inhibit cysteine proteases are antibodies and antigen-binding fragments or derivatives thereof (Fab's, (Fab)2, scFv, Fd, Fv) which bind to and inhibit cysteine proteases, particularly those that bind to and inhibit members of the cathepsin family (e.g. cathepsin L and/or cathepsin W).
Fusions between a single or multiple Kunitz domain-containing sequences and an antibody (or antigen-binding fragment or derivative thereof) may be in any configuration, such as fusion of the Kunitz domain-containing sequence to: a) the N-term of the light chain of an antigen binding fragment or full length antibody, b) the C-term of the light chain of an antigen binding fragment or full length antibody, c) the N-term of the heavy chain of an antigen binding fragment or full length antibody, or d) the C-term of heavy chain of an antigen binding fragment or full length antibody. Antibodies can be arranged as Fab's, (Fab)2, scFv, Fd, Fv. For example, it is can be sufficient to use a protein that includes the light chain variable domain and the heavy chain variable domain.
Peptides which inhibit may also be incorporated into the proteins of the invention. Such inhibitory peptides may be fused to a Kunitz domain or to a protein containing multiple Kunitz domains and either antibody or albumin components that will ensure long residence in blood. Peptides that inhibit metalloproteinases could be fused to either end of a protein containing one or more therapeutic Kunitz domains. Another exemplary configuration is the insertion of an inhibitory peptide between Kunitz domains (e.g., the peptide may be used to replace a inter-Kunitz domain linker in an engineered version of human TFPI, or it may be used as a linker to connect two separate Kunitz domains).
Antibodies and/or peptides which bind proteases may be identified by selection of antibody or peptide libraries with the target protease (e.g., U.S. 2005/0180977 and Huang et al., J Biol Chem. 2003, 278:15532-40). Such selection is typically coupled with a screening step to identify those library members that inhibit the target protease as well. Such methods are similar to the selection and screening methods disclosed herein, as will be understood by practitioners in the art.
As referred to herein, an “engineered” protein sequence (e.g., an engineered Kunitz domain) is a protein that has a non-naturally occurring sequence. In some embodiments, an engineered Kunitz domain will vary from a naturally occurring Kunitz domain by at least 2, 3, 4, 5, 6, 7, 8, 9, or amino acids.
The comparison of sequences and determination of percent identity between two sequences can be performed using BLAST (Altschul et al. (1990) J. Mol. Biol. 215:403-410), particularly BLAST 2 Sequences as described by Tatusova and Madden (1999, FEMS Microbiol. Lett. 174:247-250) and as implemented by the National Center for Biotechnology Information (available via the world wide web at ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). Parameters for comparison of two nucleotide sequences (e.g., BLASTN) are Reward for a match: 1; Penalty for a mismatch: −2; Open gap penalty: 5; extension penalty gap: 2; gap x_dropoff: 50; expect: 10.0; word size: 11. Parameters for comparison of two amino acid sequences (e.g., BLASTP) are Matrix: BLOSUM62; Open gap penalty: 11; extension gap penalty: 1; gap x_dropoff: 50; expect: 10.0; and word size 3.
As used herein, reference to hybridization under “low stringency,” “medium stringency,” “high stringency,” or “very high stringency” conditions describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: (a) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); (b) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (c) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and (d) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.
Kunitz Domains
A “Kunitz domain” is a polypeptide domain having at least 51 amino acids and containing at least two, and perhaps three, disulfides. The domain is folded such that the first and sixth cysteines, the third and fifth cysteine, and optionally the second and fourth form disulfide bonds (e.g., in a Kunitz domain having 58 amino acids, cysteines can be present at positions corresponding to amino acids 5, 14, 30, 38, 51, and 55, according to the number of the BPTI sequence provided below, and disulfides can form between the cysteines at position 5 and 55, 14 and 38, and 30 and 51), or, if two disulfides are present, they can form between 5 and 55 and 30 and 51. The spacing between respective cysteines can be within 7, 5, 4, 3, 2, or 1 amino acids of the following spacing between positions corresponding to: 5 to 55, 14 to 38, and 30 to 51, according to the numbering of the BPTI sequence provided herein (Table 7). The BPTI sequence can be used as a reference to refer to specific positions in any generic Kunitz domain. Comparison of Kunitz domains of interest to BPTI can be performed by identifying the best fit alignment in which the number of aligned cysteines is maximized.
The three dimensional structure (at high resolution) of the BPTI Kunitz domain is known. One of the X-ray structures of BPTI is deposited in the Brookhaven Protein Data Bank as “6PTI”. The three dimensional structure of some BPTI homologues (Eigenbrot et al., (1990) Protein Engineering, 3(7):591-598; Hynes et al., (1990) Biochemistry, 29:10018-10022) are likewise known. At least seventy natural Kunitz domain sequences are known. Known human homologues include three Kunitz domains of TFPI (Wun et al., (1988) J. Biol. Chem. 263(13):6001-6004; Girard et al., (1989) Nature, 338:518-20; Novotny et al, (1989) J. Biol. Chem., 264(31):18832-18837; in some of these references, TFPI is identified as LACI (Lipoprotein Associated Coagulation Inhibitor) or EPI (Extrinsic Pathway Inhibitor)), two Kunitz domains of Inter-α-Trypsin Inhibitor, APP-I (Kido et al., (1988) J. Biol. Chem., 263(34):18104-18107), a Kunitz domain from collagen, and three Kunitz domains of TFPI-2 (Sprecher et al., (1994) PNAS USA, 91:3353-3357). TFPI is a human serum glycoprotein with a molecular weight of 39 kDa (amino acid sequence in Table 1) containing three Kunitz domains.
Kunitz domains interact with target protease using, primarily, amino acids in two loop regions (“binding loops”). The first loop region is between about residues corresponding to amino acids 11-21 of BPTI. The second loop region is between about residues corresponding to amino acids 31-42 of BPTI. An exemplary library of Kunitz domains varies one or more amino acid positions in the first and/or second loop regions. Particularly useful positions to vary include: positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 with respect to the sequence of BPTI. At least some of these positions are expected to be in close contact with the target protease.
The “framework region” of a Kunitz domain is defined as those residues that are a part of the Kunitz domain, but specifically excluding residues in the first and second binding loops regions, e.g., about residues corresponding to amino acids 11-21 of BPTI and 31-42 of BPTI. The framework region can be derived from a human Kunitz domain, e.g., TFPI domain 1. Exemplary frameworks can include at least one, two, or three lysines. In one embodiment, the lysines are present at positions corresponding to the positions found in the framework of LACI, or within at least three, two, or one amino acid(s) from such a position.
Conversely, residues that are not at these particular positions or which are not in the loop regions may tolerate a wider range of amino acid substitutions (e.g., conservative and/or non-conservative substitutions) than these amino acid positions.
BPTI is the prototypic Kunitz domain with 58 amino acids and cysteine residues at position 5, 14, 30, 38, 51, and 55. These form disulfides 5:55, 14:38, and 30:51.
There are phenylalanine residues at 33 and 45 in almost all Kunitz domains. There are glycine residues at positions 12 and 37 in almost all Kunitz domains. There is the possibility of an extra residue between positions 9 and 10, called 9a. If 9a is present, 12 need not be glycine. Insertion of up to three residues after position 26 is observed in some natural Kunitz domains; these inserted residues can be numbered 26a, 26b, and 26c. Insertion of up to three residues after position 42 is observed in some natural Kunitz domains (numbered 42a, 42b, and 42c). Cysteine residues other than those named are observed (e.g., as seen in bungarotoxin) and can lead to disulfides linking the Kunitz domain to other proteins.
Most Kunitz domains have 58 amino acids with Cys at positions 5, 14, 30, 38, 51, and 55. These domains are termed “canonical Kunitz domains” herein. All canonical Kunitz domains have essentially identical main-chain 3D structures. The first Kunitz domain of TFPI is a canonical Kunitz domain. We have constructed a phage-display library of TFPI-K1 Kunitz domains (see, e.g., U.S. Pat. No. 6,333,402). Moreover, it is possible to select a Kunitz domain having the TFPI-K1 framework for binding to a particular target protease. Then residues 13, 15-21, 34, 39-40 can be transferred into any other canonical Kunitz domain such that the recipient Kunitz domain obtains affinity for the particular target protease.
One class of non-canonical Kunitz domains has an extra amino acid at position 9a. In this class of Kunitz domains, residue 12 need not be glycine and the 3D structure in that region is different from that of canonical Kunitz domains. In TFPI-2, the second Kunitz domain has this non-canonical feature. To re-engineer TFPI-2 Kunitz domain2, a preferred embodiment is to build a library of TFPI-2 Kunitz domain2s and select the desired binding properties. Alternatively, one could change residues 8-12 and 39-43 to be canonical. That is, we would delete S9a and change D12 to G. At 13 and 39 we would put the amino acids needed for binding and change 40-45 to GNANNF (SEQ ID NO:203) since these are the most common amino acids at these positions.
When a Kunitz domain binds to a serine protease, only a small number of the amino acids of the Kunitz domain contact the serine protease. The exact number depends on the serine protease and the Kunitz domain, but is usually in the range 12 to 20. In the case of BPTI and trypsin, 13 residues (T11, G12, P13, C14, K15, A16, R17, I18, I19, G36, G37, C38, and R39) of BPTI have at least one atom positioned within 4 Å of an atom in trypsin. Residues 12, 14, 16, 37, and 38 are highly conserved in canonical Kunitz domains. In BPTI, residue 34 is valine which comes within 5 Å of an atom of trypsin. Charge (or lack thereof) and/or a bulky side chain at this position could influence binding, thus substitution of a large or charged residue at 34 could influence binding.
There are about 20 residues of trypsin (using chymotrypsinogen numbering) that come within 4 Å of an atom in BPTI: Y39, H40, F41, C42, H57, N97, L99, Y151, D189, S190, C191, Q192, G193, D194, S195, S214, W215, G216, G219, and G226. Of these positions, C42, H57, G193, D194, S195, S214, are absolutely conserved among trypsin-homologous serine proteases. Positions 39, 41, 97, 99, 151, and 192 are highly variable. Position 40 is often histidine but shows considerable variability. Position 189 is most often D, but S, G, A, and T occur in known serine proteases. Position 190 is often S, but G, V, T, A, I, and P are observed. Position 191 is most often C, but F and Y are observed. Position 215 is most often W, but F, Y, G, N, S, and K are observed. Position 216 is most often G, but V, S, and K are observed. Position 219 is most often G, but S, T, E, P, D, N, and T are observed, as is deletion of the residue. Position 226 is often G, but T, D, S, E, and A are observed. These residues are the ones most directly responsible for the substrate specificity of a particular serine protease because they contact the substrate. For other proteases, the number of amino acids that would contact a Kunitz domain could be more or less than 20.
BPTI and other Kunitz domains inhibit serine proteases by binding in the protease catalytic site but resisting cleavage and release. For a Kunitz domain to inhibit a serine protease, it must fit tightly into the catalytic site. In some cases, the bond between residue 15 of the Kunitz domain and residue 16 is cleaved, but the cleaved Kunitz domain does not release from the protease.
BPTI is not especially specific and inhibits a variety of serine proteases with affinity better than 10 nM. For example, its affinities for different proteases are: (human urine kallikrein (4 nM), porcine pancreatic kallikrein (0.55 nM), human plasmin (0.89 nM), activated protein C (1 μM) (Taby, et al., 1990, Thromb Res, 59:27-35) thrombin (30 μM) (Pintigny and Dachary-Prigent, 1992, Eur J Biochem, 207:89-95), and bovine trypsin (0.06 pM)).
Other Kunitz domains can be highly specific. For example, DX-88 inhibits human plasma kallikrein with KD≈30 μM while its affinity for (human urine kallikrein (>100 μM), porcine pancreatic kallikrein (27 μM), human C1ractivated (>20 μM), C1sactivated (>20 μM), human plasmin (137 nM), human neutrophil elastase (>20 μM), human plasma F.XIIa (>20 μM), human plasma F.XIa (10 nM), human thrombin (>20 μM), human trypsin (>20 μM), and human chymotrypsin (>20 μM)) is less by a factor at least 250,000. The amino acid sequence of DX-88 is shown in Table 5 and DNA encoding DX-88 is shown in Table 6. Table 8 shows the DX-88 gene with restriction sites.
Engineered Kunitz domains are further disclosed in U.S. Pat. Nos. 6,333,402, 5,663,143, 6,953,674, each of which is incorporated by reference.
A Kunitz domain can be located within a larger protein, e.g., there can be sequences present N- and/or C-terminal to the Kunitz domain.
TFPI:
The sequence of tissue-factor pathway inhibitor (TFPI; also termed LACI) (GeneBank NM—006287) is shown in Table 3. The kinetics of TFPI binding to Factor Xa was published in 1993 (Huang, et al., 1993, J Biol Chem, 268:26950-5). The sequences of TFPI proteins are known for Danio rerio (zebrafish), Canis familiaris (dog), Macaca mulatta (rhesus macaque), Ogctolagus cuniculus (rabbit), Pan troglodytes (chimpanzee), Mus musculus (mouse), Gallus gallus (chicken), and Homo sapiens (man). TFPI contains three Kunitz domains. The first Kunitz domain binds and inhibits factor VIIa in the factor VIIa/tissue factor complex, Kunitz domain2 binds and inhibits factor Xa, and Kunitz domain-3 is involved in binding to cells (Piro and Broze, 2004, Circulation, 110:3567-72). The cell binding by Kunitz domain-3 can be reduced by changing the P1 arginine to leucine (Piro and Broze, 2004, Circulation, 110:3567-72).
Human TFPI may be modified to provide an engineered protein that inhibits at least two different human proteases (e.g., proteases other than factor VIIa and factor X2) and that has a plurality of Kunitz domains. Many of the modified proteins of the present invention will be very similar to human TFPI. A protein that includes TFPI or its Kunitz domains can be altered so that it does not bind to cells as binding a polypeptide to cell surfaces can increase antigenicity (Leonetti, et al., 1998, J Immunol, 160:3820-7). For example, modification of human TFPI to delete the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (SEQ ID NO:145) while retaining Kunitz domain 3 reduces cell binding and other consequent cell signaling events. In a preferred embodiment, the therapeutic protein has no effect on the expression of any genes and does not bind to cells.
Mine et al. (Biochemistry (2002) 41:78-85.) have reported that three lysine residues in the third KuDom of TFPI (which correspond to K241, K260, and K268 in Table 1) form a positively-charged cluster that may bind heparin and foster binding to cells. In one embodiment of the invention, one or more of K241, K260, and K268 are changed to an amino acid that is not positively charged. At position 29, K is the most common AA type and Q is second most common, thus K241Q is a preferred mutation for removal of positive charge in KuDom 3 of TFPI. At position 48, E is the most common AA type. Thus K260E is a preferred mutation for removal of positive charge in KuDom 3 of TFPI. At position 56, G is the most common AA type. Thus K268G is a preferred mutation for removal of positive charge in KuDom 3 of TFPI. Removal of at least one of the three lysines will reduce the possibility of the TFPI variant binding to glycosaminoglycans.
TFPI is cleaved a particular sites by metalloproteinases, e.g., by MMP-8 after S174 and after K20. P18, L19, and L21 conform to substrate 3 in Table 42. In one embodiment, the protein used is based on TFPI, but includes modifications in the linker sequences to prevent MMP cleavage. For example, the sites of cleavage by MMPs are changed to avoid such cleavage. In certain cases S174 and K20 are altered, e.g., to alanine. In another embodiment, the sites are left as is. In most cases, the engineered Kunitz domains will have very high affinity for their targets, thus engineering TFPI-based proteins to reduce or eliminate sensitivity to MMP digestion may not be necessary.
TFPI itself does not have especially high affinity for factor Xa and factor VIIa. Petersen et al. (Petersen, et al., 1996, Eur J Biochem, 235:310-6) report that Kunitz domain 1 binds factor VIIa/TF with KD=250 nM and Kunitz domain 2 binds factor Xa with KD=90 nM. Engineered Kunitz domain proteins based on TFPI will have much higher affinity for their targets (e.g., TFPI Kunitz domains may be engineered to increase affinity for factor Xa and factor VIIa to a KD of less than or equal to 10 nM or 1 nM). The basic C-terminal segment (Linker 4) is also involved in binding F.Xa (Cunningham, et al., 2002, Biochem J, 367:451-8). For example, a protein described herein has a KD of greater than 10 nM or 100 nM for one or both of factor VIIa and factor Xa.
TFPI-2:
The amino acid sequence of TFPI-2 is shown in Table 11. Note that the second Kunitz domain is non-canonical, having a residue 9a and residues 42a and 42b. The linkers are shorter than in TFPI. The C-terminal linker is quite basic, but linkers 1, 2, and 3 are much shorter than the corresponding linkers in TFPI. TFPI-2 may be engineered to be a multiple protease inhibitor by remodeling Kunitz domain 1 and Kunitz domain 3 in the same way that the Kunitz domains of TFPI are remodeled in Examples 2, 3, and 5. Kunitz domain 2 could either be forced to be canonical or one could make a library of Kunitz domain 2 derivatives, select a member having the desired binding properties, and remodel Kunitz domain 2 as needed. TFPI-2, like TFPI, binds cell surfaces. Preferred derivatives of TFPI-2 are modified to abolish binding to cells and effects on gene expression.
Table 12 shows a DNA sequence that would cause display of TFPI-2 Kunitz domain 2 on the stump of M13 III on M13-derived phage. The sequence of the parental phage vector is shown in Table 13. The restriction sites that bound the display cassette of Table 12 are bold in Table 13. To generate a TFPI-2 Kunitz domain 2 library, the DNA between PstI and BspEI is synthesized with the diversity shown in Table 12. Table 14 shows oligonucleotides (ONs) that can be used for this synthesis. ON1U96 and ON71L97 are complementary at their 3′ ends where underscored. ON1U21T2 and ON147L21 are identical to the 5′ ends of the long oligonucleotides and can be used to prime PCR amplification of the long oligonucleotides. There are PstI and BspEI sites near the ends of the final product. The double-stranded DNA is cut with PstI and BspEI and ligated to similarly cleaved vector. The theoretical diversity of the library is 6.7×1014 amino acid sequences. Actual libraries can include at least 109, 1010, or 1011 transformants. One could also use codon-based mutagenesis to synthesize the mixtures shown in Table 12.
Hepatocyte Growth Factor Activator Inhibitor-1 and -2:
Additional examples of a mammalian proteins that include multiple Kunitz domains are: Hepatocyte Growth Factor Activator Inhibitor-1 (HAI-1) and Hepatocyte Growth Factor Activator Inhibitor-2 (HAI-2). Each contains two Kunitz domains.
HAI-1 and HAI-2 are cell bound proteins. Derivatives that are not associated with cells can be prepared, e.g., by producing a form of the protein that includes only the Kunitz domain and intervening sequences. The regions of HAI-1 and HAI-2 responsible for cell binding can be inactivated, e.g., by deletion.
Bikunin:
Table 39 shows four amino acid sequences for “humin bikunin” reported in U.S. Pat. No. 6,583,108 (the '108 patent). The SEQ ID NOs of '108 are shown as “SEQ ID NO:123”, “SEQ ID NO:124”, and “SEQ ID NO:125” have differences in the signal sequence and in amino acid sequence that follows the second Kunitz domain. Table 38 shows affinity data from the '108 patent for the two Kunitz domains of bikunin against a variety of serine proteases.
Protease Targets
Proteases are involved in a wide variety of biological processes, including inflammation and tissue injury. Serine proteases produced by inflammatory cells, including neutrophils, are implicated in various disorders, such as pulmonary emphysema. As such, multiple different proteases can contribute to a single disorder.
Examples of particular proteases include the following. Neutrophil elastase is a serine protease produced by polymorphonuclear leukocytes with activity against extracellular matrix components and pathogens. Pulmonary emphysema is characterized by alveolar destruction leading to a major impairment in lung function. Human neutrophil elastase consists of approximately 238 amino acid residues, contains 2 asparagine-linked carbohydrate side chains, and is joined together by 4 disulfide bonds (GeneBank Entry P08246). It is normally synthesized in the developing neutrophil as a proenzyme but stored in the primary granules in its active form, ready with full enzymatic activity when released from the granules, normally at sites of inflammation (Gullberg U, et al. Eur J Haematol. 1997; 58:137-153; Borregaard N, Cowland J B. Blood. 1997; 89:3503-3521).
Other exemplary protease targets include: plasmin, tissue kallikrein, plasma kallikrein, Factor VIIa, Factor XIa, thrombin, urokinase, trypsin 1, trypsin 2, pancreatic chymotrypsin, pancreatic elastase, tryptase, and Factor IIa. Classes of relevant proteases include: proteases associated with blood coagulation, proteases associated with fibrinolysis, proteases associated with complement, proteases that digest extracellular matrix components, proteases that digest basement membranes, and proteases associated with endothelial cells. For example, the protease is a serine protease. Proteins described herein that inhibit one or more proteases can be engineered to inhibit one or more proteases within these classes.
Identifying Kunitz Domains and Other Protease Inhibitors
A variety of methods can be used to identify a Kunitz domain that binds to and/or inhibits a protease, e.g., a human protease. These methods can be used to identify natural and non-naturally occurring Kunitz domains that can be used as components of a protein that inhibits a plurality of proteases, e.g., a plurality of human proteases. Other protease inhibitors can be similarly identified, e.g., by varying known protease inhibitors or protease resistant proteins, by using libraries of randomized peptides or proteins, or using libraries of antibodies e.g., as described in US 2004-0071705 (including ¶ 102-112). As will be understood by those of skill in the art, while the description of methods of identifying inhibitors of proteases herein are largely directed towards Kunitz domains, the methods can be adapted for identifying peptide-based or antibody-based protease inhibitors.
For example, a Kunitz domain can be identified from a library of proteins in which each of a plurality of library members includes a varied Kunitz domain. Various amino acids can be varied in the domain. See, e.g., U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,663,143, and U.S. Pat. No. 6,333,402. Kunitz domains can be varied, e.g., using DNA mutagenesis, DNA shuffling, chemical synthesis of oligonucleotides (e.g., using codons as subunits), and cloning of natural genes. See, e.g., U.S. Pat. No. 5,223,409 and U.S. 2003-0129659.
Libraries of Kunitz domains can be generated by varying one or more binding loop amino acid residues of a Kunitz domain described herein, e.g., a Kunitz domain having a framework described herein, e.g., a modified or naturally occurring framework region. In one embodiment, the residues that are varied are varied among a plurality of amino acids.
The library can be an expression library that is used to produce proteins. The proteins can be arrayed, e.g., using a protein array. U.S. Pat. No. 5,143,854; De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/98534, WO 01/83827, WO 02/12893, WO 00/63701, WO 01/40803 and WO 99/51773.
Kunitz domains can also be displayed on a replicable genetic package, e.g., in the form of a phage library such as a phage display, yeast display library, ribosome display, or nucleic acid-protein fusion library. See, e.g., U.S. Pat. No. 5,223,409; Garrard et al. (1991) Bio/Technology 9:1373-1377; WO 03/029456; and U.S. Pat. No. 6,207,446. Binding members of such libraries can be obtained by selection and screened in a high throughput format. See, e.g., U.S. 2003-0129659.
Selection from Display Libraries
This section describes exemplary methods of selecting members of a display library to identify a Kunitz domain that inhibits a protease. The methods disclosed herein can also be modified and used in combination with other types of libraries, e.g., an expression library or a protein array, and so forth. Kunitz domains can be displayed on phage, especially on filamentous phage (e.g., M13). In a preferred embodiment, the library contains members that have a variety of amino acid types at the positions of the Kunitz domain that are likely to contact a serine protease where they can affect the binding. Using immobilized or immobilizable (e.g., by modification with one member of a binding pair, such as avidin or streptavidin) serine protease as a target, one can select those members of the library that have high affinity for the serine protease of interest.
In an exemplary display library selection, a phage library is contacted with and allowed to bind to the target protease which may be an active or an inactivated form of the protease (e.g., mutant or chemically inactivated protein) or a fragment thereof. To facilitate separation of binders and non-binders in the selection process, it is often convenient to immobilize the protease on a solid support, although it is also possible to first permit binding to the target protease in solution and then segregate binders from non-binders by coupling the target protease to a support. By way of illustration, when incubated in the presence of the protease, phage displaying a Kunitz domain that interacts with target protease form complexes with the target protease immobilized on a solid support whereas non-binding phage remain in solution and are washed away with buffer. Bound phage may then be liberated from the protease by a number of means, such as changing the buffer to a relatively high acidic or basic pH (e.g., pH 2 or pH 10), changing the ionic strength of the buffer, adding denaturants, adding a competitor, adding host cells which can be infected (Hogan et al. (2005) Biotechniques 38(4):536, 538.), or other known means.
For example, to identify protease-binding peptides, protease can be adsorbed to a solid surface, such as the plastic surface of wells in a multi-well assay plate. Subsequently, an aliquot of a phage display library is added to a well under appropriate conditions that maintain the structure of the immobilized protease and the phage, such as pH 6-7. Phage that display polypeptides that bind the immobilized protease are bound to the protease and are retained in the well. Non-binding phage can be removed (e.g., by washing with buffer). It is also possible to include a blocking agent or competing agent during the binding of the phage library to the immobilized protease.
Phage bound to the immobilized protease may then be eluted by washing with a buffer solution having a relatively strong acid pH (e.g., pH 2) or an alkaline pH (e.g., pH 8-9). The solutions of recovered phage that are eluted from the protease are then neutralized and may, if desired, be pooled as an enriched mixed library population of phage displaying protease binding peptides. Alternatively the eluted phage from each library may be kept separate as a library-specific enriched population of protease binders. Enriched populations of phage displaying protease binding peptides may then be grown up by standard methods for further rounds of selection and/or for analysis of peptide displayed on the phage and/or for sequencing the DNA encoding the displayed binding peptide.
Recovered phage may then be amplified by infection of bacterial cells, and the screening process may be repeated with the new pool of phage that is now depleted in non-protease binders and enriched in protease binders. The recovery of even a few binding phage may be sufficient to carry the process to completion. After a few rounds of selection (e.g. 2 or 3), the gene sequences encoding the binding moieties derived from selected phage clones in the binding pool are determined by conventional methods, revealing the peptide sequence that imparts binding affinity of the phage to the target. An increase in the number of phage recovered after each round of selection and the recovery of closely related sequences indicate that the screening is converging on sequences of the library having a desired characteristic.
After a set of binding polypeptides is identified, the sequence information may be used to design secondary libraries. For example, the secondary libraries can explore a smaller segment of sequence space in more detail than the initial library. In some embodiments, the secondary library includes proteins that are biased for members having additional desired properties, e.g., sequences that have a high percentage identity to a human protein.
Iterative Selection.
Display library technology may be used in an iterative mode. A first display library is used to identify one or more Kunitz domains that bind to a target protease. These identified Kunitz domains are then varied using a mutagenesis method to form a second display library. Higher affinity proteins are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.
In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. For Kunitz domains, many positions near the binding interface are known. Such positions include, for example, positions 13, 14, 15, 16, 17, 18, 19, 31, 32, 34, 38, and 39 with respect to the sequence of BPTI. Some exemplary mutagenesis techniques include: synthesis of DNA with mixed nucleotides, error-prone PCR (Leung et al. (1989) Technique 1:11-15), recombination, DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391; termed “nucleic acid shuffling”), RACHITTTM (Coco et al. (2001) Nature Biotech. 19:354), site-directed mutagenesis (Zoller et al. (1987) Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and incorporation of degenerate oligonucleotides (Griffiths et al. (1994) EMBO J 13:3245). The mixtures of nucleotides used in synthesis can be, for example, NNK which allows any base at the first two positions of a varied codon and G or T at the final base and allows all 20 amino-acid types to appear. Alternatively, varied codons that exclude some amino-acid types and perhaps stop codons may be used. NNK provides 32 codons, one of which is TAG, a stop. NNK also provides three codons for each of Ser, Leu, and Arg; two codons for each of Val, Ala, Pro, Thr, and Gly; the other twelve amino-acid types have a single codon. The varied codon NNT provides 15 amino-acid types and no stops. NNG provides 13 amino-acid types and one stop.
Mutagenesis may also be carried out in a more limited or targeted fashion. For example, if the parental amino-acid in a position of interest is Ser, a codon mix with (0.85 T, 0.05 C, 0.05 A, 0.05 G) at the first base, (0.85 C, 0.05 T, 0.05 A, 0.5 G) at the second base and (0.85 T, 0.05 C, 0.05 A, 0.05 G) at the third base may be employed. Most of the DNA molecules will have TCT encoding Ser, but others will have codons that differ by one, two, or three bases, providing a diversity of amino-acid types.
In one example of iterative selection, the methods described herein are used to first identify Kunitz domains from a display library that bind a target protease with at least a minimal binding specificity, e.g., an equilibrium dissociation constant for binding of less than 100 nM, 10 nM, or 1 nM. The nucleic acid sequences encoding the Kunitz domains that are initially identified are used as a template nucleic acid for the introduction of variations, e.g., to identify a second Kunitz domain that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial protein Kunitz domain.
Selecting or Screening for Specificity.
The display library selection and screening methods described herein can include a selection or screening process that discards display library members that bind to a non-target molecule, e.g., a protease other than the target protease. In one embodiment, the non-target molecule is a protease that has been inactivated by treatment with an irreversibly bound inhibitor, e.g., a covalent inhibitor.
In one implementation, a so-called “negative selection” step or “depletion” is used to discriminate between the target and a related, but distinct or an unrelated non-target molecules. The display library or a pool thereof is contacted with the non-target protease. Members of the sample that do not bind the non-target protease are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target protease.
In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target protease. For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target protease.
Single Kunitz Domains with Multiple Specificities
Single Kunitz domains with more than one specificity are obtained by alternating selection of libraries using the target serine proteases. Members of a library of Kunitz domains are first selected for binding to the first target serine protease, and those library members that bind the first target are selected and screened against the second target serine protease. This alternating selection and screening may be carried out on as many target serine proteases as desired. Additionally, the selection may be repeated, such that the library members which bind to all of the target proteases are then screened for the desired binding. Two to four rounds of repeated selections are commonly performed.
For example, alternating selection can be used to obtain a single Kunitz domain that inhibits both human kallikrein 1 (h.K1, also known as tissue kallikrein) and murine kallikrein 1 (m.K1). As described above, a library would be selected first against one of the targets (e.g., hK1), then the Kunitz domains identified in the first selection are then selected against the other target (e.g., mK1). A Kunitz domain that inhibits a human serine protease and its counterpart from another mammalian species (e.g., murine) is useful because its characteristics can be evaluated in an animal model of disease.
In another example, a Kunitz domain is selected that inhibits both human plasma kallikrein (h.pKal) and human tissue kallikrein by alternating selection with the two targets. Such an inhibitor would be advantageous in a disease, such as arthritis, where both human plasma kallikrein and human tissue kallikrein are excessively active. In one embodiment, the Kunitz domain differs from human bikunin (e.g., as described in U.S. Pat. No. 6,583,108) by at least two, three, five, or ten amino acids.
Transferring Specificity Determinants
Natural proteins that have more than one Kunitz domain can be modified to provide a protein with at least two engineered Kunitz domains that inhibit, respectively, two different human proteases, e.g., plasma kallikrein (pKal) and human neutrophil elastase (hNE). For example, residues from the protease binding loops of DX-88 can be transferred to one Kunitz domain of the recipient protein and residues from the protease binding loops of DX-890 can be transferred into another of Kunitz domains. The resulting protein inhibits plasma kallikrein and human neutrophil elastase.
In one embodiment, residues that contribute to binding and/or specificity are substituted into the Kunitz domains of a pre-existing protein, e.g., a naturally occurring protein such as a serum protein. Several serum proteins contain two or three Kunitz domains. Each of these Kunitz domains could be engineered to inhibit one target. For example, the extracellular compartment of human tissue contains tissue-factor pathway inhibitor (TFPI, also known as lipoprotein-associated coagulation inhibitor (LACI)), tissue-factor pathway inhibitor-2 (TFPI-2), hepatocyte growth factor activator inhibitor-1 (HAI-1), hepatocyte growth factor activator inhibitor-2 (HAI-2, also known as placental bikunin), and bikunin (also known as inter-alpha trypsin inhibitor). One strategy is to use one of the multi-Kunitz domain human proteins as a template and change only those residues that confer needed affinity and specificity.
The residues that confer affinity and specificity can be transferred from one Kunitz domain to another with high retention of the desired properties by substitution of a few residues. For example, DX-88 is based on Kunitz domain 1 of TFPI and has five amino acids within the Kunitz domain changed based on selection from a phage-displayed library. DX-88 is a 30 pM inhibitor of human plasma kallikrein (h.pKal) while TFPI Kunitz domain 1 has hardly any affinity for h.pKal. Table 7 shows the amino acid sequences of several Kunitz domains, some wildtype, some selected from phage libraries, some designed, and some designed, built and tested. BPTI, TFPI-K1, TFPI-K2, and TFPI-K3 are naturally occurring Kunitz domains. DX-88 and DX-1000 were selected from a library of TFPI Kunitz domain 1 derivatives and inhibit human plasma kallikrein and human plasmin respectively. TF890 is designed by comparison with DX-890 and should inhibit human neutrophil elastase with high affinity. TFPI-K2—88 is a modification of TFPI-K2 which should inhibit human plasma kallikrein. TFPI-K3—88 is a derivative of TFPI Kunitz domain 3 which should inhibit human plasma kallikrein. ITI-K2 is the second Kunitz domain of bikunin. DX-890 is a potent inhibitor of human neutrophil elastase that was designed bases on inhibitor selected from a library of BPTI derivatives for binding to human neutrophil elastase. TFPI-K2—890 is a derivative of TFPI Kunitz domain 2 that was designed to inhibit human neutrophil elastase. TFPI-K3—890 is a derivative of TFPI Kunitz domain 3 that was designed to inhibit human neutrophil elastase.
(*)New engineered Kunitz domain sequences may be selected using display library technology. The ‘base’, or ‘scaffold’ sequence can be any Kunitz domain-containing protein, but in certain embodiments, the ‘base’ sequence is derived from a naturally-occurring protein that contains two or more Kunitz domains (e.g., TFPI, for example, TFPI K1). Any serine protease of interest may be used as the target, including neutrophil elastase (e.g., human neutrophil elastase), proteinase 3 (e.g., human proteinase 3), cathepsin G (e.g., human cathepsin G), tryptase (e.g., human tryptase), plasma kallikrein (e.g., human plasma kallikrein), neutrophil elastase (e.g., human neutrophil elastase), kallikrein 6 (e.g., human kallikrein 6), kallikrein 8 (e.g., human kallikrein 8), kallikrein 10 (e.g., human kallikrein 10), tissue kallikrein (e.g., human tissue kallikrein), plasmin (e.g., human plasmin), hepsin (e.g., human hepsin), matriptase (e.g., human matriptase), endotheliase 1 (e.g., human endotheliase 1), and endotheliase 2 (e.g., human endotheliase 2).
In some embodiments, the recipient Kunitz domain is modified so that residues positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 (numbered with respect to the sequence of BPTI) are the same as the residues in the originating Kunitz domain. Alternatively, one could vary residues 11, 13, 15-19, 31, 32, 34, 39, and 40. In another embodiment, one could vary 11-19, 31, 32, 34, and 38-40; in this case one allows the 14-38 disulfide to be changed to other amino-acid types. In other embodiments, the recipient Kunitz domain is modified so that residues in the first and second binding loops are the same as the residues in the originating Kunitz domain.
Modifying and Varying Polypeptides
It is also possible to vary a Kunitz domain or other protein described herein to obtain useful variant that has similar or improved or altered properties. Typically, a number of variants are possible. A variant can be prepared and then tested, e.g., using a binding assay described herein (such as fluorescence anisotropy).
One type of variant is a truncation of a Kunitz domain or other protein described herein. In this example, the variant is prepared by removing one or more amino acid residues of the domain from the N or C terminus. In some cases, a series of such variants is prepared and tested. Information from testing the series is used to determine a region of the domain that is essential for protease binding and/or stability in serum. A series of internal deletions or insertions can be similarly constructed and tested. For Kunitz domains, it can be possible to remove, e.g., between one and five residues or one and three residues that are N-terminal to C5, the first cysteine, and between one and five residues or one and three residues that are C-terminal to C55, the final cysteine, wherein each of the cysteines corresponds to a respectively numbered cysteine in BPTI.
Another type of variant is a substitution. In one example, the Kunitz domain is subjected to alanine scanning to identify residues that contribute to binding activity and or stability. In another example, a library of substitutions at one or more positions is constructed. The library may be unbiased or, particularly if multiple positions are varied, biased towards an original residue. In some cases, the substitutions are all conservative substitutions.
In another example, each lysine is replaced by arginine and the binding affinity is measured. Thus, one may obtain a binding protein having no lysines and retaining the selected binding properties. Proteins lacking lysine residues with the polypeptide chain provide only a single site for pegylation (the amino terminus), and thus provide a more homogenous product upon pegylation.
Another type of variant includes one or more non-naturally occurring amino acids. Such variant domains can be produced by chemical synthesis or modification. One or more positions can be substituted with a non-naturally occurring amino acid. In some cases, the substituted amino acid may be chemically related to the original naturally occurring residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic) or an isostere of the original residue.
It is also be possible to include non-peptide linkages and other chemical modifications. For example, part or all of the Kunitz domain or other protein may be synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol. 13:132-4).
Protein Production
Recombinant Production of Polypeptides.
Standard recombinant nucleic acid methods can be used to express a protein described herein (e.g., a protein that includes one or more Kunitz domains). Generally, a nucleic acid sequence encoding the protein is cloned into a nucleic acid expression vector. If the protein is sufficiently small, e.g., the protein is a peptide of less than 60 amino acids, the protein can be synthesized using automated organic synthetic methods.
The expression vector for expressing the protein can include a segment encoding the protein and regulatory sequences, for example, a promoter, operably linked to the coding segment. Suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. See, for example, the techniques described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, N.Y. (1989).
Expression of proteins that contain no sites for N-linked glycosylation can be carried out in bacteria, yeast, or mammalian cells. Proteins having sites for N-linked glycosylation can be expressed in bacteria to obtain non-glycosylated versions or in yeast or mammalian cells if glycosylation is desired. For use in humans, glycosylated versions are preferably produced in mammalian cells such as CHO or HEK293T cells.
Scopes (1994) Protein Purification: Principles and Practice, New York: Springer-Verlag and other texts provide a number of general methods for purifying recombinant (and non-recombinant) proteins.
Synthetic Production of Peptides.
A polypeptide can also be produced by synthetic means. See, e.g., Merrifield (1963) J. Am. Chem. Soc., 85: 2149. For example, the molecular weight of synthetic peptides or peptide mimetics can be from about 250 to about 8,0000 Daltons. A peptide can be modified, e.g., by attachment to a moiety that increases the effective molecular weight of the peptide. If the peptide is oligomerized, dimerized and/or derivatized, e.g., with a hydrophilic polymer (e.g., to increase the affinity and/or activity of the peptides), its molecular weights can be greater and can range anywhere from about 500 to about 50,000 Daltons.
Pegylation and Other Modifications
A single Kunitz domain has low molecular mass and at least in some cases are rapidly filtered by the kidneys. Increasing molecular mass can increase serum residence.
A variety of moieties can be covalently attached to the protein to increase the molecular weight of a protein that includes a Kunitz domain. In one embodiment, the moiety is a polymer, e.g., a water soluble and/or substantially non-antigenic polymer such as a homopolymer or a non-biological polymer. Substantially non-antigenic polymers include, e.g., polyalkylene oxides or polyethylene oxides. The moiety may improve stabilization and/or retention of the Kunitz domain in circulation, e.g., in blood, serum, lymph, or other tissues, e.g., by at least 1.5, 2, 5, 10, 50, 75, or 100 fold. One or a plurality of moieties can be attached to protein that includes a Kunitz domain. For example, the protein is attached to at least three moieties of the polymer. In one embodiment, particularly if there are no lysines in the protease binding loops, each lysine of the protein can be attached to a moiety of the polymer.
Suitable polymers can vary substantially by weight. For example, it is possible to use polymers having average molecular weights ranging from about 200 Daltons to about 40 kDa, e.g., 1-20 kDa, 4-12 kDa or 3-8 kDa, e.g., about 4, 5, 6, or 7 kDa. In one embodiment, the average molecular weight of individual moieties of the polymer that are associated with the compound are less than 20, 18, 17, 15, 12, 10, 8, or 7 kDa. The final molecular weight can also depend upon the desired effective size of the conjugate, the nature (e.g. structure, such as linear or branched) of the polymer, and the degree of derivatization.
A non-limiting list of exemplary polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. The polymer can be a hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polylactic acid; polyglycolic acid; polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, cellulose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon. In some embodiments, the polymer includes a variety of different copolymer blocks.
Additional exemplary methods of attaching more than one polymer moiety to a protein are described in US 2005-0089515.
Another method of increasing the molecular weight of a protein is by making multimers of the protein (e.g., producing the protein such that at least two copies of the protein are fused, typically in tandem, but head to head fusions are also possible). Multimers may be produced by chemical crosslinking or by expression of a construct encoding the multimer. Because long repeats of DNA may be unstable, it is preferred that the polynucleotides encoding the different copies of the protein have different sequences.
Characterization of Binding Interactions
The binding properties of a protein (e.g., a protein that includes one or more Kunitz domains) can be readily assessed using various assay formats. For example, the binding properties can be assayed by quantitative measurement of the ability to inhibit the target and non-target proteases. Alternatively, for example, the binding property of a protein can be measured in solution by fluorescence anisotropy (Bentley et al., (1985) J. Biol. Chem. 260(12):7250-56; Shimada et al., (1985) J. Biochem. 97(6):1637-44; Terpetschnig et al., (1995) Anal. Biochem. 227(1):140-7; Durkop et al., (2002) Anal. Bioanal. Chem. 372(5-6):688-94), which provides a convenient and accurate method of determining a dissociation constant (KD) or association constant (Ka) of the protein for a particular target protease. In one such procedure, the protein to be evaluated is labeled with fluorescein. The fluorescein-labeled protein is mixed in wells of a multi-well assay plate with various concentrations of the particular target protease or non-target protease. Fluorescence anisotropy measurements are carried out using a fluorescence polarization plate reader.
The binding interactions can also be analyzed using an ELISA assay. For example, the protein to be evaluated is contacted to a microtitre plate whose bottom surface has been coated with the target protease, e.g., a limiting amount of the target protease. The molecule is contacted to the plate. The plate is washed with buffer to remove non-specifically bound molecules. Then the amount of the protein bound to the plate is determined by probing the plate with an antibody that recognizes the protein. For example, the protein can include an epitope tag. The antibody can be linked to an enzyme such as alkaline phosphatase, which produces a colorimetric product when appropriate substrates are provided. In the case where a display library member includes the protein to be tested, the antibody can recognize a region that is constant among all display library members, e.g., for a phage display library member, a major phage coat protein.
A binding interaction between a protein and a particular target protease can be analyzed using surface plasmon resonance (SPR). For example, after sequencing of a display library member present in a sample, and optionally verified, e.g., by ELISA, the displayed protein can be produced in quantity and assayed for binding the target using SPR. SPR or real-time Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander, S, and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (KD), and kinetic parameters, including kon and koff, for the binding of a protein (e.g., a protein include Kunitz domains) to a target protease. Such data can be used to compare different proteins. Information from SPR can also be used to develop structure-activity relationship (SAR). For example, if the proteins are all mutated variants of a single parental antibody or a set of known parental antibodies, variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow koff.
Additional methods for measuring binding affinities include nuclear magnetic resonance (NMR), and binding titrations (e.g., using fluorescence energy transfer).
Other solution measures for studying binding properties include fluorescence resonance energy transfer (FRET), NMR, X-ray crystallography, molecular modeling, and measuring bound vs. free molecules. Measurement of bound vs. free molecules can be accomplished with a KinExA instrument from Sapidyne Instruments Inc., Boise, Id.
Characterization of Protease Inhibition
It may be useful to characterize the ability of a protein described herein to inhibit target proteases, as well as non-target proteases.
Kunitz domains can be selected for their potency and selectivity of inhibition of the target proteases. In one example, target protease and a substrate are combined under assay conditions permitting reaction of the protease with its substrate. The assay is performed in the absence of the protein being tested, and in the presence of increasing concentrations of the protein being tested. The concentration of test protein at which the protease activity is 50% inhibited is the IC50 value (Inhibitory Concentration) or EC50 (Effective Concentration) value for that test protein. Proteins having lower IC50 or EC50 values are considered more potent inhibitors of the target protease than those proteins having higher IC50 or EC50 values. Preferred proteins according to this aspect have an IC50 value of 100 nM, 10 nM, 1 nM, or 0.1 nM or less as measured in an in vitro assay for inhibition of protease activity.
A test protein can also be evaluated for selectivity toward different protease(s). A test protein is assayed for its potency toward a panel of proteases and other enzymes and an IC50 value is determined for each. A protein that demonstrates a low IC50 value for the target protease, and a higher IC50 value for other proteases within the test panel (e.g., trypsin, plasmin, chymotrypsin), is considered to be selective toward the target protease. Generally, a protein is deemed selective if its IC50 value is at least one order of magnitude less than the next smallest IC50 value measured in the panel of enzymes.
It is also possible to evaluate Kunitz domain activity in vivo or in samples (e.g., pulmonary lavages or serum) of subjects to which a compound described herein has been administered.
Pharmaceutical Compositions & Treatments
Also featured is a composition, e.g., a pharmaceutically acceptable composition, that includes a protein described herein (e.g., a protein that inhibits at least two human proteases). In one embodiment, the protein includes one or more Kunitz domains. For example, the protein inhibits a plurality of target proteases. The pharmaceutical composition can include a pharmaceutically acceptable carrier.
Exemplary carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
The pharmaceutical composition can include a pharmaceutically acceptable salt, e.g., a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
The pharmaceutical composition may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of antibodies to humans. A common mode of administration is parenteral (e.g., intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion). In one embodiment, the compound is administered by intravenous infusion or injection. In another embodiment, the compound is administered by intramuscular or subcutaneous injection. Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
The proteins described herein can be administered by a variety of methods known in the art. For many applications, the route/mode of administration is intravenous injection or infusion. For example, for therapeutic applications, the compound can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or 7 to 25 mg/m2. Alternatively, the dose could be 100 μg/Kg, 500 μg/Kg, 1 mg/Kg, 5 mg/Kg, 10 mg/Kg, or mg/Kg. The route and/or mode of administration will vary depending upon the desired results.
In certain embodiments, the protein is prepared with a carrier that protects against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical formulation is a well-established art, and is further described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).
Also within the scope of the invention are kits comprising a protein described herein (e.g., a protein that inhibits at least two human proteases) and instructions for use, e.g., treatment, prophylactic, or diagnostic use. In one embodiment, the kit includes (a) the compound, e.g., a composition that includes the compound, and, optionally, (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the compound for the methods described herein.
In one embodiment, the informational material can include instructions to administer the compound in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions for identifying a suitable subject, e.g., a human, e.g., a human having, or at risk for a disorder characterized by excessive elastase activity. The informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the compound and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.
In addition to the compound, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or a second agent for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the compound. In such embodiments, the kit can include instructions for admixing the compound and the other ingredients, or for using the compound together with the other ingredients.
The kit can include one or more containers for the composition containing the compound. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the compound. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the compound. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
A protein described herein (e.g., a protein that inhibits at least two human proteases) can be administered, alone or in combination with, a second agent to a subject, e.g., a patient, e.g., a patient, who has a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition toward a disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. The treatment may also delay onset, e.g., prevent onset, or prevent deterioration of a condition.
A therapeutically effective amount can be administered, typically an amount of the compound which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects. A therapeutically effective dosage preferably modulates a measurable parameter, favorably, relative to untreated subjects. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder.
Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a compound described herein is 0.1-20 mg/Kg, more preferably 1-10 mg/Kg. The compound can be administered by parenteral (e.g., intravenous or subcutaneous) infusion at a rate of less than 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 50 mg/m2 or about 5 to 20 mg/m2. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions (e.g., the supervising physician), and that dosage ranges set forth herein are only exemplary.
Therapeutic Uses
The proteins of the invention may be used in therapies for the treatment or prophylaxis of disorders mediated by two or more proteases. Likewise, the proteins of the invention may be used for the manufacture of a medicament for the treatment of disorders mediated by two or more proteases.
Rheumatoid Arthritis (RA):
Human plasma kallikrein, human tissue kallikrein (h.K1), and human neutrophil elastase have all be implicated in RA (Volpe-Junior, et al., 1996, Inflamm Res, 45:198-202; Cassim, et al., 2002, Pharmacol Ther, 94:1-34; Colman, 1999, Immunopharmacology, 43:103-8; Dela Cadena, et al., 1995, Faseb J, 9:446-52; Rahman, et al., 1995, Ann Rheum Dis, 54:345-50; Tremblay, et al., 2003, Curr Opin Investig Drugs, 4:556-65). Thus, a method of treating rheumatoid arthritis (e.g., stabilizing or improving total Sharp score, the Sharp erosion score, or the HAQ disability index) by administering to a subject having (or suspected of having) RA an effective amount of a protein that inhibits at least two proteases from the group of human plasma kallikrein, human tissue kallikrein (h.K1), and human neutrophil elastase is provided.
MMPs 1, 2, 3, 9, and 13 have also been implicated in RA (Burrage et al. (2006) Front Biosci. 11:529-43). Accordingly, also provided is a method of treating RA (e.g., stabilizing or improving total Sharp score, the Sharp erosion score, or the HAQ disability index) by administering an effective amount of a protein which inhibits (a) at least two proteases from the group of human plasma kallikrein, human tissue kallikrein (h.K1), and human neutrophil elastase, (b) at least one protease from the group of MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13.
RA can be assessed by a variety of clinical measures. Some exemplary indicia include the total Sharp score (TSS), Sharp erosion score, and the HAQ disability index. A protein of the invention is administered to treat RA, e.g., to improve an assessment on one or more of these clinical measures.
Pulmonary Disorders:
Lung inflammatory disorders involve a number of different proteases, including human neutrophil elastase, proteinase 3, cathepsin G, tryptase, MMP-12, and MMP-9. Accordingly, the invention provides methods for treatment and/or prophylaxis of pulmonary disorders involving inflammation (e.g., improving or stabilizing PFT scores or slowing, preventing, or reducing a deterioration in PFT scores, or for COPD patients by reducing the destructive index score by at least 10, 20, 30 or 40% or to at least within 50, 40, 30, or 20% of normal destructive index scores for the age- and gender-matched normal population) by administering to a subject having (or suspected of having) a pulmonary disorder involving inflammation an effective amount of a protein which inhibits the activity of two or more (or all) of these proteases. Exemplary pulmonary disorders involving inflammation including asthma, emphysema, cystic fibrosis, and chronic obstructive pulmonary disease (COPD), particularly acute exacerbations of COPD. In particular, human neutrophil elastase, proteinase 3, cathepsin G, and MMP-12 have been associated with COPD, and thus proteins for the treatment of COPD inhibit at least two proteases from the group: human neutrophil elastase, proteinase 3, and cathepsin G, and, in some embodiments additionally inhibit MMP-9 and/or MMP-12.
A variety of clinical measures are available to assess pulmonary diseases and the efficacy of treatment thereof. Pulmonary function tests (PFT) such as forced expiratory volume in one second (FEV), forced expiratory volume in six second (FEV), forced vital capacity (FVC), peak expiratory flow, the ratio of FEV1 to FVC, expressed as a percentage (FEV1/FVC), the forced expiratory flow less the average forced expiratory flow during the mid (25-75%) portion of the FVC (FEF25-75), and thin section computed tomography may be used to assess pulmonary diseases and the efficacy of treatment. The “destructive index,” which is a measure of alveolar septal damage and emphysema, may be particularly useful in monitoring COPD and the therapeutic effect of the proteins and methods herein. See, e.g., Am Rev Respir Dis 1991 July; 144(1):156-59.
Multiple Sclerosis:
Multiple sclerosis (MS) may involve human kallikrein 6 (hK6), human kallikrein 8 (hK8), and human kallikrein 10 (hK10). Accordingly, the invention provides methods of treating multiple sclerosis (e.g., reducing or stabilizing EDSS score, reducing the intensity or frequency of exacerbations, or reducing the size, number, or rate of increase in size or number or sclerotic lesions) by administering to a subject having (or suspected of having) MS an effective amount of a protein that inhibits two or more (e.g., all three) of these proteases. Patients having MS may be identified by criteria recognized in the medical arts as establishing a diagnosis of clinically definite MS, such as the criteria defined by the workshop on the diagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983).
Efficacy of the treatment of multiple sclerosis may be examined using any method or criteria recognized in the art. Three commonly used criteria are: EDSS (extended disability status scale, Kurtzke, Neurology 33:1444, 1983), appearance of exacerbations or MRI (magnetic resonance imaging).
Spinal Cord Injury:
Spinal chord injury may be exacerbated by hK6, human neutrophil elastase, and other serine proteases. Accordingly, the invention provides methods of treating spinal cord injury (e.g., improving gross or fine motor skills, increasing the activities of daily living (ADLs) which can be performed by the patient, improvement in reflex responses, or improvement in complex locomotion behavior) by administering to a subject with a spinal cord injury an effective amount of a protein that inhibits at least the two serine proteases hK6 and human neutrophil elastase.
Behavioural assessments often fail to define the exact nature of motor deficits or to evaluate the return of motor behaviour following spinal cord injury. In addition to the assessment of gross motor behaviour, it is appropriate to use complex tests for locomotion to evaluate the masked deficits in the evaluation of functional recovery after spinal cord injury. Suresh et al. have designed sensitive quantitative tests for reflex responses and complex locomotor behaviour as a combined behavioural score (CBS) to assess the recovery of function in the Bonnet monkey (Macaca radiata) (J Neurol Sci. (2000) 178(2):136-52.). Since these measurements are non-invasive, they may be adapted for use on human patients.
Inflammatory Bowel Disease:
Inflammatory bowel diseases (IBD) include two distinct disorders, Crohn's disease and ulcerative colitis (UC). Ulcerative colitis occurs in the large intestine, while in Crohn's, the disease can involve the entire GI tract as well as the small and large intestines. For most patients, IBD is a chronic condition with symptoms, which include intermittent rectal bleeding, crampy abdominal pain, weight loss and diarrhea, lasting for months to years. Human tissue kallikrein (h.K1), human plasma kallikrein and human neutrophil elastase have been implicated in both Crohn's disease and ulcerative colitis (Colman, 1999, Immunopharmacology, 43:103-8; Devani et al., (2005) Dig. Liver Dis. 37(9):665-73; Kuno et al., (2002) J. Gastroenterol. 37(Suppl 14):22-32; Stadnicki et al., (1997) Dig. Dis. Sci. 42(11):2356-66). Accordingly, the invention also provides methods of treatment and/or prophylaxis of Crohn's disease and/or ulcerative colitis (e.g., reducing or eliminated rectal bleeding, abdominal pain/cramping, weight loss, or diarrhea, improving hematocrit/eliminating improving anemia, or improving Clinical Activity Index for Ulcerative Colitis scores in UC patients) by administering to a subject an effective amount of a protein which inhibits at least two (or all three) of human tissue kallikrein (h.K1), human plasma kallikrein and human neutrophil elastase. The subject has, is suspected of having, or is at risk of developing an IBD.
Many of the symptoms of IBD (e.g., rectal bleeding, abdominal pain/cramping, diarrhea, weight loss, hematocrit/anemia) can be quantitated using standard methodologies. Additionally, a clinical index such as the Clinical Activity Index for Ulcerative Colitis may be used to monitor IBD. (see also, e.g., Walmsley et al. Gut. 1998 July; 43(1):29-32 and Jowett et al. (2003) Scand J Gastroenterol. 38(2):164-71).
Pancreatitis
Acute pancreatitis is characterized by sudden intrapancreatic activation of pancreatic zymogens, resulting in an essentially autolytic destruction of the pancreas if unchecked. The major enzymes involved are human trypsins 1 and 2, human pancreatic chymotrypsin, and human pancreatic elastase. Accordingly, the invention provides a method for the treatment of acute pancreatitis (e.g., stabilizing or improving Ranson score, Acute Physiology and Chronic Health Evaluation (APACHE II) score, or Balthazar computed tomography severity index (CTSI)) by administering to a subject having (or suspected of having) acute pancreatitis a protein which inhibits at least two of the proteases human trypsin 1, human trypsin 2, human pancreatic chymotrypsin, and human pancreatic elastase.
Cancer
A number of serine proteases are involved in cancer and tumorigenic pathways. Hepsin and matriptase are involved in the activation of hepatocyte growth factor. Hepatocyte growth factor (HGF) is a growth factor for many tumors and blocking its release will deprive the tumor cells of a needed growth factor. In addition, matriptase can also cleave pro-uPA into uPA, the enzyme responsible for the generation of plasmin from plasminogen. Plasmin is implicated in the activation of various matrix metalloproteinases (MMPs). Upon conversion from plasminogen to active plasmin, plasmin remains associated with the surface of a variety of epithelial cancer cells. MMP activity is important in cleaving a variety of matrix proteins that allow cancer cells to escape from one site or to invade at new sites. In addition, matriptase can also cleave pro-uPA into uPA, the enzyme responsible for the generation of plasmin from plasminogen. Endotheliases 1 and 2 have also been implicated in cancer.
Accordingly, the disclosure provides methods of treating (e.g., slowing, eliminating, or reversing tumor growth, preventing or reducing, either in number or size, metastases, reducing or eliminating tumor cell invasiveness, providing an increased interval to tumor progression, or increasing disease-free survival time) cancer (e.g., breast cancer, including Her2+, Her2−, ER+, ER−, Her2+/ER+, Her2+/ER−, Her2−/ER+, and Her2−/ER− breast cancer), head and neck cancer, oral cavity cancer, laryngeal cancer, chondrosarcoma, ovarian cancer, testicular carcinoma, melanoma, brain tumors (e.g., astrocytomas, glioblastomas, gliomas)) by administering an effective amount of an engineered multi-Kunitz domain protein of the invention that inhibits at least two proteases involved in cancer, for example at least two proteases from the group of human plasmin, human hepsin, human matriptase, human endotheliase 1, and human endotheliase 2.
The disclosed methods are useful in the prevention and treatment of solid tumors, soft tissue tumors, and metastases thereof. Solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of the various organ systems, such as those of lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary (e.g., renal, urothelial, or testicular tumors) tracts, pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal cancers, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine. Additional exemplary solid tumors include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastrointestinal system carcinomas, colon carcinoma, pancreatic cancer, breast cancer, genitourinary system carcinomas, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, endocrine system carcinomas, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Metastases of the aforementioned cancers can also be treated or prevented in accordance with the methods described herein.
EXAMPLES
Example 1
Expression of TFPI in Eukaryotic Cells
Table 1 shows the amino acid sequence of LACI (also known as tissue-factor pathway inhibitor (TFPI)). Table 2 shows the amino acid sequence of the different Kunitz domains within LACI. The positions marked with “|” (11, 13, 15-19, 34, 39-40) are examples of residues that can be varied to change the protease specificity of the domain. Positions marked with “^” (21 and 32) may also be varied.
The positions marked “$” can contribute to the structure of the Kunitz domain. In some implementations, these positions are kept invariant. In other implementations, they are varied, but to a limited degree, e.g., to allow for conservative substitutions.
LACI contains three potential N-linked glycosylation (Nx(S/T)) sites. The Asn (N) residues are shown bold and underscored in Table 1. In Kunitz domain2, N25 (numbering from the beginning of the domain) is a potential glycosylation site. This position is distant from the residues that contact serine proteases and glycosylation at this site is unlikely to interfere with protease binding or inhibition. Glycosylatioin of a second Asn in Linker 3 is also unlikely to affect activity. The third potential site in Kunitz domain 3 is distant from the binding site and glycosylation at this site will not prevent binding to or inhibition of a protease.
Table 3 shows the cDNA that encodes TFPI. The DNA encoding the putative signal sequence runs from base 133 to base 216 (underlined). Table 32 shows the cDNA for rabbit TFPI. There a shorter signal sequence is identified. The mature protein is encoded by bases 217-1044. Table 4 shows the annotated DNA sequence of a vector, pFTFPI_V1, having a modified sequence of TFPI that it would be expressed in CHO or HEK293T cells. The modifications provide restriction sites so that parts of TFPI can be readily altered. Other vectors and other signal sequences can be used. The DNA sequence has been modified to introduce a number of restriction sites so that parts of TFPI can be removed or modified easily. Altered bases are shown as uppercase, bold, or underscored. On each line of Table 4, every character that follows an exclamation point (!) is a comment. The naked DNA sequence in this table and other tables of similar appearance can be recovered by dropping the exclamation points and all characters that follow them on that line.
The DNA sequence in Table 4 can be constructed using the human TFPI cDNA and the oligonucleotides in Table 15. First the cDNA is used as template and ONMatureT and ON1019L27 are used to amplify the coding region, a 829 base segment. This DNA can be used as a template for ten PCR reactions that use primers 1: (ONSfiT, ONSacHinL), 2: (ONSacHinU, ONMluIL), 3: (ONMluIU, ONBBBL), 4: (ONBBBU, ONXhoBamL), 5: (ONXhoBamU, ONBsmBIL), 6: (ONBsmBIU, ONApaSalL), 7: (ONApaSalU, ONSac2L), 8: (ONSac2U, ONBspE2L), 9: (ONBspE2U, ONBssH2L), and 10: (ONBssH2U, ONfinalL). The ten products (which have 20 or more bases of overlap with one other product) are mixed and amplified with the primers ONSfiT and ONfinalL to produce a 874 base product. This nucleic acid is cut with SfiI and XbaI and ligated to a similarly cut expression vector.
Example 2
A TFPI Derivative that Inhibits h.pKal and Human Neutrophil Elastase
Human neutrophil elastase and human plasma kallikrein may both be involved in the inflammatory response associated with coronary by-pass surgery (CBPS). Thus a protein that inhibits both human neutrophil elastase and human plasma kallikrein may have synergistic beneficial effects when used for treating patients, e.g., before, during or after coronary by-pass surgery.
An inhibitor that is highly potent and specific for each of human plasma kallikrein (h.pKal) and human neutrophil elastase is encoded by the gene displayed in Table 9. Altered amino acids are shown bold and mutated bases are shown bold and uppercase. Restriction sites are shown bold. Any other signal sequence functional in eukaryotic cells could be used. This protein is named TF88890. This gene can be expressed in the same vector as was used for full-length TFPI shown in Table 4 or in any suitable expression vector. One cuts the expression vector with SfiI and XbaI and ligates the DNA shown in Table 9 to the digested vector DNA. Table 10 shows the oligonucleotides that can be used to make changes in TFPI Kunitz domain 1 and Kunitz domain 2. The procedure is as follows.
PCR of pFTFPI_V1 with ON1U23 and ON718L21 gives a 522 base product, PNK1, comprising the mature sequence up to S174. Using PNK1 as template, ON1U23 and ONK1Bot give a 141 base product, PNK2, that includes changes around residues 13-20 of TFPI Kunitz domain1. Within Kunitz domain 1, the changes are K15R, R17A, I18H, I19P, and F21W. PCR of PNK1 with ONK1T and ONK2B1 gives a 265 base product, PNK3, that overlaps the first product by 57 bases. This PCR introduces changes into Kunitz domain 2: I13P, R15I, G16A, Y17F, I18F, T19P, and Y21W. PCR of PNK1 with ONK2T1 and ONK2B2 give PNK4 of length 104 which comprises an overlap of 51 bases with PNK3 and the changes in TFPI Kunitz domain2. This introduces the changes R32L, K34P, and L39Q. PCR of PNK1 with ONK2T2 and ON718L21 give PNK5 with length 165. Mix PNK2, PNK3, PNK4, and PNK5 and use ONSfiTop and BotAmp to amplify a 573 base fragment. Cut the final PCR product with SfiI and XbaI and ligate to similarly cleaved expression vector. The new vector is called pFTFPI_V2.
The molecule encoded by pFTFPI_V2, TF88890, has the following expected characteristics. The first Kunitz domain will inhibit human plasma kallikrein and the second Kunitz domain will inhibit human neutrophil elastase (hNE). The mature protein should be 174 amino acids and may be glycosylated at N117 and N167. N117 is at position 26 in Kunitz domain 2. This site is located on a different side of the protein from the protease binding site, and, accordingly, is not expected to interfere with protease binding. The N167 is in Linker 3 and is not expected to interfere with inhibition of hNE. The protein is terminated at S174. MMP-8 and MMP12 cleave wildtype TFPI at this site. Thus, the presence of a protein ending with this sequence is less likely to cause an immune response because just such a peptide results from MMP-8 or MMP12 cleavage of wildtype TFPI. The expected molecular weight of TF88890 is 19 KDa plus the glycosylation. TF88890 is not expected to bind cell surfaces unlike TFPI because the region of TFPI implicated in cell-surface binding is absent. The molecular mass of TF88890 is approximately 14 KDa and would have a short residence in serum. A short residency may be useful for treating patients undergoing coronary bypass surgery. However, increased residency time can be engineered, e.g., by attaching one or more polymer moieties (e.g., PEG moieties).
Alternative designs for TF88890 are shown below in Tables 20-25. An additional alternative design is to use the sequence shown in Table 9 wi bases 508-579 deleted. This construct would be TF88890S and would comprise 150 amino acids with two potential N-linked sites of glycosylation. TF88890S would inhibit human plasma kallikrein and human neutrophil elastase. The sequences in these tables are exemplary mature amino acid sequences. To produce a protein including these sequence by secretion, a suitable signal sequence can be added for expression in eukaryotic cells (such as mammalian (e.g., CHO or HEK293T) or yeast cells (e.g., Pichia pastoris), or bacterial cells (such as E. coli or Caulobacter crescentus).
The protein can be designed so that it does not include more than two glycosylation sites, e.g., the protein can be devoid of N-linked glycosylation sites.
Kunitz domains can be joined with a linker, e.g., a hydrophilic, flexible linker (e.g., by the linker: GGSGGSSSSSG (SEQ ID NO:147)). Linkers that lack T-cell epitopes are particularly useful. All ligands for MHC molecules contain at least one or two amino-acid types picked from: (a) medium to large hydrophobic (VILMFWY (SEQ ID NO:148)), or (b) charged (EDRKH (SEQ ID NO:149)). Accordingly, linkers (e.g., of twelve amino acids) that do not contain any of these amino acids avoids creation of an MHC-I neo-epitope, including epitopes that may arise from juxtaposition of residues from adjoining Kunitz domains. Where more than one linker is used (e.g., for a protein with at least three Kunitz domains), the linkers between the Kunitz domains could be different.
For example, TF88890-2 is an alternative to the design of TF88890 and is shown in Table 20. TF88890-2 contains two copies of TFPI-Kunitz domain 1, the first modified to make it into an inhibitor of human plasma kallikrein (h.pKal) and the second modified to make it an inhibitor of human neutrophil elastase.
The amino acid sequence of mature TF88890-3 is shown in Table 21. TF88890-3 contains three copies of TFPI-Kunitz domain 1, the first and third modified to make it into an inhibitor of human plasma kallikrein and the second modified to make it an inhibitor of human neutrophil elastase. The instances of TFPI-Kunitz domain 1 are joined by hydrophilic, flexible linkers (GGSGGSSSSSG (SEQ ID NO:147)) which is not expected to provide any T-cell epitope. The first or third Kunitz domain could be modified to inhibit a different protease, giving a tri-specific inhibitor. The increased molecular mass is expected to increase the residence time in serum.
The amino acid sequence of mature TF88890-4 is shown in Table 22. TF88890-4 contains four copies of TFPI-Kunitz domain 1, the first and third modified to make it into an inhibitor of h.pKal and the second and third modified to make it an inhibitor of human neutrophil elastase. The instances of TFPI-Kunitz domain 1 are joined by hydrophilic, flexible linkers (GGSGGSSSSSG) (SEQ ID NO:147) which is not expected to provide any T-cell epitope. The linkers need not all be the same. One could have all four Kunitz domains directed to different proteases.
The amino acid sequence of mature TF88890-5 is shown in Table 23. TF88890-5 contains four copies of TFPI-Kunitz domain 1, the first and third modified to make it into an inhibitor of h.pKal and the second and third modified to make it an inhibitor of human neutrophil elastase. The instances of TFPI-Kunitz domain 1 are joined by hydrophilic, flexible linkers (GGSGNGSSSSGGSGSG (SEQ ID NO:150)) which is not expected to provide any T-cell epitope. The linkers shown each contain an N-linked glycosylation site (NGS). If produced in eukaryotic cells, TF88890-5 should carry glycosylation at three sites which should increase solubility and serum residence time. The linkers need not all be the same. One could have all four Kunitz domains directed to different proteases.
Example 3
An Inhibitor of Human Plasmin, h.pKal, and Human Neutrophil Elastase Based on TFPI
Table 16 shows the amino acid sequence of a protein derived from human TFPI and named PlaKalEl01. The sequence KGGLIKTKRKRKKQRVKIAYEEIFVKNM (SEQ ID NO:151) is deleted from the C-terminus of w.t. TFPI; this sequence is involved with binding to cells and the effects that TFPI has on gene expression. Removal of the sequence and changes in Kunitz domain 3 provides a protein that neither binds cells nor modulates cellular gene expression. In Kunitz domain 1, we have changed residue 15 of Kunitz domain 1 from Lys to Arg. At residue 17, we have changed Ile to Arg. At residue 18, Met is changed to Phe. At residue 19, we change Lys to Asp. At residue 21 we change Phe to Trp. These changes produce a Kunitz domain identical to DX-1000, a potent inhibitor of human plasmin.
In Kunitz domain2 we make the following changes (all denoted by position within the Kunitz domain): I13P, G16A, Y17A, I18H, T19P, Y21W, K34I, and L38E. These changes make a Kunitz domain that is very similar to DX-88 in the part of the Kunitz domain that contacts human plasma kallikrein. This domain is highly likely to be a potent and specific inhibitor of human plasma kallikrein.
In Kunitz domain 3, we make the following changes: L13P, R15I, N17F, E18F, N19P, K34P, S36G, G39Q. Thus, Kunitz domain is very similar to DX-890 which is a highly specific inhibitor of human neutrophil elastase. As reported by Piro and Broze (Circulation 2004, 110:3567-72.), removal of the highly basic C-terminal tail greatly reduces binding to cells. Piro and Broze also reported that changing R15 of Kunitz domain 3 to Leu greatly reduced cell binding.
A gene encoding PlaKalEl01 can be constructed as follows. Oligonucleotides shown in Table 17 are used as primers with the cDNA of human TFPI (shown in Table 3). The oligonucleotides Get_TFPI_top and ON937L24 are first used to amplify the coding sequence from codon 1 to codon 276, thus truncating the 3′ terminal codons. The 828 base product is called PKE_P1 and is used as template for six PCR reactions: a) Get_TFPI_top and MutK1:310L43 make PKE_P2 (220 bases), b) MutK1:310U43 and MutK2:517L56 make PKF_P3 (263 bases), c) MutK2:517U56 and MutK2:577L47 make PKF_P4 (107 bases), d) MutK2:577U47 and MutK3:792L55 make PKF_P5 (270 bases), e) MutK3:792U55 and MutK3:867L31 make PKF_P6 (106 bases), and f) MutK3:867U31 and ONLowCapPKE make PKE_P7 (114 bases). PKE_P2 through PKE_P7 are mixed and PCR amplified with primers ONLowCapPKE and ONNcoI to give a 861 base product having NcoI and XbaI sites at the ends. This fragment is cut with SfiI and XbaI and ligated to similarly cleaved expression vector having an expression cassette as shown in Table 9 (e.g., pFcFuse) for expression in eukaryotic cells.
Example 4
A Modification of PlaKalEl01 that is Resistant to Cleavage by MMPs
Known substrates for MMP-8 are shown in Table 42. The preference for P at P3, G at P1, and I or L at P1′ should be noted. Known substrates for MMP-12 are shown in Table 43.







TABLE 42







Substrates of MMP-8 (From MEROPS(http://


merops.sanger.ac.uk/))



























SEQ












ID


Substrate
P4
P3
P2
P1
P1′
P2′
P3′
P4′
Ref
NO:





1
G772
P
Q
G
I
A
G
Q
1
152


 


2
Dpn
P
Q
G
I
Q
G

1
153


 


3
Dpn
P
L
G
L
W
A
Dr
1
154


 


4
G
P
Q
G
I
W
G
Q
1
155


 


5
Mca
P
L
G
L
Dpa
A
R
1
156





Substrates: 1: collagen alpha 1(I) chain (Homo sapiens); cleavage after G775.


2: Dnp-P-Q-G-|-I-A-G-Q-dR-OH (SEQ ID NO: 201)


3: Dnp-P-L-G-|-L-W-A-dR-NH2 (SEQ ID NO: 154)


4: G-P-Q-G-|-I-W-G-Q (SEQ ID NO: 155)


5: Mca-P-L-G-|-L-Dpa-A-R-NH2 (SEQ ID NO: 156)


Ref 1 = Tschesche, H., Pieper, M. & Wenzel, H. Neutrophil collagenase. In Handbook of Proteolytic Enzymes, 2 edn (Barrett, A. J., Rawlings, N. D. & Woessner, J. F. eds), p. 480-486, Elsevier, London (2004)













TABLE 43







Some known substrate cleavages for MMP-12

























SEQ











ID


Substrate
P4
P3
P2
P1
P1′
P2′
P3′
P4′
NO:





1
R
P
F
Q
V
K
D
T
157


 


2
G
A
M
F
L
E
A
I
158


 


3
E
A
I
P
M
S
I
P
159


 


4
L
V
E
A
L
Y
L
V
160


 


5
E
A
L
Y
L
V
C
G
161





Substrates


1: alpha1-proteinase inhibitor (Homo sapiens)


2: alpha1-proteinase inhibitor


3: alpha1-proteinase inhibitor


4: insulin B chain (oxidized)


5: insulin B chain (oxidized)


Reference for all: Shapiro, S. D., Hartzell, W. O. & Senior, R. M. Macrophage elastase. In Handbook of Proteolytic Enzymes, 2 edn (Barrett, A. J., Rawlings, N. D. & Woessner, J. F. eds), p. 540-544, Elsevier, London (2004)






Table 30 shows the amino acid sequence of PlaKalEl02. This protein differs from PlaKalEl01 in that codon 49 is changed from a codon encoding Leu to a codon encoding Pro, codon 112 is changed from a codon encoding Ile to a codon encoding Pro, and codon 203 is changed from a codon encoding Thr to a codon encoding Pro to prevent cleavage by MMP-8 or MMP-12. Many proteases have difficulty cleaving before Pro and none of the known substrates of MMP-8 or MMP-12 have Pro at the P1′ position.
Example 5
Half Life, Metalloproteinases, and Cysteine Proteases
The half-life of a protein (by way of example, the protein PlaKalEl01) may be extended by methods including: 1) PEGylation, 2) fusion to Fc (e.g., the Fc domain of an IgG) at either the amino or carboxy terminus of Fc (e.g., as PlaKalEl01::Fc or Fc::PlaKalEl01), 3) fusion to human serum albumin (or a fragment thereof) at the amino or carboxy terminus (e.g., as PlaKalEl01::hSA or hSA::PlaKalEl01, or 4) making multimers of the protein until the molecular mass is large enough to give an acceptable half-life.
Table 18 shows the amino acid sequence of a PlaKalEl::GGSGGSGGSGGSGG (SEQ ID NO:162)::hSA fusion protein. Although the molecules are large, the half life is very likely to be on the order of weeks, similar to IgGs or hSA.
A PlaKalEl01 dimer is shown in Table 19. After Linker 5, a 13 amino acid glycine-rich hydrophilic linker avoiding known antigenic sequences is inserted. This is followed by another copy of the mature PlaKalEl01 sequence. The resulting protein has a 509 mature amino acid sequence, PKEPKE02. The increased molecular mass is expected to give the molecule a much longer half-life than PlaKalEl01. PKEPKE02 and related proteins are expected to have molecular masses in excess of 60 kDa and may have half lives of several days.
PKEPKE02 has two inhibitory sites for each of human plasmin, h.pKal, and human neutrophil elastase. The Kunitz domains of this protein may be further engineered to create a protein that specifically inhibits up to six different serine proteases. Additionally, the protein may be modified by fusion with one or more short peptide sequences that inhibit: a) other serine protease, b) metalloproteinases, or c) cysteine proteinases.
A gene encoding PKEPKE02 is inserted after a functional signal sequence in a vector suitable for expression in mammalian cells or yeast, e.g., secretory expression. For example, a DNA sequence encoding PKEPKE02 is inserted between the SfiI and XbaI sites of an expression vector having an expression cassette similar to the one shown in Table 9. It is also possible to employ the signal sequence as shown in Table 9 and follow the amino acid sequence shown with one or two stop codons, as in Table 9.
Table 31 shows the sequence of Elsix, a protein comprising six Kunitz domains designed to inhibit human neutrophil elastase. The sequence is derived from a dimer of TFPI. The mutations are indicated in bold. In Kunitz domain 1 the mutations are K151, I17F, M18F, K19P, and E39Q. In Kunitz domain 2, the mutations are I13P, R15I, G16A, Y17F, I18F, T19P, and L39Q. In Kunitz domain 3, the mutations are L13P, R15I, N17F, E18F, N19P, S36G, and G39Q. Kunitz domain 4 is a repeat of Kunitz domain 1; Kunitz domain 5 is a repeat of Kunitz domain 2, and Kunitz domain 6 is a repeat of Kunitz domain 3. The linker that joins the two halves is composed of Gly and Ser. There are six possible glycosylation sites shown as n, one at N25 of Kunitz domain 2, one in the Linker2 at N17, one at N44 in Kunitz domain 3, and three more in the second copy of modified TFPI.
Table 24 shows the mature amino acid sequence of TF890-6. This molecule has six identical Kunitz domains, each designed to inhibit human neutrophil elastase. TFPI Kunitz domain 1 is modified by the mutations K15I, I17F, M18F, K19P, and E39Q. There are more than 1. E 24 DNA sequences that encode this amino acid sequence. Preferably, each instance of the Kunitz domain would be different to avoid possible genetic instability. The linkers are each twelve amino acids long and are composed of glycine and serine. The length is such that the carboxy terminal amino acid of one Kunitz domain in conjunction with the amino terminal amino acids of the next Kunitz domain would not form an epitope for MHC-I. Peptides composed entirely of glycine and serine are unlikely to make tight-binding MHC-I epitopes.
For expression by secretion, a sequence encoding a signal sequence can be added before the DNA that encodes TF890-6. For eukaryotic expression by secretion, it is possible to add a eukaryotic signal sequence and express the protein in Pichia pastoris or mammalian cells. Since TF890-6 includes 408 amino acids, the molecular mass is approximately 44.8 KDa which could give the protein an increased serum residence time relative to proteins having fewer Kunitz domains.
Table 25 shows the amino acid sequence of TF890-8 which comprises 548 amino acids and would have an approximate molecular mass of 60.3 KDa. TF890-8 contains eight Kunitz domains of identical amino acid sequence. The linkers are made of glycine and serine and are all of length 12 amino acids. This molecule is monospecific for human neutrophil elastase, but may have a much greater half-life than DX-890 (a single Kunitz domain with molecular mass of approximately 7 KDa).
To avoid having multiple repeats of identical DNA, one can engineer the gene for (TF890-linker)n (n can be 1 to 10) as follows. Table 28 shows DNA that encodes a short linker, a modified version of TFPI Kunitz domain 1 (numbered 1-58), and more linker (lettered a-l). These sequences are flanked by a primer annealing sequence that includes sequences that facilitate restriction enzymes digestion. At the end of Table 28 are oligonucleotides that can be used with PCR to produce dsDNA. ONUPTF890 and ONLPTF890 are upper and lower strand PCR primers. ONUATF890 and ONUCTF890 are top strands and ONLBTF890 is a lower strand. The dsDNA is cut with Bsu36I which cleaves each strand after CC. Thus the overlap is 5′-TCA-3′ in the upper strand and 5′-TGA-3′ in the lower strand. This means that ligations will put top strands together. Had we used an enzyme that produces a palindromic overhang, top and bottom strands would be randomly joined. After cleavage, the long inserts are ligated into a vector having a compatible Bsu36I site under conditions that favor insertion of multiple inserts. Using priming sites outside the Bsu36I insertion site, we screen isolates to find those with 3, 4, 5, 6, 7, or 8 inserts. The vector can be designed to allow expression in eukaryotic cells, preferably mammalian cells. As shown in Table 28, the codons for residues S3-F20, I25-R47, and C51-X_h are varied in a way that maintains the encoded Kunitz domain amino acid sequence but maximized the DNA diversity. Residues in the linker can be either G or S. Thus, chains of units will have only short segments of exact identity.
Example 6
Stabilization of Kunitz Domains
Kunitz domains with disulfide bonds can oligomerize at high concentration and 37° C. on prolonged storage. Oligomerization may result form disulfide exchange between monomers. The C14-C38 disulfide is exposed on the surface of canonical Kunitz domains and can exchange with cysteines on other Kunitz domains to form intermolecular bonds. Accordingly, Kunitz domains that inhibit target proteases but are less likely to form oligomers can be designed by removal of the C14-C38 disulfide. In particular, the cysteine residues at these positions can be mutated to amino acids other than cysteine, e.g., alanine, glycine, or serine. In one embodiment, the mutations are C14G or C38V. Such a mutated Kunitz domain may be less stable with respect to melting but more stable with respect to multimerization.
TFPI Kunitz domain 1 can be engineered into a potent inhibitor of human neutrophil elastase by the mutations K151, I17F, M18F, K19P, and E39Q. To eliminate the 14-38 disulfide, the mutations C14G and C38V as found by Hagihara et al. (Hagihara, et al., 2002, J Biol Chem, 277:51043-8) may be made. This could decrease the binding to human neutrophil elastase. Table 26 shows a display cassette for TFPI Kunitz domain 1 in which the mutations K15I, I17F, M18F, and K19P have been made in order to confer affinity for human neutrophil elastase. In addition, positions 12, 13, 14, 38, and 39 are allowed to be any amino acid except Cys. This gives a diversity of 2,476,099. Note that NHK encodes amino acids FLSY<stop>PHQIMTNKVADE (SEQ ID NOS:146 and 202) while BGG encodes amino acids WRG. By using both of these codons, we allow all amino acids except Cys. ONTAAAxC has a NcoI restriction site, and codons up to T27 with NHK at codons 12, 13, and 14. ONTAABxC covers the same range except that 12 and 13 are NHK and 14 is BGG. ONTABAxC has BGG at 13 and NHK at 12 and 14. ONTABBxC has BGG at both 13 and 14 and NHK at 12. ONTBAAxC, ONTBABxC, ONTBBAxC, and ONTBBBxC all have BGG at 12 and NHK or BGG at 13 and 14 in the same pattern as the first four oligonucleotides. Thus 12, 13, and 14 are allowed to be any AA except C. ONLoAAxC covers codons R20 to E49 (where the XbaI site is) plus scab DNA. Because ONLoAAxC is a lower strand, it has the reverse complements of NHK and BGG, namely MDN and ccV. Table 27 shows oligonucleotides that would implement this strategy. The eight long oligonucleotides are mixed and the short oligonucleotides are used as primers to generate DNA that extends from NcoI to XbaI. The PCR product is cleaved with NcoI and XbaI and is ligated to a version of DY3P82 containing TFPI Kunitz domain 1 or a derivative thereof having NcoI and XbaI sites. Table 29 contains the annotated sequence of DY3P82TFPI1, a phage vector that displays TFPI Kunitz domain1 on the stump of M13 III. DY3P82TFPI1 has a wildtype M13 iii gene and a display gene regulated by PLac so that a) expression of the display can be suppressed with glucose and induced with IPTG, and b) most phage display one or fewer Kunitz domain::III stump proteins. This allows distinguishing very good binders from merely adequate binders. We select binders to human neutrophil elastase from the library of phage. Since DX-890 binds human neutrophil elastase with KD of about 3 pM, very stringent washes are used. In addition, DX-890 may be used as an eluting agent.
It is expected that Kunitz domains that lack the 14-38 disulfide and that inhibit target proteases can be selected. Once an amino acid sequence is determined that gives high-affinity binding and inhibition of human neutrophil elastase, one can construct a library of multimers as given in Example 5. This approach can be applied to any Kunitz domain and to any target protease, and to plural target proteases. For example, if human plasma kallikrein is the target, it is possible to vary the sequence of DX-88 at positions 12, 13, 14, 38, and 39 excluding only Cys from the varied amino acids.
Example 7
A Derivative of Rabbit TFPI for PK and Immunogenicity Studies
Table 32 shows the amino acid sequence of rabbit TFPI. Table 33 shows the cDNA of rabbit TFPI. Table 34 shows RaTFPKE01, an engineered derivative of rabbit TFPI. In Kunitz domain 1, the mutations are Y17R, I18F, K19D, and F21W which are designed to confer human plasmin inhibitory activity on Kunitz domain 1. In Kunitz domain 2, the mutations are I13P, G16A, Y17A, I18H, T19P, and L39E which are designed to confer inhibition of h.pKal on Kunitz domain 2. In Kunitz domain 3, the mutations are L13P, Q15I, N17F, E18F, I19P, S36G, and G39Q which are designed to confer human neutrophil elastase inhibition on Kunitz domain 3. The C-terminal basic peptide has been truncated. This protein can be produced in CHO cells in the same manner as PlaKalEl01. The protein should have a molecular mass of about 30 KDa plus glycosylation. There are six possible Nx(S/T) sites but one is NPT and is unlikely to be glycosylated. Because we have altered the third Kunitz domain and removed the lysine-rich C-terminal peptide, RaTFPKE01 should have reasonable half-life and should not bind to cells.
Table 36 shows a gene that encodes RaTFPKE01. The sequence is arranged to show: a) signal sequence, b) first linker from rabbit TFPI, c) Kunitz domain 1 with cysteine codons underlined and mutations shown uppercase bold, d) the second linker, e) Kunitz domain 2, f) third linker, g) Kunitz domain 3, and g) final linker. Table 37 shows oligonucleotides that can be used to construct the gene of Table 36 from rabbit TFPI cDNA. One first uses rabbit TFPI cDNA and ONrSfi and TruncLo to produce a 776 base product, PrTF1. PrTF1 is used as a template with ONrSfi and ONrK1Bot to produce PrTF2, a 162 base oligonucleotide. PrTF1 is used as a template with ONrK1Top and ONrK2Bot1 to produce PrTF3, a 239 base oligonucleotide. PrTF1 is used as a template with ONrK2To1 and ONrK2B2 to produce PrTF4, a 103 base oligonucleotide. PrTF1 is used as a template with ONrK2T2 and ONrK3B1 to produce PrTF5, a 236 base oligonucleotide. PrTF1 is used as a template with ONrK3T1 and ONrK3B2 to produce PrTF6, a 99 base oligonucleotide. PrTF1 is used as a template with ONrK3T2 and ONrXbaIL to produce PrTF7, a 117 base oligonucleotide. PrTF2-7 are mixed and ONrSfi and ONrXbaIL are used as primers to produce PrTF8, a 790 base oligonucleotide which is cut with SfiI and XbaI and ligated to a similarly cut expression vector.
Table 40 shows the amino acid sequence of mature RaTFPKE04, a derivative of rabbit TFPI. RaTFPKE04 has all of the mutations of RaTFPKE01 and has C14G and C38V in each of the Kunitz domains. The purpose is to make a protein that is less likely to aggregate than RaTFPKE01 might be.
Table 35 shows the amino acid sequence of RaTFPKE02, a 509 AA protein formed by joining two copies of mature RaTFPKE01 with a linker=GGSGSSGSGGSGSSG (SEQ ID NO:163). The mature protein should have molecular mass of about 56 Kda plus glycosylation. RaTFPKE02 should have a long half-like in rabbits and should not be immunogenic. Table 41 shows the amino acid sequence of mature RaTFPKE05. RaTFPKE05 is similar to RaTFPKE02 but in each Kunitz domain, C14 is changed to G and C38 is changed to V. RaTFPKE05 is less likely to aggregate than is RaTFPKE02.
Example 8
An Inhibitor for hNE, hPr3, and hGatG
Neutrophils release three serine proteases when activated: neutrophil elastase (hNE, also known as leukocyte elastase), proteinase 3 (Pr3), and cathepsin G (CatG). To block the damage caused by excessive neutrophil activation, one can use an agent that inhibits two or more of these proteases, e.g., all three proteases, each with high affinity and specificity. Kunitz-domain that inhibit human neutrophil elastase are known. Inhibitors of CatG can be obtained by selection from a library of Kunitz domains (U.S. Pat. No. 5,571,698). Inhibitors to PR3 could be obtained from a Kunitz domain library in a similar manner.
In one embodiment, the amino acid resides that confer Pr3 inhibitory activity can then be transferred to another Kunitz domain, e.g., a Kunitz domain of a multi-domain protein, e.g., a protein that includes three Kunitz domains. The amino acid residues that confer CatG inhibitory activity can likewise be transferred to another Kunitz domain of the protein. The amino acid residues that confer hNE inhibitory activity can be transferred to the third domain.
In another embodiment, Kunitz domains that inhibit neutrophil elastase, proteinase 3, and cathepsin G are physically associated with each other, e.g., by forming a fusion protein that includes the different Kunitz domains.
In some embodiments, the engineered protein does not bind to cells and can have one or more of the following attributes (e.g., two, three or four of the following): (a) ability to function in the presence of neutrophil defensins, (b) ability to resist degradation by MT6-MMP (Leukolysin), (c) ability to inhibit proteases bound to anionic surfaces, (d) ability to reduce production of IL-8 from neutrophils and TNFα and IL-1β from monocytes.
Example 9
Pharmacokinetics in Animals
The following methods can be used to evaluate the pharmacokinetics (PK) of proteins such as proteins that include Kunitz domains in animals, e.g., mice and rabbits.
The protein to be tested is labeled with iodine (125I) using the iodogen method (Pierce). The reaction tube is rinsed with reaction buffer (25 mM Tris, 0.4 M NaCl, pH 7.5). The tube is emptied and then replaced with 0.1 ml of reaction buffer and 12 μL of carrier free iodine-126, about 1.6 mCi. After six minutes, the activated iodine is transferred to a tube containing the protein to be tested. After nine minutes, the reaction is terminated with 25 μL of saturated tyrosine solution. The reaction can be purified on a 5 ml D-salt polyacrylamide 6000 column in Tris/NaCl. HSA can be used to minimize sticking to the gel.
A sufficient number of mice (about 36) are obtained. The weight of each animal is recorded. In the case of mice, the animals are injected in the tail vein with about 5 μg of the protein to be tested. Samples are recovered at each time point per animal, with four animals per time point, at approximately 0, 7, 15, 30, and 90 minutes, 4 hours, 8 hours, 16 hours, and 24 hours post injection. Samples (about 0.5 ml) are collected into anti-coagulant (0.02 ml EDTA). Cells are spun down and separated from plasma/serum. Samples can be analyzed by radiation counting and SEC peptide column on HPLC with inline radiation detection.
For rabbits, the material is injected into the ear vein. Samples can be collected at 0, 7, 15, 30, 90 minutes, 4, 8, 16, 24, 48, 72, 96, 120, and 144 hours post-injection. Samples can be collected and analyzed as for mice.
Data can be fit to a bi-exponential (equation 1) or a tri-exponential (equation 2) decay curve describing “fast”, “slow”, and “slowest” phases of in vivo clearance:

y=Aexp(−αt)+Bexp(−βt)  Equation 1

y=Aexp(−αt)+Bexp(−βt)+Cexp(−γt)  Equation 2

Where:

y=Amount of label remaining in plasma at time=t post-administration

A=Total label in “fast” clearance phase

B=Total label in “slow” clearance phase

C=Total label in “slowest” clearance phase

α=“Fast” clearance phase decay constant

β=“Slow” clearance phase decay constant

γ=“Slowest” clearance phase decay constant

t=Time post administration

The α, β, and γ phase decay constants can be converted to half-lives for their respective phases as:

α Phase Half-life=0.69(1/α)

β Phase Half-life=0.69(1/β)

γ Phase Half-life=0.69(1/γ)

In the case where the data are fit using the bi-exponential equation, the percentages of the total label cleared from in vivo circulation through the α and β phases are calculated as:

% α Phase=[A/(A+B)]×100

% β Phase=[B/(A+B)]×100

In the case where the data are fit using the tri-exponential equation, the percentages of the total label cleared from in vivo circulation through the α and β phases are calculated as:

% α Phase=[A/(A+B+C)]×100

% β Phase=[B/(A+B+C)]×100

% γ Phase=[C/(A+B+C)]×100

Example 10
A Gene that Encodes TIFP Minus the Positively Charged C-Terminal Region
Table 50 shows a gene that has been optimized for expression in CHO cells and which encodes hTFPImC1. hTFPImC1 comprises: a) a signal sequence, b) a short linker to foster cleavage by signal peptidase, c) a six-his tag to facilitate purification of the encoded multi-KuDom protein, d) a thrombin cleavage site, e) codons corresponding to TFPI 2-270. This gene can be expressed in mammalian cells, such as CHO cells. Codons 271-304 have been eliminated. Residues 271-304 are involved with causing TFPI to bind to cell surfaces, to be internalized, and to alter gene expression. These effects are unwanted in a therapeutic protease inhibitor. hTFPImC1 can be purified on a copper-chelating resin via the His6 tag. The His tag is removable by treatment with thrombin, which should cleave after R38.
Table 53 shows the DNA of a vector that contains the gene of Table 50 so that it can be expressed in eukaryotic cells. Table 56 shows the amino-acid sequence of hTFPImC1, The Asn residues where N-linked glycosylation is expected are shown as bold N with a “*” above.
hTFPImC1 was expressed in HEK293T cells transformed with the vector shown in Table 53. The protein was purified by affinity chromatography, and a sample was analyzed by gel chromatography, which showed an apparent molecular mass of  ̃42.5 kDa.
Cell surface binding of hTFPImC1 was tested by flow cytometry, which showed no detectable (above background) cell surface binding.
Example 11
A Modified TFPI, hTFPI[PpKhNE]
Table 51 shows an annotated gene for hTFPI[PpKhNE]. hTFPI[PpKhNE] has the first KuDom of TFPI modified so that it will inhibit human plasmin (hPla), the second KuDom modified so that it will inhibit human plasma kallikrein (h.pKal), and the third KuDom modified so that it will inhibit human neutrophil elastase (hNE). To achieve these specificities, the following mutations have been made. In the first KuDom, we made the mutations K15R, I17R, M18F, K19D, and F21W to confer anti plasmin activity. In the second KuDom, we made the mutations I13P, G16A, Y17A, I18H, T19P, Y21W, K34I, and L39E to confer anti plasma kallikrein activity. In the third KuDom, we made the mutations L13P, R15I, N17F, E18F, N19P, K34P, S36G, and G39Q to confer anti human neutrophil elastase activity. Table 54 shows the DNA sequence of a vector that allows expression of the gene shown in Table 51 in eukaryotic cells such as CHO cells. Table 57 shows the amino-acid sequence of hTFPI[PpKhNE] with annotation. For each of the KuDoms, the w.t. AAs are shown below the actual AA sequence. Because the protein has been truncated and the third KuDom has been modified, it is not expected to bind to cell surfaces (see Example 10, above). It is expected that hTFPI[PpKhNE] will strongly inhibit human plasmin (h.Pla), human plasma kallikrein (h.pKal), and human neutrophil elastase (hNE).
Example 12
Pla301B, a Triple Inhibitor of Human Plasmin
Table 52 shows a gene that encodes Pla301B, a protein derived from human TFPI and having three KuDoms, each of which is designed to inhibit h.Pla. Table 55 shows the sequence of a vector that allows the expression of Pla301B in eukaryotic cells. Table 58 shows the amino-acid sequence of Pla301B. To confer anti h.Pla activity on Pla301B, we made the following mutations. In the first KuDom, we made the mutations K15R, I17R, M18F, K19D, F21W. In the second KuDom, we made the mutations I13P, G16A, Y17R, I18F, T19D, Y21W, and K34I. In the third KuDom, we made the mutations L13P, N17R, E18F, N19D, F21W, K34I, S36G, and G39E. Each KuDom should now be a potent inhibitor of h.Pla.
Human plasmin is known to be activated by urokinase-type plasminogen activator (uPA). uPA is often bound to cell surfaces by the urokinase-type plasminogen activator receptor (uPA-R). h.Pla that is activated by the uPA-uPA-R complex often binds to the plasmin receptor (Pla-R).
Pla301B was expressed in HEK293T cells with the vector shown in Table 55. The protein was assayed for plasmin inhibition and was found to be active.
Example 13
A Multiple KuDom Inhibitor that Inhibits Plasmin, Matriptase, and Hepsin
HAI-1 inhibits matriptase (Herter et al., Biochem J. 2005 390(Pt 1):125-36.) via Kunitz domain I (Kirchhofer et al., FEBS Lett. 2005 579(9):1945-50.) (the first Kunitz domain of HAI-1B is identical to the first Kunitz domain of HAI-1). HAI-2, domain 1 inhibits hepsin (Parr and Jiang, Int J Cancer. 2006 Sep. 1; 119(5):1176-83.). Table 61 shows an amino-acid sequence of PlaMatHep, a protein designed to inhibit plasmin, matriptase, and hepsin. In the first KuDom of TFPI, we have mutated K15 to R, I17 to R, M18 to F, K19 to D, and F21 to W. This corresponds to DX-1000 (a potent inhibitor of plasmin) shown in Table 7. In the second KuDom, we have mutated I13 to R, Y17 to S, I18 to F, T19 to P, Y21 to W, E31 to K, R32 to S, and K34 to V. The mutated domain is shown in Table 7 as TFPI-K2matr with w.t. AA in lower case and mutations in upper case. These changes should make the second domain of PlaMatHep be a potent inhibitor of matriptase. In the third domain of TFPI, we have made the mutations R11 to V, L13 to R, N17 to S, E18 to M, N19 to P, F21 to W, K34 to V, S36 to G, E42 to S, and K48 to E. The K48E mutation will reduce the chance of binding glycoaminoglycans. These changes are shown in Table 7 as TFPI-K3hep and should make the third domain of PlaMatHep be a potent inhibitor of hepsin. A gene to express the protein shown in Table 61 is readily constructed from the genes of other TFPI derivatives shown herein.
Tables







TABLE 1







Human LACI (TFPI) (SEQ ID NO: 1)









AAs
Function
AA sequence





1-28
signal
         1    1    2    2




1   5    0    5    0    5




MIYTMKKVHALWASVCLLLNLAPAPLNA


 


29-49
Linker 1
 3    3    4    4   4




 0    5    0    5   9




DSEEDEEHTIITDTELPPLKL


 


50-107
KuDom 1
                                                  1    1 




5    5    6    6    7    7    8    8    9    9    0    0




0    5    0    5    0    5    0    5    0    5    0    5




MHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD


 


108-120
Linker 2
  1    1    1




  1    1    2




  0    5    0




NANRIIKTTLQQE


 


121-178
KuDom 2
1   1    1    1    1    1    1    1    1    1    1    1




2   2    3    3    4    4    5    5    6    6    7    7




1   5    0    5    0    5    0    5    0    5    0    5




KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG


 


179-212
Linker 3
 1    1    1    1    2    2    2




 8    8    9    9    0    0    1




 0    5    0    5    0    5    0




PNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFEFH


 


213-270
KuDom 3
  2    2    2    2    2    2    2    2    2    2    2    2




  1    2    2    3    3    4    4    5    5    6    6    7




  5    0    5    0    5    0    5    0    5    0    5    0




GPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG


 


271-304
Linker 4
2   2    2    2    2    2    3   3




7   7    8    8    9    9    0   0 




1   5    0    5    0    5    0   4 




FIQRISKGGLIKTKRKRKKQRVKIAYEEIFVKNM
















TABLE 2





AA sequences of the KuDoms in human LACI (TFPI)



























TABLE 3





cDNA of human LACI (TFPI) (SEQ ID NO: 2)
















LOCUS
NM_006287             1431 bp    mRNA    linear   PRI 08-JUN-2005


DEFINITION
Homo sapiens tissue factor pathway inhibitor



(lipoprotein-associated coagulation inhibitor) (TFPI), mRNA.


ACCESSION
NM_006287


VERSION
NM_006287.3 GI: 40254845







sig_peptide     133..216


                /gene=“TFPI”


        /note=“lipoprotein-associated coagulation inhibitor signal peptide”


     mat_peptide     217..1044


                     /gene=“TFPI”


                     /product=“lipoprotein-associated coagulation inhibitor”


ORIGIN


   1  ggcgggtctg cttctaaaag aagaagtaga gaagataaat cctgtcttca atacctggaa


 


  61  ggaaaaacaa aataacctca actccgtttt gaaaaaaaca ttccaagaac tttcatcaga


 


 121  gattttactt agatgattta cacaatgaag aaagtacatg cactttgggc ttctgtatgc


 


 181  ctgctgctta atcttgcccc tgcccctctt aatgctgatt ctgaggaaga tgaagaacac


 


 241  acaattatca cagatacgga gttgccacca ctgaaactta tgcattcatt ttgtgcattc


 


 301  aaggcggatg atggcccatg taaagcaatc atgaaaagat ttttcttcaa tattttcact


 


 361  cgacagtgcg aagaatttat atatggggga tgtgaaggaa atcagaatcg atttgaaagt


 


 421  ctggaagagt gcaaaaaaat gtgtacaaga gataatgcaa acaggattat aaagacaaca


 


 481  ttgcaacaag aaaagccaga tttctgcttt ttggaagaag atcctggaat atgtcgaggt


 


 541  tatattacca ggtattttta taacaatcag acaaaacagt gtgaacgttt caagtatggt


 


 601  ggatgcctgg gcaatatgaa caattttgag acactggaag aatgcaagaa catttgtgaa


 


 661  gatggtccga atggtttcca ggtggataat tatggaaccc agctcaatgc tgtgaataac


 


 721  tccctgactc cgcaatcaac caaggttccc agcctttttg aatttcacgg tccctcatgg


 


 781  tgtctcactc cagcagacag aggattgtgt cgtgccaatg agaacagatt ctactacaat


 


 841  tcagtcattg ggaaatgccg cccatttaag tacagtggat gtgggggaaa tgaaaacaat


 


 901  tttacttcca aacaagaatg tctgagggca tgtaaaaaag gtttcatcca aagaatatca


 


 961  aaaggaggcc taattaaaac caaaagaaaa agaaagaagc agagagtgaa aatagcatat


 


1021  gaagaaattt ttgttaaaaa tatgTGAatt tgttatagca atgtaacatt aattctacta


 


1081  aatattttat atgaaatgtt tcactatgat tttctatttt tcttctaaaa tcgttttaat


 


1141  taatatgttc attaaatttt ctatgcttat tgtacttgtt atcaacacgt ttgtatcaga


 


1201  gttgcttttc taatcttgtt aaattgctta ttctaggtct gtaatttatt aactggctac


 


1261  tgggaaatta cttattttct ggatctatct gtattttcat ttaactacaa attatcatac


 


1321  taccggctac atcaaatcag tcctttgatt ccatttggtg accatctgtt tgagaatatg


 


1381  atcatgtaaa tgattatctc ctttatagcc tgtaaccaga ttaagccccc c
















TABLE 4





The annotated DNA sequence of pFTFPI_V1















!pFTFPI_V1       5705                CIRCULAR


!


! Non-cutters


!AfeI AGCgct         AflII Cttaag           AgeI Accggt


!ApaLI Gtgcac        AscI GGcgcgcc          BglII Agatct


!BlpI GCtnagc        BsgI ctgcac            BsiWI Cgtacg


!BstXI CCANNNNNntgg  Bsu36I CCtnagg         EcoNI CCTNNnnnagg


(SEQ ID NO: 164)                            (SEQ ID NO: 165)


!EcoRV GATatc        EspI GCtnagc           FseI GGCCGGcc


!HpaI GTTaac         MfeI Caattg            NheI Gctagc


!NotI GCggccgc       PacI TTAATtaa          PmeI GTTTaaac


!PpuMI RGgwccy       PshAI GACNNnngtc       PstI CTGCAg


                     (SEQ ID NO: 187)


!SbfI CCTGCAgg       SgfI GCGATcgc          SgrAI CRccggyg


!SnaBI TACgta        Spel Actagt            Sse8387I CCTGCAgg


!SwaI ATTTaaat       XcmI CCANNNNNnnnntgg


                     (SEQ ID NO: 166)


!


! cutters


!


! Enzymes that cut more than 3 times.


!


!AatII GACGTc                  5 


!BanII GRGCYc                  6 


!BciVI GTATCCNNNNNN            4 (SEQ ID NO: 167)


!BglI GCCNNNNnggc              5 (SEQ ID NO: 168)


!BpmI CTGGAG                   4 


!BsaAI YACgtr                  4 


!BsiHKAI GWGCWc                5 


!BsmFI GGGACNNNNNNNNNNnn      10 (SEQ ID NO: 169)


!BsmI NGcattc                  4 


!BsrFI Rccggy                  5 


!BssSI Cacgag                  4 


!EarI CTCTTCNnnn               5 (SEQ ID NO: 170)


!Eco57I CTGAAG                 5 


!FauI nNNNNNNGCGGG            11 (SEQ ID NO: 171)


!HgiAI GWGCWc                  5 


!PsiI TTAtaa                   4 


!StyI Ccwwgg                   4 


!


! Enzymes that cut from 1 to 3 times.


!


! $ = DAM site, * = DCM site, & = both


!










!EcoRI Gaattc
1
   6



!NdeI CAtatg
2
 259
1513


!BtrI CACgtg
1
 628



!PmlI CACgtg
1
 628



!BmgBI GACgtg
1
 645



!MscI TGGcca
2
 649
2845


!BtgI Ccrygg
2
 655
1288


!DsaI Ccrygg
2
 655
1288


!NcoI Ccatgg
1
 655



!Pf1MI CCANNNNntgg
1
 655
(SEQ ID NO: 172)


!NruI TCGcga
1
 688



!SfiI GGCCNNNNnggcc
1
 695
(SEQ ID NO: 173)


!Eco0109I RGgnccy
1
 702



!ApaI GGGCCc
2
 703
1161


!Bsp120I Gggccc
2
 703
1161


!PspOMI Gggccc
2
 703
1161


!Ec11361 GAGctc
1
 756



!SacI GAGCTc
1
 756



!HindIII Aagctt
1
 771



!NsiI ATGCAt
3
 777
2347 2419


!BspHI Tcatga
3
 826
4587 5595


!MluI Acgcgt
1
 855



!BstEII Ggtnacc
1
 894



!BspDI ATcgat
1
 904



!BsaBI GATNNnnatc
1
 907
(SEQ ID NO: 174)


!BsrGI Tgtaca
1
 939



!AvaI Cycgrg
3
1008
   1785     2622


!TliI Ctcgag
1
1008



!XhoI Ctcgag
1
1008



!BseRI GAGGAGNNNNNNNNNN
2
1011&
   2582   (SEQ ID NO: 175)


!BamHI Ggatcc
1
1016&



!SexAI Accwggt
2
1044*
   2368*


!BsmBI Nnnnnngagacg
1
1119
 (SEQ ID NO: 176)


!AccI GTmkac
2
1179*
   3735


!HincII GTYrac
1
1179*



!Sall Gtcgac
1
1179*



!SacII CCGCgg
1
1288



!BspEI Tccgga
1
1371



!BssHII Gcgcgc
1
1421



!StuI AGGcct
2
1463
   2598


!XbaI Tctaga
1
1548$



!BclI Tgatca
1
1561$



!BbsI GAAGAC
1
1756



!SphI GCATGc
3
1770
   2345 2417


!XmaI Cccggg
2
1785
   2622


!PvuII CAGctg
2
1818
   2869 


!NaeI GCCggc
3
1969
   3265 3548


!NgoMIV Gccggc
3
1969
   3265 3548


!DraIII CACNNNgtg
1
2072



!DrdI GACNNNNnngtc
3
2115
   2786 3969 (SEQ ID NO: 177)


!BstAPI GCANNNNntgc
2
2340
   2412 (SEQ ID NO: 178)


!AvrII Cctagg
2
2508
   2601


!BspMI Nnnnnnnnngcaggt
1
2652 
 (SEQ ID NO: 179)


!BspMI ACCTGCNNNNn
2
3024
 (SEQ ID NO: 180)


!EagI Cggccg
1
2671



!KasI Ggcgcc
1
2764



!FspI TGCgca
2
2865
   4980


!Pf1FI GACNnngtc
1
2880



!Tth111I GACNnngtc
1
2880



!SapI gaagagc
2
3114
   3324


!RsrII CGgwccg
1
3280



!BstBI TTcgaa
1
3446



!BstZ17I GTAtac
1
3735



!AlwNI CAGNNNctg
1
4278



!AhdI GACNNNnngtc
1
4755
 (SEQ ID NO: 181)


!Eam1105I GACNNNnngtc
1
4755
 (SEQ ID NO: 181)


!BsaI nnnnngagacc
1
4822
 (SEQ ID NO: 182)


!PvuI CGATcg
1
5127$



!ScaI AGTact
1
5238



!BcgI cgannnnnntgc
1
5276
 (SEQ ID NO: 183)


!Acc65I Ggtacc
1
5705



!KpnI GGTACc
1
5705








!------------------------------------------------------------------------


 


     1  GTACCGAATT CACATTGATT ATTGAGTAGT TATTAATAGT AATCAATTAC GGGGTCATTA


 


    61  GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG CCCGCCTGGC


 


   121  TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC CATAGTAACG


 


   181  CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGACTATT TACGGTAAAC TGCCCACTTG


 


   241  GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA TGACGGTAAA


 


   301  TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC TTGGCAGTAC


 


   361  ATCTACGTGT TAGTCATCGC TATTACCATA GTGATGCGGT TTTGGCAGTA CATCAATGGG


 


   421  CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG


 


   481  AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA


 


   541  TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG AGCTTTCTGG


 


   601  CTAACTAGAG AACCCACTGC TTACTGGCAC GTGGAAATTA ATACGACGTG GCCACC


!


!          Signal sequence...............................


!          M   G   W   S   C   I   I   L   F   L   V   A


  657     aTG Gga tgg agc tgt atc atc ctc ttc ttg gTC GCG


!                                                  NruI...


!


!         Signal sequence............    Mature TFPI.....


!                                        1   2 


!          T   A   T   G   A   H   S     D   S 


  693     AcG GCC aca ggG GCC cac tcc   gac tct!


!    NruI.. SfiI.............


!                     Eco0109I.


!                      ApaI....(Bsp120I)


!


!        Linker 1...................................................


!         3   4   5   6   7   8   9  10  11  12  13  14  15  16  17


!         E   E   D   E   E   H   T   I   I   T   D   T   E   L   P


  720    gag gaa gat gaa gaa cac aca att atc aca gat acg gag CtC cca


!                                                        SacI...


!


!      Linker 1........     KuDom 1...................................


!       18  19  20  21     22  23  24  25  26  27  28  29  30  31  32


!       P   L   K   L       M1  H   S   F   C5  A   F   K   A   D  D11


  765  cca ctg aaG ctt     atg cat tca ttt tgt gca ttc aag gcg gat gat


!              HindIII(1/1)NsiI...(1/3)


!                 |


!  Secondary cleavage site of MMP-8


!    Cleavage site of MMP-12 (Belaaouaj, et al.,


     2000, J Biol Chem, 275:27123-8)


!


!        KuDom 1....................................................


!        33  34  35  36  37  38  39  40  41  42  43  44  45  46  47


!         G   P  C14  K   A   I   M   K  R20  F   F   F   N   I   F


  810    ggc cca tgt aaa gca atc atg aaa aga ttt ttc ttc aat att ttc


!                             BspHI...


!


!        KuDom 1....................................................


!        48  49  50  51  52  53  54  55  56  57  58  59  60  61  62


!         T   R   Q  C30  E   E   F   I   Y   G   G  C38  E   G   N


  855    acG cgT cag tgc gaa gaa ttt ata tat ggg gga tgt gaa ggT aaC


!        MluI...                                           BstEII...


!


!        KuDom 1....................................................


!        63  64  65  66  67  68  69  70  71  72  73  74  75  76  77


!         Q   N   R  F45  E   S   L   E  E50  C   K   K   M  C55  T


  900    cag aat cga ttt gaa TCt ctg gaa gag tgc aaa aaa atg tgt aca


! BstEII..    BspDI...                                       BsrGI..


!                 BsaBI........


!


!      KuDom 1     Linker 2...........................................


!      78  79      80  81  82  83  84  85  86  87  88  89  90  91  92


!       R  D58      N   A   N   R   I   I   K   T   T   L   Q   Q   E


  945  aga gat     aat gca aac agg att ata aag aca aca ttg caa caa gaa


!


! Cleavage site of MMP-12 (Belaaouaj, et al., 2000,


  J Biol Chem, 275:27123-8)


!


!        Kudom 2....................................................


!        93  94  95  96  97  98  99  100 101 102 103 104 105 106 107


!         K   P   D   F   C5  F   L   E   E   D   P   G   I  C14  R


  990    aag cca gat ttc tgc ttt CtC gaG gaG gat cct gga ata tgt cga


!                               XhoI...   BamHI...


!


!        Kudom 2....................................................


!        108 109 110 111 112 113 114 115 116 117 118 119 120 121 122


!         G   Y   I   T  R20  Y   F   Y   N   N*  Q   T   K   Q  C30


 1035    ggt tat att acc agg tat ttt tat aac aat cag aca aaa cag tgt


!                    SexAI....(not U)


!


!        Kudom 2....................................................


!        123 124 125 126 127 128 129 130 131 132 133 134 135 136 137


!         E   R   F   K   Y   G   G  C38  L   G   N   M   N   N  F45


 1080    gaa cgt ttc aag tat ggt gga tgc ctg ggc aat atg aac aat ttt


!


!      Kudom 2............................................     Linker 3


!      138 139 140 141 142 143 144 145 146 147 148 149 150     151 152


!       E   T   L   E   E  C51  K   N   I  C55  E   D  G58      P   N


 1125  gag acG ctg gaa gaa tgc aag aac att tgt gaa gat ggG     ccC aat


!      BsmBI....-----                                  ApaI.......


!                                                      Bsp120I....


!


!        Linker 3...................................................


!        153 154 155 156 157 158 159 160 161 162 163 164 165 166 167


!         G   F   Q   V   D   N   Y   G   T   Q   L   N   A   V   N*


 1170    ggt ttc cag gtC gaC aat tat gga acc cag ctc aat get gtg aat


!                    SalI...


!


!        Linker 3...................................................


!        168 169 170 171 172 173 174 175 176 177 178 179 180 181 182


!         N   S   L   T   P   Q   S   T   K   V   P   S   L   F   E


 1215    aac tcc ctg act ccg caa tca acc aag gtt ccc agc ctt ttt gaa


!                                   |


!                          Cleavage site of MMP8


!      Cleavage site of MMP-12 (Belaaouaj, et al., 2000, J Biol Chem, 


       275:27123-8)


!


!     Linker 3     KuDom 3............................................


!      183 184     185 186 187 188 189 190 191 192 193 194 195 196 197


!       F   H       G   P   S   W   C5  L   T   P   A   D   R   G   L


 1260  ttt cac     ggt ccc tca tgg tgt ctc act ccC gcG gac aga gga ttg


!                                              SacII..


!


!        KuDom 3....................................................


!       198 199 200 201 202 203 204 205 206 207 208 209 210 211 212


!       C14  R   A   N   E   N  R20  F   Y   Y   N   S   V   I  G28


 1305   tgt cgt gcc aat gag aac aga ttc tac tac aat tca gtc att ggg


!


!        KuDom 3....................................................


!        213 214 215 216 217 218 219 220 221 222 223 224 225 226 227


!         K  C30  R   P   F   K   Y   S   G  C38  G   G   N   E   N


 1350    aaa tgc cgc cca ttt aag taT TCC gga tgt ggg gga aat gaa aac


!                                    BspEI..


!


!        KuDom 3....................................................


!        228 229 230 231 232 233 234 235 236 237 238 239 340 241 242


!         N*  F   T   S   K   Q   E  C51  L   R   A  C55  K   K  G58


 1395    aat ttt act tcc aaa caa gaa tgt ctg CgC gca tgt aaa aaa ggt


!                                          BssHII..


!


!        Linker 4...................................................


!        243 244 245 246 247 248 249 250 251 252 253 254 255 256 257


!         F   I   Q   R   I   S   K   G   G   L   I   K   T   K   R


 1440    ttc atc caa aga ata tca aaa gga ggc cta att aaa acc aaa aga


!


!        Linker 4...................................................


!        258 259 260 261 262 263 264 265 266 267 268 269 270 271 272


!         K   R   K   K   Q   R   V   K   I   A   Y   E   E   I   F


 1485    aaa aga aag aag cag aga gtg aaa ata gca tat gaa gaa att ttt


!


!        Linker 4.......


!        273 274 275 276


!         V   K   N   M   . (SEQ ID NO: 7).


 1530    gtt aaa aat atg taa tga TCTAGA


!                                XbaI..


 1554       AGCTC GCTGATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC


 


 1609  CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA


 


 1669  AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT CTATTCTGGG GGGTGGGGTG


 


 1729  GGGCAGGACA GCAAGGGGGA GGATTGGGAA GACAATAGCA GGCATGCTGG GGATGGCCCG


 


 1789  GGCTCTATGG CTTCTGAGGC GGAAAGAACC AGCTGGGGCT CTAGGGGGTA TCCCCACGCG


 


 1849  CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT GACCGCTACA


 


 1909  CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT CGCCACGTTC


 


 1969  GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGCATCCCTT TAGGGTTCCG ATTTAGTGCT


 


 2029  TTACGGCACC TCGACCCCAA AAAACTTGAT TAGGGTGATG GTTCACGTAG TGGGCCATCG


 


 2089  CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA CGTTCTTTAA TAGTGGACTC


 


 2149  TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT ATTCTTTTGA TTTATAAGGG


 


 2209  ATTTTGGGGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAA ATTTAACGCG


 


 2269  AATTAATTCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TCCCCAGGCA


 


 2329  GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTGCTCA


 


 2389  GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC


 


 2449  CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC


 


 2509  CTAGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCTGC CTCTGAGCTA


 


 2569  TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA GCTCCCGGGA


 


 2629  GGTCCACAAT GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA


 


 2689  GGCTATTCGG CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC


 


 2749  GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA


 


 2809  ATGAACTCCA GGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG


 


 2869  CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG GGCGAAGTGC


 


 2929  CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG


 


 2989  ATGCAATGCG GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA


 


 3049  AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC


 


 3109  TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGCGTA


 


 3169  TGCCCGACGG CGAGGATCTC GTCGTGACTC ATGGCGATGC CTGCTTGCCG AATATCATGG


 


 3229  TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT


 


 3289  ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG


 


 3349  ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC


 


 3409  GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC


 


 3469  GCCCAACCTG CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT


 


 3529  CGGAATCGTT TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA


 


 3589  GTTCTTCGCC CACCCCAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG


 


 3649  CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA


 


 3709  ACTCATCAAT GTATCTTATC ATGTCTGTAT ACCGGATCTT TCCGCTTCCT CGCTCACTGA


 


 3769  CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT


 


 3829  ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA


 


 3889  AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC


 


 3949  TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA


 


 4009  AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC


 


 4069  GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCAATGCTC


 


 4129  ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTCCACGA


 


 4189  ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC


 


 4249  GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG


 


 4309  GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG


 


 4369  GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG


 


 4429  CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA


 


 4489  GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA


 


 4549  CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT


 


 4609  CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA


 


 4669  GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG


 


 4729  TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA


 


 4789  GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC


 


 4849  AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC


 


 4909  TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC


 


 4969  AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC


 


 5029  GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC


 


 5089  CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT


 


 5149  GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC


 


 5209  ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG


 


 5269  TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG


 


 5329  CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT


 


 5389  CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCTCCCAACT GATCTTCAGC


 


 5449  ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA


 


 5509  AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA


 


 5569  TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA


 


 5629  AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCAGATC


 


 5689  GACGGATCGG GAGATCG (SEQ ID NO: 6)
















TABLE 5





The AA sequence of DX-88, an inhibitor of human plasma 


kallikrein.















(SEQ ID NO: 9)


           1    1    2    2    3    3    4    4    5    5  5 


ab1...5....0....5....0....5....0....5....0....5....0....5..8


EAMHSFCAFKADDGPCRAAHPRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD
















TABLE 6





DNA encoding DX-88















(SEQ ID NO: 8)


GAA GCT ATG CAC TCT TTC TGT GCT TTC AAG GCT GAC


 


GAC GGT CCG TGC AGA GCT GCT CAC CCA AGA TGG TTC


 


TTC AAC ATC TTC ACG CGA CAA TGC GAG GAG TTC ATC


 


TAC GGT GGT TGT GAG GGT AAC CAA AAC AGA TTC GAG


 


TCT CTA GAG GAG TGT AAG AAG ATG TGT ACT AGA GAC
















TABLE 7





Comparison of Various BPTI-related KuDoms_SEQ ID NOs: 188, 3, 9,


129, 189, 4, 190, 5, 191, 188, 192, 128, 4, 193, 5, 194, 195, 4, 196, 197,


5 and 198, respectively.













 











w.t. = wild-type


sel. = selected


des. = designed


DBT = designed, built, and tested













TABLE 8





DX: 88 gene















! DX88 gene


!       a b 1 2 3


!       E A M H S


       gag gct atg cac tct


!     |.....mature DX88 ....


!      derived from human LACI-K1


!


!      4   5   6   7   8   9  10  11  12  13  14  15  16  17  18


!      F   C   A   F   K   A   D   D   G   P   C   R   A   A   H


 1224 ttc tgt get ttc aag gct gac gaC GGT CCG tgc aga gct gct cac


!                                   | RsrII  |


!     19  20  21  22  23  24  25  26  27  28  29  30  31  32  33


!      P   R   W   F   F   N   I   F   T   R   Q   C   E   E   F


 1269 cca aga tgg ttc ttc aac atc ttc acg cga caa tgc gag gag ttc


!


!     34  35  36  37  38  39  40  41  42  43  44  45  46  47  48


!      I   Y   G   G   C   E   G   N   Q   N   R   F   E   S   L


 1314 atc tac ggt ggt tgt gag GGT AAC Caa aac aga ttc gag tct cta


!                            | BstEII |


!


!     49  50  51  52  53  54  55  56  57  58 


!      E   E   C   K   K   M   C   T   R   D  (SEQ ID NO: 200)


 1359 gag gag tgt aag aag atg tgt act aga gat (SEQ ID NO: 199)


!LACI domain---------------------------------


!
















TABLE 9





Gene encoding TF88890 for expression in pFTFPI_V2















!Human mature TFPI(1-174) with changes in KuDom1 and KuDom2


!


!         Signal sequence from pFcFuse3..................


!         M   G   W   S   C   I   I   L   F   L   V   A


   1     aTG Gga tgg agc tgt atc atc ctc ttc ttg gtc gcg


!                                                 NruI...


!


!        Signal sequence............


!         T   A   T   G   A   H   S


  37     acg gcc aca ggg gcc cac tcc


!   NruI.. SfiI.............


!            BglI..........


!                    Eco0109I.


!                     ApaI....(Bsp120I)


!


!       Mature modified TFPI


!         1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!        D   S   E   E   D   E   E   H   T   I   I   T   D   T   E


  58    gat tct gag gaa gat gaa gaa cac aca att atc aca gat acg gag


!


!                                KuDom 1...........................


!        16  17  18  19  20  21  22  23  24  25  26  27  28  29  30


!        L   P   P   L   K   L   M   H   S   F   C   A   F   K   A


 103    ttg cca cca ctg aaa ctt atg cat tca ttt tgt gca ttc aag gcg


!                               NsiI...


!


!        KuDom 1.... Now like DX-88


!        31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!        D   D   G   P   C   R   A   A   H   P   R   W   F   F   N


 148    gat gat ggc cca tgt aGa gca GCc CAT CCT aga tGG ttc ttc aat


!


!        46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!        I   F   T   R   Q   C   E   E   F   I   Y   G   G   C   E


 193    att ttc act cga cag tgc gaa gaa ttt ata tat ggg gga tgt gaa


!


!        61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!        G   N   Q   N   R   F   E   S   L   E   E   C   K   K   M


 238    gga aat cag aAT CGA Ttt gaa agt ctg gaa gag tgc aaa aaa atg


!                    BspDI...


!


!        End KuDom1.... Linker.....................................


!        76  77  78  79  80  81  82  83  84  85  86  87  88  89  90


!        C   T   R   D   N   A   N   R   I   I   K   T   T   L   Q


 283    TGT ACA aga gat aat gca aac agg att ata aag aca aca ttg caa


!       BsrGI..


!


!                KuDom 2...........................................


!        91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!        Q   E   K   P   D   F   C5  F   L   E   E   D   P  G12 P13


 328    caa gaa aag cca gat ttc tgc ttt ttg gaa gaa gat cct gga CCa


!


!      KuDom 2.... Now like DX-890........................


!       106 107 108 109 110 111 112 113 114 115 116 117 118 119 120


!       C14  I   A   F   F   P   R   W   F   Y   N   N   Q   T   K 


 373    tgt ATC gCt tTt Ttt Ccc agg tGG ttt tat aac aat cag aca aaa


!


!       121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!        Q   C   E   L   F   P   Y   G   G   C   Q   G   N   M   N


 418    cag tgt gaa cTt ttc CCg tat ggt gga tgc cAg ggc aat atg aac


!


!        KuDom 2...................................................


!       136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!        N   F   E   T   L   E   E   C   K   N   I   C   E   D   G


 463    aat ttt gag aca ctg gaa gaa tgc aag aac att tgt gaa gat ggt


!


!       Linker.....................................................


!       151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!        P   N   G   F   Q   V   D   N   Y   G   T   Q   L   N   A


 508    ccg aat ggt ttc cag gtg gat aat tat gga acc cag ctc aat get


!


!       Linker.............................


!       166 167 168 169 170 171 172 173 174


!        V   N   N   S   L   T   P   Q   S  (SEQ ID NO: 11)


 553    gtg aat aac tcc ctg act ccg caa tca


!                                          |


!                                      Cleavage site of MMP8


!                                     Cleavage site of MMP-12


!          (Belaaouaj, et al., 2000, J Biol Chem, 275:27123-8)


!


 580                    taa tga tctaga (SEQ ID NO: 10)


!                                 XbaI...


!


! Peptide Mol. Wt. = 20061
















TABLE 10





ONs for Remodeling of KuDom 1 and KuDom 2


of mature TFPI(1-174)


(All ONs are writen 5′ to 3′.)


















 1
ON1U23
GAT TCT GAG GAA GAT
(SEQ ID NO: 12)




GAA GAA CA



 


 2
ON718L21
TGA TTG CGG AGT CAG
(SEQ ID NO: 13)




GGA GTT



 


 3
ONK1Bot
GAA AAT ATT GAA GAA
(SEQ ID NO: 14)




ccA TCT agg atg Ggc





TGC TcT ACA TGG GCC





ATC ATC CGC CTT



 


 4
ONK1T
AAG GCG GAT GAT GGC
(SEQ ID NO: 15)




CCA TGT AgA GCA gcC





cat cct AGA Tgg TTC





TTC AAT ATT TTC



 


 5
ONK2B1
T GTT ATA AAA ccA
(SEQ ID NO: 16)




CCT GGg AAa AaA AgC





gat ACA Tgg TCC AGG





ATC TTC TT



 


 6
ONK2T1
AA GAA GAT CCT GGA
(SEQ ID NO: 17)




ccA TGT atc GcT TtT





tTT cCC AGG Tgg TTT





TAT AAC A



 


 7
ONK2B2
CAT ATT GCC CtG GCA
(SEQ ID NO: 18)




TCC ACC ATA Cgg GAA





AaG TTC ACA CTG TTT



 


 8
ONK2T2
AAA CAG TGT GAA CtT
(SEQ ID NO: 19)




TTC ccG TAT GGT GGA





TGC CaG GGC AAT ATG



 


 9
ON_SfiITop
TTAATCTTGCGGCCACAGG
(SEQ ID NO: 20)




GGCCCACTCT GAT TCT





GAG GAA GAT GAA GAA





CA



 


10
BotAmp
CAAAAAGGCT TCTAGA
(SEQ ID NO: 21)




TCA TTA TGA TTG CGG





AGT CAG GGA GTT






Mutations are shown lowercase, bold.


Restriction sites are UPPERCASE, BOLD.













TABLE 11







TFPI-2 (SEQ ID NO: 22)









Codons
Function
AA sequence





 1-24
signal
MDPARPLGLSILLLFLTEAALGDA


 


25-31
Linker 1
AQEPTGN


 




          1    1    2    2    3    3    4      4    5    5  5




1   5    a0    5    0    5    0    5    0  ab  5    0    5  8


32-89
KuDom 1
NAEICLLPL-DYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNA--NNFYTWEACDDACWRI


 


90-91
Linker 2
EK


 


 92-162
KuDom 2
VPKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFPDEATCMGFCAPK


 


163
Linker 3
K


 


164-221
KuDom 3
IPSFCYSPK-DEGLCSANVTRYYFNPRYRTCDAFTYTGCGGND--NNFVSREDCKRACAKA


 


222-245
Linker 4
LKKKKKMPKLRFASRIRKIRKKQF
















TABLE 12





Library of TFPI2 KuDom2















! M13_iii::TFPI-2 K2 for DY3P82


! 


!                 M13 signal sequence...................|     1   2


!                                             S   M   A   D   V   P


                                            |tCC|ATG|Gct|gat|gtt|cct|


!                                             NcoI....


!


!                                            X1  X1  X3      X3  X4


!         3   4   5   6   7   8   9  9a  10  11  12  13  14  15  16


!         K   V   C   R   L   Q   V   S   V   D   D   Q   C   E   G


        |aag|gtt|tgt|cgt|CTG|CAG|gtt|tct|gtt|gac|gat|caa|tgt|gag|ggt|


!                        PstI...


!


!        X3  X3  X3  X5


!        17  18  19  20  21  22  23  24  25  26  27  28  29  30  31


!         S   T   E   K   Y   F   F   N   L   S   S   M   T   C   E


        |tct|act|gaa|aag|tat|ttt|ttc|aac|CtG|AGC|TCt|atg|act|tgc|gaa|


!                                          SacI....


!


!                 X3  X7              X3  X8      X9 X10


!        32  33  34  35  36  37  38  39  40  41  42  42a 42b 43  44


!         K   F   F   S   G   G   C   H   R   N   R   I   E   N   R


        |aag|ttc|ttt|tct|ggt|ggt|tgc|cat|cgt|aac|cgt|atc|gag|aAC|CGG|


!                                                             Agel...


!


!        45  46  47  48  49  50  51  52  53  54  55  56  57  58


!         F   P   D   E   A   T   C   M   G   F   C   A   P   K   S


        |TtT|CCG|GAc|gag|gct|act|tgt|atg|ggt|ttc|tgt|gct|cct|aag|tct|


! AgeI.... BspEI...


!


!         A   D   A   S  (SEQ ID NO: 24)


        |gct|gac|GCT|AGC|  (SEQ ID NO: 23);


!                NheI...


modified version: nucleic acid sequence (SEQ ID NO: 143) and amino acid 


sequence (SEQ ID NO: 144).


!


!








! Mixed ON version
Codon based


! X1 = RRK [DENKSRGG]
D,E,N,K,S,R,G,V,L,F


! X3 = NNK [FLLLIMVVSSPPITAAY.HQNKDECWRRSRGG]
A,D,E,F,G,H,I,K,L,M,N,P,


(no C)
Q,R,S,T,V,W,Y


! X4 = GRt [AG]
A,G


! X5 = ARG [KR]
K,R


! X7 = TMT [SY]
S,Y


! X8 = RRK [DENKSRGG]
D,E,N,K,S,R,Q,G,V,L,F


! X9 = RRK [DENKSRGG]
D,E,N,K,S,R,Q,G,V,L,F


! X10 = RNK [IMVVTTAANKDESRGG]
I,M,V,L,A,T,S,Y,G,D,R,F
















TABLE 13





DNA of a Display Phage displaying TFPI2 KuDom2 (SEQ ID NO: 25)















LOCUS     DY3P82_22   8931             CIRCULAR


ORIGIN


 


   1  AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT


 


  61  ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT


 


 121  CGTTCGCAGA ATTGGGAATC AACTGTTATA TGGAATGAAA CTTCCAGACA CCGTACTTTA


 


 181  GTTGCATATT TAAAACATGT TGAGCTACAG CATTATATTC AGCAATTAAG CTCTAAGCCA


 


 241  TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG


 


 301  TTGGAGTTTG CTTCCGGTCT GGTTCGCTTT GAAGCTCGAA TTAAAACGCG ATATTTGAAG


 


 361  TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT


 


 421  CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA


 


 481  TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT


 


 541  AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT


 


 601  GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT


 


 661  AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG


 


 721  ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT


 


 781  TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA


 


 841  CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT


 


 901  CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG


 


 961  AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC


 


1021  TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC


 


1081  GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT


 


1141  CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT


 


1201  CAAAGATGAG TGTTTTAGTG TATTCTTTTG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA


 


1261  GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT


 


1321  CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA


 


1381  CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA


 


1441  TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA


 


1501  ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT


 


1561  TTTTTGGAGA TTTTCAACGT GAAAAAATTA TTATTCGCAA TTCCTTTAGT TGTTCCTTTC


 


1621  TATTCTCACT CCGCTGAAAC TGTTGAAAGT TGTTTAGCAA AATCCCATAC AGAAAATTCA


 


1681  TTTACTAACG TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGCTGT


 


1741  CTGTGGAATG CTACAGGCGT TGTAGTTTGT ACTGGTGACG AAACTCAGTG TTACGGTACA


 


1801  TGGGTTCCTA TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA GGGTGGCGGT


 


1861  TCTGAGGGTG GCGGTTCTGA GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT


 


1921  ATTCCGGGCT ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA


 


1981  AACCCCGCTA ATCCTAATCC TTCTCTTGAG GAGTCTCAGC CTCTTAATAC TTTCATGTTT


 


2041  CAGAATAATA GGTTCCGAAA TAGGCAGGGG GCATTAACTG TTTATACGGG CACTGTTACT


 


2101  CAAGGCACTG ACCCCGTTAA AACTTATTAC CAGTACACTC CTGTATCATC AAAAGCCATG


 


2161  TATGACGCTT ACTGGAACGG TAAATTCAGA GACTGCGCTT TCCATTCTGG CTTTAATGAG


 


2221  GATTTATTTG TTTGTGAATA TCAAGGCCAA TCGTCTGACC TGCCTCAACC TCCTGTCAAT


 


2281  GCTGGCGGCG GCTCTGGTGG TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG CTCTGAGGGT


 


2341  GGCGGTTCTG AGGGTGGCGG CTCTGAGGGA GGCGGTTCCG GTGGTGGCTC TGGTTCCGGT


 


2401  GATTTTGATT ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT


 


2461  GAAAACGCGC TACAGTCTGA CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT


 


2521  GCTGCTATCG ATGGTTTCAT TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT


 


2581  GGTGATTTTG CTGGCTCTAA TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT


 


2641  TTAATGAATA ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT


 


2701  TTTGTCTTTG GCGCTGGTAA ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA


 


2761  TTCCGTGGTG TCTTTGCGTT TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCTACG


 


2821  TTTGCTAACA TACTGCGTAA TAAGGAGTCT TAATCATGCC AGTTCTTTTG GGTATTCCGT


 


2881  TATTATTGCG TTTCCTCGGT TTCCTTCTGG TAACTTTGTT CGGCTATCTG CTTACTTTTC


 


2941  TTAAAAAGGG CTTCGGTAAG ATAGCTATTG CTATTTCATT GTTTCTTGCT CTTATTATTG


 


3001  GGCTTAACTC AATTCTTGTG GGTTATCTCT CTGATATTAG CGCTCAATTA CCCTCTGACT


 


3061  TTGTTCAGGG TGTTCAGTTA ATTCTCCCGT CTAATGCGCT TCCCTGTTTT TATGTTATTC


 


3121  TCTCTGTAAA GGCTGCTATT TTCATTTTTG ACGTTAAACA AAAAATCGTT TCTTATTTGG


 


3181  ATTGGGATAA ATAATATGGC TGTTTATTTT GTAACTGGCA AATTAGGCTC TGGAAAGACG


 


3241  CTCGTTAGCG TTGGTAAGAT TCAGGATAAA ATTGTAGCTG GGTGCAAAAT AGCAACTAAT


 


3301  CTTGATTTAA GGCTTCAAAA CCTCCCGCAA GTCGGGAGGT TCGCTAAAAC GCCTCGCGTT


 


3361  CTTAGAATAC CGGATAAGCC TTCTATATCT GATTTGCTTG CTATTGGGCG CGGTAATGAT


 


3421  TCCTACGATG AAAATAAAAA CGGCTTGCTT GTTCTCGATG AGTGCGGTAC TTGGTTTAAT


 


3481  ACCCGTTCTT GGAATGATAA GGAAAGACAG CCGATTATTG ATTGGTTTCT ACATGCTCGT


 


3541  AAATTAGGAT GGGATATTAT TTTTCTTGTT CAGGACTTAT CTATTGTTGA TAAACAGGCG


 


3601  CGTTCTGCAT TAGCTGAACA TGTTGTTTAT TGTCGTCGTC TGGACAGAAT TACTTTACCT


 


3661  TTTGTCGGTA CTTTATATTC TCTTATTACT GGCTCGAAAA TGCCTCTGCC TAAATTACAT


 


3721  GTTGGCGTTG TTAAATATGG CGATTCTCAA TTAAGCCCTA CTGTTGAGCG TTGGCTTTAT


 


3781  ACTGGTAAGA ATTTGTATAA CGCATATGAT ACTAAACAGG CTTTTTCTAG TAATTATGAT


 


3841  TCCGGTGTTT ATTCTTATTT AACGCCTTAT TTATCACACG GTCGGTATTT CAAACCATTA


 


3901  AATTTAGGTC AGAAGATGAA ATTAACTAAA ATATATTTGA AAAAGTTTTC TCGCGTTCTT


 


3961  TGTCTTGCGA TTGGATTTGC ATCAGCATTT ACATATAGTT ATATAACCCA ACCTAAGCCG


 


4021  GAGGTTAAAA AGGTAGTCTC TCAGACCTAT GATTTTGATA AATTCACTAT TGACTCTTCT


 


4081  CAGCGTCTTA ATCTAAGCTA TCGCTATGTT TTCAAGGATT CTAAGGGAAA ATTAATTAAT


 


4141  AGCGACGATT TACAGAAGCA AGGTTATTCA CTCACATATA TTGATTTATG TACTGTTTCC


 


4201  ATTAAAAAAG GTAATTCAAA TGAAATTGTT AAATGTAATT AATTTTGTTT TCTTGATGTT


 


4261  TGTTTCATCA TCTTCTTTTG CTCAGGTAAT TGAAATGAAT AATTCGCCTC TGCGCGATTT


 


4321  TGTAACTTGG TATTCAAAGC AATCAGGCGA ATCCGTTATT GTTTCTCCCG ATGTAAAAGG


 


4381  TACTGTTACT GTATATTCAT CTGACGTTAA ACCTGAAAAT CTACGCAATT TCTTTATTTC


 


4441  TGTTTTACGT GCAAATAATT TTGATATGGT AGGTTCTAAC CCTTCCATAA TTCAGAAGTA


 


4501  TAATCCAAAC AATCAGGATT ATATTGATGA ATTGCCATCA TCTGATAATC AGGAATATGA


 


4561  TGATAATTCC GCTCCTTCTG GTGGTTTCTT TGTTCCGCAA AATGATAATG TTACTCAAAC


 


4621  TTTTAAAATT AATAACGTTC GGGCAAAGGA TTTAATACGA GTTGTCGAAT TGTTTGTAAA


 


4681  GTCTAATACT TCTAAATCCT CAAATGTATT ATCTATTGAC GGCTCTAATC TATTAGTTGT


 


4741  TAGTGCTCCT AAAGATATTT TAGATAACCT TCCTCAATTC CTTTCAACTG TTGATTTGCC


 


4801  AACTGACCAG ATATTGATTG AGGGTTTGAT ATTTGAGGTT CAGCAAGGTG ATGCTTTAGA


 


4861  TTTTTCATTT GCTGCTGGCT CTCAGCGTGG CACTGTTGCA GGCGGTGTTA ATACTGACCG


 


4921  CCTCACCTCT GTTTTATCTT CTGCTGGTGG TTCGTTCGGT ATTTTTAATG GCGATGTTTT


 


4981  AGGGCTATCA GTTCGCGCAT TAAAGACTAA TAGCCATTCA AAAATATTGT CTGTGCCACG


 


5041  TATTCTTACG CTTTCAGGTC AGAAGGGTTC TATCTCTGTT GGCCAGAATG TCCCTTTTAT


 


5101  TACTGGTCGT GTGACTGGTG AATCTGCCAA TGTAAATAAT CCATTTCAGA CGATTGAGCG


 


5161  TCAAAATGTA GGTATTTCCA TGAGCGTTTT TCCTGTTGCA ATGGCTGGCG GTAATATTGT


 


5221  TCTGGATATT ACCAGCAAGG CCGATAGTTT GAGTTCTTCT ACTCAGGCAA GTGATGTTAT


 


5281  TACTAATCAA AGAAGTATTG CTACAACGGT TAATTTGCGT GATGGACAGA CTCTTTTACT


 


5341  CGGTGGCCTC ACTGATTATA AAAACACTTC TCAGGATTCT GGCGTACCGT TCCTGTCTAA


 


5401  AATCCCTTTA ATCGGCCTCC TGTTTAGCTC CCGCTCTGAT TCTAACGAGG AAAGCACGTT


 


5461  ATACGTGCTC GTCAAAGCAA CCATAGTACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG


 


5521  GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT


 


5581  TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC


 


5641  GGGGGCTCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTG


 


5701  ATTTGGGTGA TGGTTGGCCA TCGCCCTGAT AGACGGTTTT TCGCCCTTTG ACGTTGGAGT


 


5761  CCACGTTCTT TAATAGTGGA CTCTTGTTCC AAACTGGAAC AACACTCAAC CCTATCTCGG


 


5821  GCTATTCTTT TGATTTATAA GGGATTTTGC CGATTTCGGA ACCACCATCA AACAGGATTT


 


5881  TCGCCTGCTG GGGCAAACCA GCGTGGACCG CTTGCTGCAA CTCTCTCAGG GCCAGGCGGT


 


5941  GAAGGGCAAT CAGCTGTTGC CCGTCTCACT GGTGAAAAGA AAAACCACCC TGGATCCAAG


 


6001  CTTGCAGGTG GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT ATTTTTCTAA


 


6061  ATACATTCAA ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT TCAATAATAT


 


6121  TGAAAAAGGA AGAGTATGAG TATTCAACAT TTCCGTGTCG CCCTTATTCC CTTTTTTGCG


 


6181  GCATTTTGCC TTCCTGTTTT TGCTCACCCA GAAACGCTGG TGAAAGTAAA AGATGCTGAA


 


6241  GATCAGTTGG GCGCACTAGT GGGTTACATC GAACTGGATC TCAACAGCGG TAAGATCCTT


 


6301  GAGAGTTTTC GCCCCGAAGA ACGTTTTCCA ATGATGAGCA CTTTTAAAGT TCTGCTATGT


 


6361  GGCGCGGTAT TATCCCGTAT TGACGCCGGG CAAGAGCAAC TCGGTCGCCG CATACACTAT


 


6421  TCTCAGAATG ACTTGGTTGA GTACTCACCA GTCACAGAAA AGCATCTTAC GGATGGCATG


 


6481  ACAGTAAGAG AATTATGCAG TGCTGCCATA ACCATGAGTG ATAACACTGC GGCCAACTTA


 


6541  CTTCTGACAA CGATCGGAGG ACCGAAGGAG CTAACCGCTT TTTTGCACAA CATGGGGGAT


 


6601  CATGTAACTC GCCTTGATCG TTGGGAACCG GAGCTGAATG AAGCCATACC AAACGACGAG


 


6661  CGTGACACCA CGATGCCTGT AGCAATGGCA ACAACGTTGC GCAAACTATT AACTGGCGAA


 


6721  CTACTTACTC TAGCTTCCCG GCAACAATTA ATAGACTGGA TGGAGGCGGA TAAAGTTGCA


 


6781  GGACCACTTC TGCGCTCGGC CCTTCCGGCT GGCTGGTTTA TTGCTGATAA ATCTGGAGCC


 


6841  GGTGAGCGTG GGTCTCGCGG TATCATTGCA GCACTGGGGC CAGATGGTAA GCCCTCCCGT


 


6901  ATCGTAGTTA TCTACACGAC GGGGAGTCAG GCAACTATGG ATGAACGAAA TAGACAGATC


 


6961  GCTGAGATAG GTGCCTCACT GATTAAGCAT TGGTAACTGT CAGACCAAGT TTACTCATAT


 


7021  ATACTTTAGA TTGATTTAAA ACTTCATTTT TAATTTAAAA GGATCTAGGT GAAGATCCTT


 


7081  TTTGATAATC TCATGACCAA AATCCCTTAA CGTGAGTTTT CGTTCCACTG TACGTAAGAC


 


7141  CCCCAAGCTT GTCGACTGAA TGGCGAATGG CGCTTTGCCT GGTTTCCGGC ACCAGAAGCG


 


7201  GTGCCGGAAA GCTGGCTGGA GTGCGATCTT CCTGACGCTC GAGCGCAACG CAATTAATGT


 


7261  GAGTTAGCTC ACTCATTAGG CACCCCAGGC TTTACACTTT ATGCTTCCGG CTCGTATGTT


 


7321  GTGTGGAATT GTGAGCGGAT AACAATTTCA CACAGGAAAC AGCTATGACC ATGATTACGC


 


7381  CAAGCTTTGG AGCCTTTTTT TTGGAGATTT TCAACGTGAA AAAATTATTA TTCGCAATTC


 


7441  CTTTAGTTGT TCCTTTCTAT TCCATGGCTG ATGTTCCTAA GGTTTGTCGT CTGCAGGTTT


 


7501  CTGTTGACGA TCAATGTGAG GGTTCTACTG AAAAGTATTT TTTCAACCTG AGCTCTATGA


 


7561  CTTGCGAAAA GTTCTTTTCT GGTGGTTGCC ATCGTAACCG TATCGAGAAC CGGTTTCCGG


 


7621  ACGAGGCTAC TTGTATGGGT TTCTGTGCTC CTAAGCCTGC TGACGCTAGC TCTGCTAGTG


 


7681  GCGACTTCGA CTACGAGAAA ATGGCTAATG CCAACAAAGG CGCCATGACT GAGAACGCTG


 


7741  ACGAGAATGC TTTGCAAAGC GATGCCAAGG GTAAGTTAGA CAGCGTCGCG ACCGACTATG


 


7801  GCGCCGCCAT CGACGGCTTT ATCGGCGATG TCAGTGGTTT GGCCAACGGC AACGGAGCCA


 


7861  CCGGAGACTT CGCAGGTTCG AATTCTCAGA TGGCCCAGGT TGGAGATGGG GACAACAGTC


 


7921  CGCTTATGAA CAACTTTAGA CAGTACCTTC CGTCTCTTCC GCAGAGTGTC GAGTGCCGTC


 


7981  CATTCGTTTT CGGTGCCGGC AAGCCTTACG AGTTCAGCAT CGACTGCGAT AAGATCAATC


 


8041  TTTTCCGCGG CGTTTTCGCT TTCTTGCTAT ACGTCGCTAC TTTCATGTAC GTTTTCAGCA


 


8101  CTTTCGCCAA TATTTTACGC AACAAAGAAA GCTAGTGATC TCCTAGGAAG CCCGCCTAAT


 


8161  GAGCGGGCTT TTTTTTTCTG GTATGCATCC TGAGGCCGAT ACTGTCGTCG TCCCCTCAAA


 


8221  CTGGCAGATG CACGGTTACG ATGCGCCCAT CTACACCAAC GTGACCTATC CCATTACGGT


 


8281  CAATCCGCCG TTTGTTCCCA CGGAGAATCC GACGGGTTGT TACTCGCTCA CATTTAATGT


 


8341  TGATGAAAGC TGGCTACAGG AAGGCCAGAC GCGAATTATT TTTGATGGCG TTCCTATTGG


 


8401  TTAAAAAATG AGCTGATTTA ACAAAAATTT AATGCGAATT TTAACAAAAT ATTAACGTTT


 


8461  ACAATTTAAA TATTTGCTTA TACAATCTTC CTGTTTTTGG GGCTTTTCTG ATTATCAACC


 


8521  GGGGTACATA TGATTGACAT GCTAGTTTTA CGATTACCGT TCATCGATTC TCTTGTTTGC


 


8581  TCCAGACTCT CAGGCAATGA CCTGATAGCC TTTGTAGATC TCTCAAAAAT AGCTACCCTC


 


8641  TCCGGCATTA ATTTATCAGC TAGAACGGTT GAATATCATA TTGATGGTGA TTTGACTGTC


 


8701  TCCGGCCTTT CTCACCCTTT TGAATCTTTA CCTACACATT ACTCAGGCAT TGCATTTAAA


 


8761  ATATATGAGG GTTCTAAAAA TTTTTATCCT TGCGTTGAAA TAAAGGCTTC TCCCGCAAAA


 


8821  GTATTACAGG GTCATAATGT TTTTGGTACA ACCGATTTAG CTTTATGCTC TGAGGCTTTA


 


8881  TTGCTTAATT TTGCTAATTC TTTGCCTTGC CTGTATGATT TATTGGATGT T
















TABLE 14





ONs to make library of TFPI2 KuDom2
















#1  ON1U21T2
(SEQ ID NO: 26)







5′ GGG TCT TCT GTA CGA GTT CTG 3′ 


 








#2  ON147L21
(SEQ ID NO: 27)







5′ CCT CCA GAG TTG ACA GAA TCC 3′ 


 








#3  ON1U96
(SEQ ID NO: 28)







5′ GGG TCT TCT GTA CGA GTT CTG CAG GTT TCT GTT RRK


   RRK NNK TGT NNK GST NNK NNK NNK ARG TAT TTT TTC


   AAC CTG AGC TCT ATG ACT TGC GAA 3′


 








#4  ON71L97
(SEQ ID NO: 29)







5′ CCTCCAGAGTTGACAGAATC CGG AAA CCG GTT CTC MNY


   MYY GTT MYY MNN GCA ACC ACC AKA MNN GAA CTT TTC


   GCA AGT CAT AGA GCT CAG GTT GA 3′





84 × 207 × 23 × 16 = 6.7 E 14 AA sequences


84 × 327 × 23 × 16 = 1.8 E 16 DNA sequences


Fraction without stops = 0.80; only TAG allowed.













TABLE 15







ONs for construction of modified tfpi gene shown 


in TABLE 4.















SEQ







ID




No.
Name
Sequence (5′-to-3′)
NO:
U/L
Len















 1
ONMature
gat tct gag gaa gat gaa gaa ca
30
U
829



T






 


 2
0N1019L2
aca tat ttt taa caa aaa ttt ctt
31
L




7
cat





 


 3
ONSfiIT
ttaatcttgcg gcc aca ggg gcc cac
32
U
 95




tcc gac tct gag gaa gat gaa gaa





 


 4
ONSacHin
cat aag Ctt cag tgg tgg GaG ctc
33
L




L
cgt atc





 


 5
ONSacHin
gat acg gag CtC cca cca ctg aaG
34
U
120



U
ctt atg





 


 6
ONMluIL
ttc gca ctg Acg Cgt gaa aa
35
L



 


 7
ONMluIU
tt ttc acG cgT cag tgc gaa
36
U
 76


 


 8
ONBBBL
tc ttc cag aGA ttc aaa tcg att 
37
L





ctg Gtt Acc ttc aca tcc





 


 9
ONBBBU
gga tgt gaa ggT aaC cag aat 
38
U
142




cga ttt gaa TCt ctg gaa ga





 


10
ONXhoBam
t tcc agg atc ctc ctc gag aaa 
39
L




L
gca gaa a





 


11
ONXhoBam
t ttc tgc ttt CtC gaG gaG gat 
40
U
142



U
cct gga a





 


12
ONBsmBIL
ttc ttc cag Cgt ctc aaa a
41
L



 


13
ONBsmBIU
t ttt gag acG ctg gaa gaa
42
U
 74


 


14
ONApaSal
c ata att Gtc Gac ctg gaa acc 
43
L




L
att Ggg Ccc atc ttc a





 


15
ONApaSal
t gaa gat ggG ccC aat ggt ttc 
44
U
148



U
cag gtC gaC aat tat g





 


16
ONSac2L
tcc tct gtc cgc ggg agt gag a
45
L



 


17
ONSac2U
t ctc act ccC gcG gac aga gga
46
U
102


 


18
ONBspE2L
cc aca tcc GGA Ata ctt aaa tg
47
L



 


19
ONBspE2U
ca ttt aag taT TCC gga tgt ggg g
48
U
 74


 


20
ONBssH2L
ttt aca tgc GcG cag aca ttc t
49
L



 


21
ONBssH2U
a gaa tgt ctg CgC gca tgt aaa
50
U
147


 


22
ONfinalL
ttgTtTCTaGaTCAtTacatatttttaacaa
51
L





aaatttcttcat





 


 3
ONSfiI
ttaatcttgcg gcc aca ggg gcc cac
52
U
874




tcc gac tct gag gaa gat gaa gaa





 


26
ONfinalL
ttgTtTCTaGaTCAtTacatatttttaacaa
53
L





aaatttcttcat





The primers are upper strand (U) or lower strand (L) as indicated in the U/L column.


“Len” indicates the length of the product obtained with human tfpi cDNA as template and the U-L pair of primers.


Uppercase BASES indicate departures from the published cDNA sequence.


Restriction sites are shown bold. Where two restriction sites overlap, one is shown as underscored.













TABLE 16







Modified Human TFPI = PlaKalEl01 (SEQ ID NO: 54)









AAs
Function
AA sequence





 1-21
Linker 1
DSEEDEEHTIITDTELPPLKL


 


22-79
KuDom 1
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD



Inhibit




plasmin



 


80-92
Linker 2
NANRIIKTTLQQE


 


 93-150
KuDom 2
KPDFCFLEEDPGPCRAAHPRWFYNnQTKQCERFIYGGCEGNMNNFETLEECKNICEDG



Inhibit




h.pKal



 


151-184
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


185-242
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFPYGGCQGNENnFTSKQECLRACKKG



Inhibit 
1   5    1    1    2    2    3    3    4    4    5    5  5



h.NE
         0    5    0    5    0    5    0    5    0    5  8


 


243-248
Linker 4
FIQRIS





Possible sites of glycosylation are shown as “n”.













TABLE 17





ONs for construction of gene encoding PlaKalEl01















(SEQ ID NO: 55)


 #1  Get_TFPI_top


5′ ATG ATT TAC ACA ATG AAG AAA GTA CAT 3′ 


 


(SEQ ID NO: 56)


 #2  ON937L24


5′ TGA TAT TCT TTG GAT GAA ACC TTT 3′ 


 


(SEQ ID NO: 57)


 #3  MutK1:310U43


5′ GAT GGC CCA TGT CGT GCA CGC TTC GAT AGA TGG TTC


 TTC AAT A 3′ 


 


(SEQ ID NO: 58)


 #4  MutK1:310L43


5′ T ATT GAA GAA CCA TCT ATC GAA GCG TGC ACG ACA


TGG GCC ATC 3′ 


 


(SEQ ID NO: 59)


 #5  MutK2:517U56


5′ GAA GAT CCT GGA CCT TGT CGA GCT GCT CAT CCC AGG


TGG TTT TAT AAC AAT CAG AC 3′


 


(SEQ ID NO:60)


 #6  MutK2:517L56


5′ GT CTG ATT GTT ATA AAA CCA CCT GGG ATG AGC AGC


TCG ACA AGG TCC AGG ATC TTC 3′


 


(SEQ ID NO: 61)


 #7  MutK2:577U47


5′ CAG TGT GAA CGT TTC ATT TAT GGT GGA TGC GAG GGC


AAT ATG AAC AA 3′


 


(SEQ ID NO: 62)


 #8  MutK2:577L47


5′ TT GTT CAT ATT GCC CTC GCA TCC ACC ATA AAT GAA


ACG TTC ACA CTG 3′ 


 


(SEQ ID NO: 63)


 #9  MutK3:792U55


5′ A GCA GAC AGA GGA CCT TGT ATT GCC TTT TTT CCC


AGA TTC TAC TAC AAT TCA GTC 3′


 


(SEQ ID NO: 64)


#10  MutK3:792L55


5′ GAC TGA ATT GTA GTA GAA TCT GGG AAA AAA GGC AAT


ACA AGG TCC TCT GTC TGC T 3′


 


(SEQ ID NO: 65)


#11  MutK3:867U31


5′ T AAG TAC GGT GGA TGT CAG GGA AAT GAA AAC 3′ 


 


(SEQ ID NO: 66)


#12  MutK3:867L31


5′ GTT TTC ATT TCC CTG ACA TCC ACC GTA CTT A 3′ 


 


(SEQ ID NO: 67)


#13  ONNcoI


5′ agattttacttC C atg Gtt tac aca atg aag aaa gt 


3′ 


 


(SEQ ID NO: 68)


#14  ONLowCapPKE


5′ ctcttgtttctagatcatta TGA TAT TCT TTG GAT GAA 


ACC TTT 3′
















TABLE 18







PlaKalEl01::(GGS)4GG::has (SEQ ID NO: 69)









AAs
Function
AA sequence





  1-28
signal
MIYTMKKVHALWASVCLLLNLAPAPLNA


 


 29-49
Linker 1
DSEEDEEHTIITDTELPPLKL


 


 50-107
KuDom 1
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD


 


108-120
Linker 2
NANRIIKTTLQQE


 


121-178
KuDom 2
KPDFCFLEEDPGPCRAAHPRWFYNnQTKQCERFIYGGCEGNMNNFETLEECKNICEDG


 


179-212
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


213-270
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFPYGGCQGNENnFTSKQECLRACKKG




1   5    1    1    2    2    3    3    4    4    5    5  5




         0    5    0    5    0    5    0    5    0    5  8


 


271-276
Linker 4
FIQRIS


 


277-290
Linker 5
GGSGGSGGSGGSGG


 


291-865
hSA
DAHKSE VAHRFKDLGE ENFKALVLIA




FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT




VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTA




FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA




CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA




EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK




ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF




LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE




FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV




SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKC




CTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ




TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV




AASQAALGL





Possible sites of glycosylation are shown as “n”.













TABLE 19







Modified Human TFPI dimer = PKEPKE02 (SEQ ID NO: 70)









AAs
Function
AA sequence





 1-21
Linker 1
DSEEDEEHTIITDTELPPLKL


 


22-79
KuDom 1
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD


 


80-92
Linker 2
NANRIIKTTLQQE


 


 93-150
KuDom 2
KPDFCFLEEDPGPCRAAHPRWFYNnQTKQCERFIYGGCEGNMNNFETLEECKNICEDG


 


151-184
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


185-242
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFKYGGCQGNENnFTSKQECLRACKKG




1   5    1    1    2    2    3    3    4    4    5    5  5




         0    5    0    5    0    5    0    5    0    5  8


 


243-248
Linker 4
FIQRIS


 


249-261
Linker 5
GGSGSSGGGSSGG


 


262-282
Linker 1
DSEEDEEHTIITDTELPPLKL


 


283-340
KuDom 1
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD


 


341-353
Linker 2
NANRIIKTTLQQE


 


354-411
KuDom 2
KPDFCFLEEDPGPCRAAHPRWFYNnQTKQCERFIYGGCEGNMNNFETLEECKNICEDG


 


412-445
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


446-503
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFKYGGCQGNENnFTSKQECLRACKKG


 


504-509

FIQRIS





Possible sites of glycosylation are shown as “n”.













TABLE 20







TF88890-2 (SEQ ID NO: 71)









 AA




 No.
Function
AA sequence





 1-58
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQCE


 
inhibition
EFIYGGCEGNQNRFESLEECKKMCTRD


 


59-70
Linker
GGSGGSSSSSGG


 


 71-128
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE


 
inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD
















TABLE 21







TF88890-3 (SEQ ID NO: 72)









AA




No.
Function
AA sequence





 1-58
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQCE



inhibition
EFIYGGCEGNQNRFESLEECKKMCTRD


 


59-70
Linker
GGSGGSSSSSGG


 


 71-128
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


129-140
Linker
GGSGGSSSSSGG


 


141-198
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQCE



inhibition
EFIYGGCEGNQNRFESLEECKKMCTRD
















TABLE 22







TF88890-4 (SEQ ID NO: 73)









AA




No.
Function
AA sequence





 1-58
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQCE



inhibition
EFIYGGCEGNQNRFESLEECKKMCTRD


 


59-70
Linker
GGSGGSSSSSGG


 


 71-128
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


129-140
Linker
GGSGGSSSSSGG


 


141-198
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQCE



inhibition
EFIYGGCEGNQNRFESLEECKKMCTRD


 


199-210
Linker
GGSGGSSSSSGG


 


211-268
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD
















TABLE 23







TF88890-5 (SEQ ID NO: 74)









AA




No.
Function
AA sequence





  1-58
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQC



inhibition
EEFIYGGCEGNQNRFESLEECKKMCTRD


 


 59-75
Linker
GGSGNGSSSSGGGSGSG


 


 76-133 
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE


 
inhibition
EFIYGGCQNQNRFESLEECKKMCTRD


 


134-150
Linker
GGSGNGSSSSGGGSGSG


 


151-208
pKal
MHSFCAFKADDGPCRAAHPRWFFNIFTRQCE


 
inhibition
EFIYGGCEGNQNRFESLEECKKMCTRD


 


209-225
Linker
GGSGNGSSSSGGGSGSG


 


226-283
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE


 
inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD
















TABLE 24







TF890-6 (SEQ ID NO: 75)









AA




No.
Function
AA sequence





  1-58
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


 59-70
Linker
GGSGGSSSSSGG


 


 71-128
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


129-140
Linker
GGSGGSSSSSGG


 


141-198
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


199-210
Linker
GGSGGSSSSSGG


 


211-268
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


269-280
Linker
GGSGGSSSSSGG


 


281-338
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


339-350
Linker
GGSGSSSGSSSG


 


351-408
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD
















TABLE 25







TF890-8 (SEQ ID NO: 76) 









AA




No.
Function
AA sequence





  1-58
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


 59-70
Linker
GGSGGSSGSSGG


 


 71-128
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


129-140
Linker
GGSGGSSSSSGG


 


141-198
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


199-210
Linker
GGSGGSGSSSGG


 


211-268
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


269-280
Linker
GGSGGSSSSGSG


 


281-338
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


339-350
Linker
GGSGSSSGSSSG


 


351-408
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


409-420
Linker
GSGSSGSSGSSG


 


421-478
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD


 


479-490
Linker
GGSSSGSGSGSG


 


491-548
h.NE
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCE



inhibition
EFIYGGCQGNQNRFESLEECKKMCTRD
















TABLE 26





Library for removal of 14-38 disulfide















! LACI-D1 display cassette for removal of 14-38 disulfide


!      Signal sequence of M13 iii---------------------------------


!       fM  K   K   L    L   F   A   I   P   L   V   V   P   F   Y


       gtg aaa aaa tta  tta ttc gca att cct tta gtt gtt cct ttc tat


!


!             S   M   A               A   E   M   H   S   F


             tCC ATG Gcg            |gcc|gag|ATG|CAT|tcc|ttc


!             NcoI . . .                     NsiI . . .


!


!     C5  A   F   K   A  D10  D  X12 X13 X14 I15  A   F   F   P  R20  F


     tgc gcc ttc aag gct gac gaC GGT CCG tgt aTT gct TTC TTc CCt cgt ttc


!


!     F   F   N  I25  F   T   R   Q  C30  E   E   F   I   Y   G   G  X38


     ttc ttc aac att ttc ACG CGT cag tgc gag GAA TTC att tac ggt ggt tgt


!                        MluI . . .          EcoRI . . .


!


!    X39 G40  N   Q   N   R  F45  E   S   L  E49


     gaa GGT AAC Cag aAC CGG Ttc gaa tcT CTA Gag


!        BstEII . . . AgeI . . .       XbaI . . .


                             AsuII . . .


!


!      E   C   K   K   M   C   T   R   D       S   A   S   S   A


(SEQ ID NO: 78)


      gaa tgt aag aag atg tgt act|cgt|gat|    tct GCT AGC tct gct


(SEQ ID NO: 77)


!                                                 NheI . . .


! an anchor protein such as IIIstump follows.


!


! N = equimolar A,C,G, and T


! H = equimolar A, C, and T


! K = equimolar T and G


! B = equimolar C, G, and T


! V = equimolar A, C, and G


! M = equimolar A and C


!  NHK encodes FLSY.PHQIMTNKVADE


!  BGG encodes WRG
















TABLE 27





Oligonucleotides for construction  


of KuDom Library missing 14-38 disulfide















! remove C14-C38 from TF890 20050727


All sequences are 5'to 3'.








1) ONTPrmr, 21 bases
(SEQ ID NO: 79)







ggcTTcAcTTAccgTgTTccA


 








2) ONLoPrm, 20 bases
(SEQ ID NO: 80)







ccAAgTAAccAcAAgTTTcT


 








3) ONTAAAxC,113 bases
(SEQ ID NO: 81)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcNHKNHKNHKATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








4) ONTAABxC,113 bases 
(SEQ ID NO: 82)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcNHKNHKBggATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








5) ONTABAxC,113 bases 
(SEQ ID NO: 83)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcNHKBggNHKATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








6) ONTABBxC,113 bases 
(SEQ ID NO: 84)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcNHKBggBggATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








7) ONTBAAxC,113 bases 
(SEQ ID NO: 85)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcBggNHKNHKATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








8) ONTBABxC,113 bases 
(SEQ ID NO: 86)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcBggNHKBggATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








9) ONTBBAxC,113 bases 
(SEQ ID NO: 87)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcBggBggNHKATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








10) ONTBBBxC,113 bases
(SEQ ID NO: 88)







ggcTTcAcTTAccgTgTTccATggcggccgAgATgcATTccTTcTgcgcc


TTcAAggcTgAcgAcBggBggBggATTgcTTTcTTcccTcgTTTcTTcTT


cAAcATTTTcAcg


 








11) ONLoAAxC,106 bases 
(SEQ ID NO: 89)







ccAAgTAAccAcAAgTTTcTAgAgATTcgAAccggTTcTggTTAccMDNM


DNAccAccgTAAATgAATTccTcgcAcTgAcgcgTgAAAATgTTgAAgAA


gAAAcg


 








12) ONLoABxC,106 bases 
(SEQ ID NO: 90)







ccAAgTAAccAcAAgTTTcTAgAgATTcgAAccggTTcTggTTAccccVM


DNAccAccgTAAATgAATTccTcgcAcTgAcgcgTgAAAATgTTgAAgAA


gAAAcg


 








13) ONLoBAxC,106 bases 
(SEQ ID NO: 91)







ccAAgTAAccAcAAgTTTcTAgAgATTcgAAccggTTcTggTTAccMDNc


cVAccAccgTAAATgAATTccTcgcAcTgAcgcgTgAAAATgTTgAAgAA


gAAAcg


 








14) ONLoBBxC,106 bases 
(SEQ ID NO: 92)







ccAAgTAAccAcAAgTTTcTAgAgATTcgAAccggTTcTggTTAccccVc


cVAccAccgTAAATgAATTccTcgcAcTgAcgcgTgAAAATgTTgAAgAA


gAAAcg
















TABLE 28





Library for genetic diversity but constant AA sequence















! Gene encoding a DX-890-like TFPI K1 domain with linker.


! Null Library - Diversity at DNA level, all encode the same AA seq.


!


    1 tgtcctgcacagggtccact


!     scab . . .


!


!       1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 


!       G   S   G   M1  H   S   F   C5  A   F   K   A  D10  D   G


   16 |TCC|TCA|GGt|atg|caT|tcN|ttY|tgY|gcN|ttY|aaR|gcN|gaY|gaY|ggN|


!       Bsu36I . . .   NsiI . . .


!


!      16  17  18  19  20  21  22  23  24  25  26  27  28  29  30


!       P  C14  I   A   F   F   P  R20  F   F   F   N  I25  F   T


   61 |ccN|tgY|atH|gcN|ttY|ttc|cct|cgt|ttc|ttt|ttc|aac|atH|ttY|ACN|


!


!      31  32  33  34  35  36  37  38  39  40  41  42  43  44  45 


!       R   Q  C30  E   E   F   I  Y35  G   G  C38  Q  G40  N   Q


  106 |CGN|caR|tgY|gaR|GAR|TTY|atH|taY|ggN|ggN|tgY|caR|ggN|aaY|caR|


!


!      46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!       N   R  F45  E   S   L   E   E  C51  K   K   M  C55  T   R


  151 |aaY|cgN|ttt|gag|tct|ctt|gaa|gag|tgY|aaR|aaR|atg|tgY|acN|cgN|


!


!      61  62  63  64  65  66  67  68  69  70  71  72  73


!      D58 X_a X_b X_c X_d X_e X_f X_g X_H G_i S_j S_k G_l (SEQ ID NO: 94)


  196 |gaY|RgY|RgY|RcY|RgY|RgY|Rgy|RgY|RgY|ggc|TCC|TCA|GGT|


!                                            BseRI . . .


!                                               Bsu36I . . .








      Cttctcgttacctggtgggc
(SEQ ID NO: 93)







!     scab . . .


 








1) ONUpPrmr890, 21 bases
(SEQ ID NO: 95)







TgTccTgcAcAgggTccAcTT


 








2) ONLoPrmr890, 21 bases
(SEQ ID NO: 96)







gcccAccAggTAAcgAgAAgA


 








3) UpperA890,103 bases
(SEQ ID NO: 97)







TgTccTgcAcAgggTccAcTTccTcAggTATgcATTcNTTYTgYgcNTTYAARgcNgAYg


AYggNccNTgYATHgcNTTYTTcccTcgTTTcTTTTTcAAcAT


 








4) LowerB890,101 bases
(SEQ ID NO: 98)







cAcTcTTcAAgAgAcTcAAANcgRTTYTgRTTNccYTgRcANccNccRTADATRAAYTcY


TcRcAYTgNcgNgTRAADATgTTgAAAAAgAAAcgAgggAA


 








5) TopStC890, 98 bases
(SEQ ID NO: 99)







TTTgAgTcTcTTgAAgAgTgYAARAARATgTgYAcNcgNgAYRgYRgYRgYRgYRgYRgY


RgYRgYggcTccTcAggTcTTcTcgTTAccTggTgggc
















TABLE 29





 A phage vector to display TFPI KuDom 1 and derivatives.















! DY3P82_TFPI1  8919 bases


!


! Non-cutters









!AatII GACGTc
Acc65I Ggtacc
AflII Cttaag


!ApaI GGGCCc
ApaLI Gtgcac
AscI GGcgcgcc


!BbsI gtcttc
BclI Tgatca
BlpI GCtnagc


!BmgBI CACgtc
BsgI ctgcac
BsiWI Cgtacg


!Bsp120I Gggccc
BspEI Tccgga
BssHII Gcgcgc










!BstXI CCANNNNNntgg
(SEQ ID NO: 164)
BstZ17I GTAtac
BtrI CACgtg









!DraIII CACNNNgtg
Ec1136I GAGctc
EcoNI CCTNNnnnagg




(SEQ ID NO: 165)


!EcoO109I RGgnccy
EcoRV GATatc
EspI GCtnagc


!FseI GGCCGGcc
HpaI GTTaac
KpnI GGTACc


!MfeI Caattg
NotI GCggccgc
PmeI GTTTaaac


!PmlI CACgtg
PpuMI RGgwccy
PspOMI Gggccc


!PstI CTGCAg
SacI GAGCTc
SapI GCTCTTC


!SbfI CCTGCAgg
SexAI Accwggt
SfiI GGCCNNNNnggcc 




(SEQ ID NO: 173)


!SgfI GCGATcgc
SgrAI CRccggyg
SphI GCATGc


!Sse8387I CCTGCAgg
StuI AGGcct
XmaI Cccggg


!




! cutters




!









! Enzymes that cut more than 3 times.


!









!BsaAI YACgtr
5










!BspMI Nnnnnnnnngcaggt
4
(SEQ ID NO: 179)









!BsrFI Rccggy
4



!BtgI Ccrygg
4



!DsaI Ccrygg
4










!FauI nNNNNNNGCGGG
9
(SEQ ID NO: 171)


!









! Enzymes that cut from 1 to 3 times.


!


! $ = DAM site, * = DCM site, & = both


!









!BsrGI Tgtaca
1
1021











!BsaBI GATNNnnatc
2
1149
3974 
(SEQ ID NO: 174)










!SnaBI TACgta
2
1268
7131











!BspHI Tcatga
3
1299
6083
7091










!BsmI GAATGCN
2
1746
7733


!-′′-NGcattc
1
7477



!BseRI GAGGAGNNNNNNNNNN
1
2008 
(SEQ ID NO: 175)


!AlwNI CAGNNNctg
1
2187



!BspDI ATcgat
2
2527
8551











!NdeI CAtatg
3
2723
3803
8515










!AfeI AGCgct
1
3039



!EarI CTCTTCNnnn
2
4074
7942 (SEQ ID NO: 170)


!-′′- Nnnnngaagag
1
6124



!PacI TTAATtaa
1
4132












!BsiHKAI GWGCWc
3
4743
5465
6336


!HgiAI GWGCWc
3
4743
5465
6336


!BsmFI Nnnnnnnnnnnnnnngtccc
2
5075
8183 
(SEQ ID No: 184)


!-′′- GGGACNNNNNNNNNNnn
1
7897
 
(SEQ ID No: 169)


!MscI TGGcca
3
5080
5715
7828










!PsiI TTAtaa
2
5356
5835


!NaeI GCCggc
2
5613
7983


!NgoMIV Gccggc
2
5613
7983


!BanII GRGCYc
1
5643



!DrdI GACNNNNnngtc
1
5750 
(SEQ ID NO: 177)


!AvaI Cycgrg
2
5816
7238


!PvuII CAGctg
1
5951



!BsmBI CGTCTCNnnnn
2
5962
7939


!BamHI Ggatcc
1
5992&












!HindIII Aagctt
3
5998
7145
7382










!BciVI GTATCCNNNNNN
1
6075 
(SEQ ID NO: 167)


!Eco57I CTGAAG
1
6236$



!SpeI Actagt
1
6255



!BcgI gcannnnnntcg
1
6396
(SEQ ID NO: 185)


!ScaI AGTact
1
6440



!PvuI CGATcg
1
6551$



!FspI TGCgca
1
6698












!BglI GCCNNNNnggc
2
6799
7491
(SEQ ID NO: 168)










!BpmI CTGGAG
2
6833
7216


!-′′- ctccag
1
8568



!BsaI GGTCTCNnnnn
1
6851
(SEQ ID NO: 186)


!AhdI GACNNNnngtc
1
6918
(SEQ ID NO: 181)


!Eam1105I GACNNNnngtc
1
6918
(SEQ ID NO: 181)


!AccI GTmkac
1
7151



!HincII GTYrac
1
7151



!SalI Gtcgac
1
7151



!TliI Ctcgag
1
7238



!XhoI Ctcgag
1
7238



!PflMI CCANNNNntgg
1
7380 
(SEQ ID NO: 172)


!NcoI Ccatgg
1
7462












!StyI Ccwwgg
3
7462
7753
8130










!EagI Cggccg
1
7468



!NsiI ATGCAt
2
7476
8171











!PshAI GACNNnngtc
2
7503
7899 
(SEQ ID NO: 187)










!RsrII CGgwccg
1
7508



!MluI Acgcgt
1
7554



!EcoRI Gaattc
2
7569
7868


!BstEII Ggtnacc
1
7593



!AgeI Accggt
1
7603



!BstBI TTcgaa
2
7608
7865


!XbaI Tctaga
1
7616



!BssSI Ctcgtg
1
7642



!NheI Gctagc
1
7653



!KasI Ggcgcc
2
7707
7788


!Pf1FI GACNnngtc
1
7767



!Tth111I GACNnngtc
1
7767



!NruI TCGcga
1
7774



!XcmI CCANNNNNnnnntgg
1
7883* 
(SEQ ID NO: 166)


!BstAPI GCANNNNntgc
1
8005
(SEQ ID NO: 178)


!SacII CCGCgg
1
8033



!AvrII Cctagg
1
8130



!Bsu36I CCtnagg
1
8177



!SwaI ATTTaaat
1
8452



!BglII Agatct
1
8604$








!--------------------------------------------------------------------------








     1
aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat


 


    61
atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact


 


   121
cgttcgcaga attgggaatc aactgttaTa tggaatgaaa cttccagaca ccgtacttta


 


   181
gttgcatatt taaaacatgt tgagctacag caTTaTattc agcaattaag ctctaagcca


 


   241
tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg


 


   301
ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag


 


   361
tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt


 


   421
cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca


 


   481
tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct


 


   541
aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt


 


   601
ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt


 


   661
aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg


 


   721
atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt


 


   781
tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca


 


   841
caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttTc


 


   902
tcgtcagggc aagccttatt cactgaatga gcagctttgt tacgttgatt tgggtaatga


 


   962
atatccggtt cttgtcaaga ttactcttga tgaaggtcag ccagcctatg cgcctggtcT


 


  1022
GTACAccgtt catctgtcct ctttcaaagt tggtcagttc ggttccctta tgattgaccg


 


  1082
tctgcgcctc gttccggcta agtaacatgg agcaggtcgc ggatttcgac acaatttatc


 


  1142
aggcgatgat acaaatctcc gttgtacttt gtttcgcgct tggtataatc gctgggggtc


 


  1202
aaagatgagt gttttagtgt attctttTgc ctctttcgtt ttaggttggt gccttcgtag


 


  1262
tggcattacg tattttaccc gtttaatgga aacttcctca tgaaaaagtc tttagtcctc


 


  1322
aaagcctctg tagccgttgc taccctcgtt ccgatgctgt ctttcgctgc tgagggtgac


 


  1382
gatcccgcaa aagcggcctt taactccctg caagcctcag cgaccgaata tatcggttat


 


  1442
gcgtgggcga tggttgttgt cattgtcggc gcaactatcg gtatcaagct gtttaagaaa


 


  1502
ttcacctcga aagcaagctg ataaaccgat acaattaaag gctccttttg gagccttttt


 


  1562
ttttggagat tttcaac







! Gene   iii (as in M13mp18)


 








  1579
gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat


 


  1624
tct cac tcc gct gaa act gtt gaa agt tgt tta gca aaa tcc cat


 


  1669
aca gaa aat tca ttt act aac gtc tgg aaa gac gac aaa act tta


 


  1714
gat cgt tac gct aac tat gag ggc tgt ctg tgg aat gct aca ggc


 


  1759
gtt gta gtt tgt act ggt gac gaa act cag tgt tac ggt aca tgg


 


  1804
gtt cct att ggg ctt gct atc cct gaa aat gag ggt ggt ggc tct


 


  1849
gag ggt ggc ggt tct gag ggt ggc ggt tct gag ggt ggc ggt act


 


  1894
aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat


 


  1939
atc aac cct ctc gac ggc act tat ccg cct ggt act gag caa aac


 


  1984
ccc gct aat cct aat cct tct ctt GAG GAG tct cag cct ctt aat


 


  2029
act ttc atg ttt cag aat aat agg ttc cga aat agg cag ggg gca


 


  2074
tta act gtt tat acg ggc act gtt act caa ggc act gac ccc gtt


 


  2119
aaa act tat tac cag tac act cct gta tca tca aaa gcc atg tat


 


  2164
gac gct tac tgg aac ggt aaa ttC AGa gaC TGc gct ttc cat tct


 


  2209
ggc ttt aat gag gat tta ttt gtt tgt gaa tat caa ggc caa tcg


 


  2254
tct gac ctg cct caa cct cct gtc aat gct ggc ggc ggc tct ggt


 


  2299
ggt ggt tct ggt ggc ggc tct gag ggt ggt ggc tct gag ggt ggc


 


  2344
ggt tct gag ggt ggc ggc tct gag gga ggc ggt tcc ggt ggt ggc


 


  2389
tct ggt tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat


 


  2434
aag ggg gct atg acc gaa aat gcc gat gaa aac gcg cta cag tct


 


  2479
gac gct aaa ggc aaa ctt gat tct gtc gct act gat tac ggt gct


 


  2524
gct atc gat ggt ttc att ggt gac gtt tcc ggc ctt gct aat ggt


 


  2569
aat ggt gct act ggt gat ttt gct ggc tct aat tcc caa atg gct


 


  2614
caa gtc ggt gac ggt gat aat tca cct tta atg aat aat ttc cgt


 


  2659
caa tat tta cct tcc ctc cct caa tcg gtt gaa tgt cgc cct ttt


 


  2704
gtc ttt Ggc gct ggt aaa cca tat gaa ttt tct att gat tgt gac


 


  2749
aaa ata aac tta ttc cgt ggt gtc ttt gcg ttt ctt tta tat gtt


 


  2794
gcc acc ttt atg tat gta ttt tct acg ttt gct aac ata ctg 


 


  2836
cgt aat aag gag tct            t aatcatgcca gttcttttgg gtattccgtt


 


  2882
attattgcgt ttcctcggtt tccttctggt aactttgttc ggctatctgc ttacttttct


 


  2942
taaaaagggc ttcggtaaga tagctattgc tatttcattg tttcttgctc ttattattgg


 


  3002
gcttaactca attcttgtgg gttatctctc tgatattAGC GCTcaattac cctctgactt


 


  3062
tgttcagggt gttcagttaa ttctcccgtc taatgcgctt ccctgttttt atgttattct


 


  3122
ctctgtaaag gctgctattt tcatttttga cgttaaacaa aaaatcgttt cttatttgga


 


  3182
ttgggataaa taatatggct gtttattttg taactggcaa attaggctct ggaaagacgc


 


  3242
tcgttagcgt tggtaagatt caggataaaa ttgtagctgg gtgcaaaata gcaactaatc


 


  3302
ttgatttaag gcttcaaaac ctcccgcaag tcgggaggtt cgctaaaacg cctcgcgttc


 


  3362
ttagaatacc ggataagcct tctatatctg atttgcttgc tattgggcgc ggtaatgatt


 


  3422
cctacgatga aaataaaaac ggcttgcttg ttctcgatga gtgcggtact tggtttaata


 


  3482
cccgttcttg gaatgataag gaaagacagc cgattattga ttggtttcta catgctcgta


 


  3542
aattaggatg ggatattatt tttcttgttc aggacttatc tattgttgat aaacaggcgc


 


  3602
gttctgcatt agctgaacat gttgtttatt gtcgtcgtct ggacagaatt actttacctt


 


  3662
ttgtcggtac tttatattct cttattactg gctcgaaaat gcctctgcct aaattacatg


 


  3722
ttggcgttgt taaatatggc gattctcaat taagccctac tgttgagcgt tggctttata


 


  3782
ctggtaagaa tttgtataac gcatatgata ctaaacaggc tttttctagt aattatgatt


 


  3842
ccggtgttta ttcttattta acgccttatt tatcacacgg tcggtatttc aaaccattaa


 


  3902
atttaggtca gaagatgaaa ttaactaaaa tatatttgaa aaagttttct cgcgttcttt


 


  3962
gtcttgcgat tggatttgca tcagcattta catatagtta tataacccaa cctaagccgg


 


  4022
aggttaaaaa ggtagtctct cagacctatg attttgataa attcactatt gactcttctc


 


  4082
agcgtcttaa tctaagctat cgctatgttt tcaaggattc taagggaaaa TTAATTAAta


 


  4142
gcgacgattt acagaagcaa ggttattcac tcacatatat tgatttatgt actgtttcca


 


  4202
ttaaaaaagg taattcaaat gaaattgtta aatgtaatta attttgtttt cttgatgttt


 


  4262
gtttcatcat cttcttttgc tcaggtaatt gaaatgaata attcgcctct gcgcgatttt


 


  4322
gtaacttggt attcaaagca atcaggcgaa tccgttattg tttctcccga tgtaaaaggt


 


  4382
actgttactg tatattcatc tgacgttaaa cctgaaaatc tacgcaattt ctttatttct


 


  4442
gttttacgtg cAaataattt tgatatggtA ggttcTaACc cttccataat tcagaagtat


 


  4502
aatccaaaca atcaggatta tattgatgaa ttgccatcat ctgataatca ggaatatgat


 


  4562
gataattccg ctccttctgg tggtttvttt gttccgcaaa stgataatgt tactcaaact


 


  4622
tttaaaatta ataacgttcg ggcaaaggat ttaatacgag ttgtcgaatt gtttgtaaag


 


  4682
tctaatactt ctaaatcctc aaatgtatta tctattgacg gctct


 


  4727
aat cta tta gtt gtt aGT GCt Cct aaa gat


 


  4757
att tta gat aac ctt cct caa ttc ctt tcA act gtt gat ttg cca


 


  4802
actgaccaga tattgattga gggtttgata tttgaggttc agcaaggtga tgctttagat


 


  4862
ttttcatttg ctgctggctc tcagcgtggc actgttgcag gcggtgttaa tactgaccgc


 


  4922
ctcacctctg ttttatcttc tgctggtggt tcgttcggta tttttaatgg cgatgtttta


 


  4982
gggctatcag ttcgcgcatt aaagactaat agccattcaa aaatattgtc tgtgccacgt


 


  5042
attcttacgc tttcaggtca gaagggttct atctctgttg gccagaatgt cccttttatt


 


  5102
actggtcgtg tgactggtga atctgccaat gtaaataatc catttcagac gattgagcgt


 


  5162
caaaatgtag gtatttccat gagcgttttt cctgttgcaa tggctggcgg taatattgtt


 


  5222
ctggatatta ccagcaaggc cgatagtttg agttcttcta ctcaggcaag tgatgttatt


 


  5282
actaatcaaa gaagtattgc tacaacggtt aatttgcgtg atggacagac tcttttactc


 


  5342
ggtggcctca ctgattataa aaacacttct caGgattctg gcgtaccgtt cctgtctaaa


 


  5402
atccctttaa tcggcctcct gtttagctcc cgctctgatt cTaacgagga aagcacgtta


 


  5462
tacgtgctcg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg


 


  5522
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt


 


  5582
cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg


 


  5642
ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga


 


  5702
tttgggtgat ggtt                    ggcca tcgccctgat agacggtttt


 


  5741
tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac


 


  5801
aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga


 


  5861
accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa


 


  5921
ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga


 


  5981
aaaaccaccc tGGATCC  AAGCTT







!                     BamHHI


!                     Insert carrying bla gene


 








  6004
gcaggtg gcacttttcg gggaaatgtg cgcggaaccc


 


  6041
ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct







!                                              BciVI


 








  6101
gataaatgct tcaataatat tgaaaaAGGA AGAgt







!         Start bla gene


 








  6136
ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt


 


  6187
tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa gat gct


 


  6238
gaa gat cag ttg ggC gcA CTA GTg ggt tac atc gaa ctg gat ctc aac agc


 


  6289
ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc


 


  6340
act ttt aaa gtt ctg cta tgt GGC GcG Gta tta tcc cgt att gac gcc ggg


 


  6391
caa gaG CAA CTC GGT CGc cgC ATA cAC tat tct cag aat gac ttg gtt gAG


 


  6442
TAC Tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa


 


  6493
tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta ctt


 


  6544
ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg


 


  6595
ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc


 


  6646
ata cca aac gac gag cgt gac acc acg atg cct gta gca atg Gca aca acg


 


  6697
tTG CGC Aaa cta tta act ggc gaa cta ctt act cta gct tcc cgg caa caa


 


  6748
tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg


 


  6799
GCC ctt ccG GCt ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt


 


  6850
gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt


 


  6901
atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga aat


 


  6952
aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg TAA ctgt







!                                                                 stop


 








  7001
cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa


 


  7061
ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt


 


  7121
cgttccactg tacgtaagac cccc


 


  7145
AAGCTT   GTCGAC tgaa tggcgaatgg cgctttgcct







!                  SalI..


!                  HincII


 








  7181
ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt


 


  7231
CCTGAcG CTCGAG







!                 XhoI..


 








  7244
                        cgcaacgc aattaatgtg agttagctca


 


  7272
ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg


 


  7322
tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca


 


  7372
tgattacg  CCaagctt  TGGa gccttttttt tggagatttt caac







!                   Pf1MI. . . 


!








!          
 1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!          
 M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y 


  7416
gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat


!         
Exact copy of M13 signal, codons 1-15.







!








!                   
          S   M   A        A   E   M   H   S   F 


  7461
         tCC ATG Gcg     |gcc|gag|ATG|CAT|tcc|ttc


!                   
          NcoI . . .







!








!         
 C5  A   F   K   A  D10  D  G12  P   C  I15  A   F   F   P  R20  F


  7488
tgc gcc ttc aag gct gac gaC GGT CCG tgt aTT gct TTC TTc CCt cgt ttc







!








!          
 F   F   N  I25  F   T   R   Q  C30  E   E   F   I   Y   G   G   C


  7539
ttc ttc aac att ttc ACG CGT cag tgc gag GAA TTC att tac ggt ggt tgt







!                             MluI . . .


!








!          
 E  G40  N   Q   N   R  F45  E   S   L   E


  7590
gaa GGT AAC Cag aAC CGG Ttc gaa tcT CTA Gag







!             BstEII . . . AgeI . . .      XbaI . . .


!                                 AsuII . . .


!








!         
 E   C   K   K   M   C   T   R   D       S   A   S 


  7623  
gaa tgt aag aag atg tgt act|cgt|gat|    tct GCT AGC







!                                                     NheI . . . 


!








!          
 S   A 


  7659
tct gct


!








! III'stump


!


! Domain 3 of III-----------------------------------------------------------


!








!         
 S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A


  7665 
agt ggc gac ttc gac tac gag aaa atg gct aat gcc aac aaa GGC GCC


!



!          
 M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G


  7713 
atG ACT GAG AAC GCT GAC GAG aat gct ttg caa agc gat gcc aag ggt 


!



!         
 K   L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F


  7761 
aag tta gac agc gTC GCG Acc gac tat GGC GCC gcc ATC GAc ggc ttt


!                          
                 NruI . . .







!








!         
 I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D


  7809
atc ggc gat gtc agt ggt tTG GCC Aac ggc aac gga gcc acc gga gac


!



!          
 F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N


  7857 
ttc GCA GGT tcG AAT TCt cag atg gcC CAg gtt gga gaT GGg gac aac


!



!         
 S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q


  7905
agt ccg ctt atg aac aac ttt aga cag tac ctt ccg tct ctt ccg cag


!



!         
 S   V   E   C   R   P   F   V   F   G   A   G   K   P   Y   E 


  7953
agt gtc gag tgc cgt cca ttc gtt ttc GGt gcc ggc aag cct tac gag


!



!          
 F   S   I   D   C   D   K   I   N   L   F   R


  8001
ttc aGC Atc gac TGC gat aAg atc aat ctt ttC CGC


!             
     BstAPI . . .                         SacII . . .


!                                                
                                            End Domain 3







!








!         
 G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V   F


  8037
GGc gtt ttc gct ttc ttg cta tac gtc gct act ttc atg tac gtt ttc


!         
start transmembrane segment







!








!          
 S   T   F   A   N   I   L     R   N   K   E   S (SEQ ID NO: 101)


  8085
agc act ttc gcc AAT ATT tta   cgc aac aaa gaa agc


!                                        
                              Intracellular anchor.


!                  
         .   .


  8121
        tag tga tct CCT AGG


!                              
                    AvrII . . .


!                                                              
                                                     | M13mp18 >


  8136
aaG CCC GCc taa tga GCG GGC ttt ttt ttt ct  ggt   ATGCAT CCTGAGG


!         
Stem . . . Loop . . . Stem . . .  MultiT . . .    NsiI   Bsu36I.


!         
| Trp terminator                              |   (2/2)


 


  8184
                   ccgat actgtcgtcg tcccctcaaa ctggcagatg


!



  8219
cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg


 


  8279
tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc


 


  8339
tggctacagg aaggccagac gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg


 


  8399
agctgattta acaaaaattt aaTgcgaatt ttaacaaaat attaacgttt acaATTTAAA


!
                                                          SwaI . . .


 


  8459
Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat


!                                                                
                                                       RBS?


 


  8518 
ATG att gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt tgc


 


!
Start gene II


  8569
tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT CTc tca aaa ata


!
                                              BglII . . .


 


  8620
gct acc ctc tcc ggc atT aat tta tca gct aga acg gtt gaa tat cat att


 


  8671
gat ggt gat ttg act gtc tcc ggc ctt tct cac cct ttt gaa tct tta cct


 


  8722
aca cat tac tca ggc att gca ttt aaa ata tat gag ggt tct aaa aat ttt


 


  8773
tat cct tgc gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt cat


 


  8824
aat gtt ttt ggt aca acc gat tta gct tta tgc tct gag gct tta ttg ctt 


 


  8875
aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt(SEQ ID NO: 100)


!
gene II continues
















TABLE 30







Modified Human TFPI = PlaKalEl02 (SEQ ID NO: 102)









AAs
Function
AA sequence





  1-21
Linker 1
DSEEDEEHTIITDTELPPLKP


 


 22-79
KuDom 1 
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD



Inhibit




plasmin



 


 80-92
Linker 2
NANRPIKTTLQQE


 


 93-150 
KuDom 2 
KPDFCFLEEDPGPCRAAHPRWFYNnQTKQCERFIYGGCEGNMNNFETLEECKNICEDG



Inhibit




h.pKal



 


151-184
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSPKVPSLFEFH


 


185-242
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFPYGGCQGNENnFTSKQECLRACKKG



Inhibit 
1   5    1    1    2    2    3    3    4    4    5    5  5



h.NE
         0    5    0    5    0    5    0    5    0    5  8


 


243-248
Linker 4
FIQRIS





Mutations from w.t. are BOLD, UPPERCASE.


Possible glycosylation sites are shown as “n”.













TABLE 31







Modified Human TFPI dimer = ELsix (SEQ ID NO: 103)









Aas
Function
AA sequence





  1-21
Linker 1
DSEEDEEHTIITDTELPPLKL


 


 22-79
KuDom 1 
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCEEFIYGGCQGNQNRFESLEECKKMCTRD


 


 80-92
Linker 2
NANRIIKTTLQQE


 


 93-150
KuDom 2
KPDFCFLEEDPGPCIAFFPRFFYNnQTKQCERFIYGGCQGNMNNFETLEECKNICEDG


 


151-184
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


185-242
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFKYGGCQGNENnFTSKQECLRACKKG




1   5    1    1    2    2    3    3    4    4    5    5  5




         0    5    0    5    0    5    0    5    0    5  8


 


243-248
Linker 4
FIQRIS


 


249-261
Linker 5
GGSGSGGSGSSGG


 


262-282
Linker 1
DSEEDEEHTIITDTELPPLKL


 


283-341
KuDom 1
MHSFCAFKADDGPCIAFFPRFFFNIFTRQCEEFIYGGCQGNQNRFESLEECKKMCTRD


 


342-354
Linker 2
NANRIIKTTLQQE


 


355-410
KuDom 2
KPDFCFLEEDPGPCIAFFPRFFYNnQTKQCERFIYGGCQGNMNNFETLEECKNICEDG


 


411-444
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


445-502
KuDom 3
GPSWCLTPADRGPCIAFFPRFYYNSVIGKCRPFKYGGCQGNENnFTSKQECLRACKKG


 


503-507

FIQRIS





Mutations from w.t. are BOLD, UPPERCASE.


Possible glycosylation sites are shown as “n”.













TABLE 32





AA Sequence of rabbit TFPI (SEQ ID NO: 104)
















LOCUS
AAB26836   299 aa linear  MAM 25-AUG-1993


DEFINITION
tissue factor pathway inhibitor; lipoprotein-associated 



coagulation inhibitor; extrinsic pathway inhibitor; TFPI; 



LACI; EPI [Oryctolagus cuniculus].


ACCESSION
AAB26836


VERSION
AAB26836.1  GI:386016







UPPERCASE BOLD Ns may be glycosylated












AAs
Function
AA sequence





  1-24
signal
mkkehifwtsiclllglvpapvss


 


 25-44
Linker 1
aaeedeftNitdikpplqkp


 


 45-102
KuDom 1
thsfcamkvddgperayikrfffnilthgceefiyggcegnenrfesleeckekcard


 


103-115
Linker 2
ypkmttkltfqkg


 


116-173
KuDom 2
kpdfcfleedpgicrgyitryfynNgskqcerfkyggclgnlnnfesleeckntceNp


 


174-207
Linker 3
tsdfqvddhrtqlntvNntlinqptkaprrwafh


 


208-265
KuDom 3
gpswclppadrglcqaneirffynaiigkcrpfkysgcggnenNftskkacitackkg


 


266-299
Linker 4
firNlskggliktkrkkkkgpvkityvetfvkkt
















TABLE 33





cDNA Sequence of rabbit TFPI
















LOCUS
S61902                 900 bp   mRNA   linera  MAM 25-AUG-1993


DEFINITION
tissue factor pathway inhibitor=Kunitz-type protease inhibitor



[rabbits, lung, mRNA, 938 nt].


ACCESSION
S61902 REGION: 21..920


VERSION
S61902.1 GI:386015


KEYWORDS
.


SOURCE
Oryctolagus cuniculus(rabbit)


   ORGANISM
Oryctolagus cuniculus



Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;



Mammalia; Eutheria; Euarchontoglires; Glires; Lagomorpha;



Leporidae; Oryctolagus.


REFERENCE
1 (bases 1 to 900)


AUTHORS
Belaaouaj, A., Kuppuswamy, M.N., Birktoft, J.J. and Bajaj, S.P.


TITLE
Revised cDNA sequence of rabbit tissue factor pathway inhibitor


JOURNAL
Thromb. Res. 69 (6), 547-553(1993)


PUBMED
8503123


REMARK
GenBank staff at the National Library of Medicine created this



entry [NCBI gibbsq 133173] from the original journal article.


FEATURES
        Location/Qualifiers


       source
        1 . . . 900



        /organism=“Oryctolagus cuniculus”



        /mol_type=“mRNA”



        /db_xref=“taxon:9986”


       gene
        <1 . . . >900



        /gene=”tissue factor pathway inhibitor,



        lipoprotein-associated coagulation inhibitor, extrinsic



        pathway inhibitor, TFPI, LACI, EPI”


       CDS
        1 . . . 900



        /gene=“tissue factor pathway inhibitor,



        lipoprotein-associated coagulation inhibitor, extrinsic



        pathway inhibitor, TFPI, LACI, EPI”



        /note=“Kunitz-type protease inhibitor;



        lipoprotein-associated coagulation inhibitor; extrinsic



        pathway inhibitor; TFPI; LACI; EPI”



        /codon_start=1



        /product=“tissue factor pathway inhibitor”



        /protein_id=“AAB26836.1”



        /db_xref=“GI:386016”



        /translation=“MKKEHIFWTSICLLLGLVPAPVSSAAEEDEFTNITDIKPPLQKP



        THSFCAMKVDDGPCRAYIKRFFFNILTHQCEEFIYGGCEGNENRFESLEECKEKCARD



        YPKMITKLIFQKGKPDFCFLEEDPGICRGYITRYFYNNQSKQCERFKYGGCLGNLNNF



        ESLEECKNTCENPTSDFQVDDHRTQLNTVNNTLINQPTKAPRRWAFHGPSWCLPPADR



        GLCQANEIRFFYNAIIGKCRPFKYSGCGGNENNFTSKKACITACKKGFIRNLSKGGLI



        KTKRKKKKQPVKITYVETFVKKT” (SEQ ID NO: 104)


ORIGIN












(SEQ ID NO: 105)



1
atgaagaaag aacacatctt ttggacgtct atatgcctgc tgcttggtct tgtccctgcc


 



61
cctgttagct cagcggccga ggaagatgaa ttcacaaaca ttacagatat taaaccgcca


 



121
ctacagaagc cgacacactc attttgtgca atgaaggtag atgatgggcc gtgcagagca


 



181
tacatcaaga gatttttttt caatattctc acccatcagt gtgaagaatt tatatatgga


 



241
ggatgtgaag ggaacgagaa tcgattcgag agtctggaag aatgcaaaga aaaatgtgca


 



301
cgagattatc caaagatgac tacaaagctg acatttcaaa aaggaaagcc tgatttctgc


 



361
tttttggaag aagatcctgg tatttgtcga ggttatatta ccagatattt ttataacaat


 



421 
caatcaaaac aatgtgaacg tttcaagtac ggtgggtgcc ttggcaatct aaacaacttt


 



481 
gagtcattgg aagaatgcaa aaacacctgt gagaatccaa cgagtgattt ccaggtggat


 



541 
gaccatagaa cccagctcaa tactgtgaat aacactttaa ttaaccagcc gaccaaggct


 



601 
cccagacgtt gggcatttca cggcccctca tggtgtctgc ccccagcaga cagaggattg


 



661 
tgtcaagcca atgagatcag attcttctac aatgcaatca tcgggaaatg ccgcccattt


 



721 
aagtacagtg gatgtggggg aaatgaaaat aatttcactt ccaaaaaagc atgtatcaca


 



781 
gcttgtaaaa aaggtttcat ccgaaatcta tcaaaaggag gactaattaa aaccaaaagg


 



841
aagaaaaaga agcagccagt gaaaataact tatgtagaaa cttttgttaa aaagacataa 
















TABLE 34







AA secuence of RaTFPKE01, a 


derivative of rabbit TFPI (SEQ ID NO: 106)









AAs
Function
AA sequence





  1-20
Linker 1
aaeedeftNitdikpplqkp


 


 21-78
KuDom 1
thsfcamkvddgpcraRFDrWffnilthgce




efiyggcegnenrfesleeckekcard


 


 79-91
Linker 2
Ypkmttkltfqkg


 


 92-149
KuDom 2
kpdfcfleedpgPcrAAHPryfynNqskqce




rfkyggcEgnlnnfesleeckntceNp


 


150-183
Linker 3
tsdfqvddhrtqlntvNntlinqptkaprrw




afh


 


184-265
KuDom 3
gpswclppadrgPcIaFFPrffynaiigkcr




pfkyGgcQgnenNftskkacitackkg


 


266-271
Linker 4
firNls
















TABLE 35 







AA sequence of RaTFPKE02, a 


derivative of rabbit TFPI (SEQ ID NO: 107)









AAs
Function
AA sequence





  1-20
Linker 1
aaeedeftNitdikpplqkp


 


 21-78
KuDom 1
thsfcamkvddgperaRFDrWffnilthgce




efiyggcegnenrfesleeckekcard


 


 79-91
Linker 2
Ypkmttkltfqkg


 


 92-149
KuDom 2
kpdfcfleedpgPcrAAHPryfynNqskqce




rfkyggcEgnlnnfesleeckntceNp


 


150-183
Linker 3
tsdfqvddhrtqlntvNntlinqptkaprrw




afh


 


184-241
KuDom 3
gpswclppadrgPcIaFFPrffynaiigkcr




pfkyGgcQgnenNftskkacitackkg


 


242-247
Linker 4
firNls


 


248-262
Linker 5
GGSGSSGSGGSGSSG


 


263-282
Linker 1
aaeedeftNitdikpplqkp


 


283-340
KuDom 1
thsfcamkvddgperaRFDrWffnilthqce




efiyggcegnenrfesleeckekcard


 


341-353
Linker 2
Ypkmttkltfqkg


 


354-411
KuDom 2
kpdfcfleedpgPcrAAHPryfynNqskqce




rfkyggcEgnlnnfesleeckntceNp


 


412-445
Linker 3
tsdfqvddhrtqlntvNntlinqptkaprrw




afh


 


446-527
KuDom 3
gpswclppadrgPcIaFFPrffynaiigkcr




pfkyGgcQgnenNftskkacitackkg


 


528-509
Linker 4
firNls





N may be glycosylated.













TABLE 36





DNA of ratfpke01 (SEQ ID NO: 108)















aTG Gga tgg agc tgt atc atc ctc ttc ttg gTC GCG


Acg gcc aca ggg gcc cac tcc


 


 gcg gcc gag gaa gat gaa ttc aca aac att aca gat att aaa ccg cca cta cag aag ccg


 


 aca cac tca ttt tgt gca atg aag gta gat gat ggg ccg tgc aga gca CGc Ttc GaT aga


 tGG ttt ttc aat att ctc acc cat cag tgt gaa gaa ttt ata tat gga gga tgt gaa ggg


 aac gag aat cga ttc gag agt ctg gaa gaa tgc aaa gaa aaa tgt gca cga gat


 


 tat cca aag atg act aca aag ctg aca ttt caa aaa gga


 


 aag cct gat ttc tgc ttt ttg gaa gaa gat cct ggt CCt tgt cga gCt GCt CAt CcT aga


 tat ttt tat aac aat caa tca aaa caa tgt gaa cgt ttc aag tac ggt ggg tgc GAG ggc


 aat cta aac aac ttt gag tca ttg gaa gaa tgc aaa aac acc tgt gag aat cca


 


 acg agt gat ttc cag gtg gat gac cat aga acc cag ctc aat act gtg aat aac act tta


 att aac cag ccg acc aag gct ccc aga cgt tgg gca ttt cac


 


 ggc ccc tca tgg tgt ctg ccc cca gca gac aga gga CCg tgt ATa gcc TTt TTC CCc aga


 ttc ttc tac aat gca atc atc ggg aaa tgc cgc cca ttt aag tac Ggt gga tgt CAg gga


 aat gaa aat aat ttc act tcc aaa aaa gca tgt atc aca gct tgt aaa aaa ggt


 


 ttc atc cga aat cta tca Taa Tga tctaga
















TABLE 37





Oligonucleotides to produce ratfpke01 from rabbit tfpi


 All oligonucleotides are 5'-to-3'



















(SEQ ID NO: 109)


 1
ONrSfi
TTAATCTTGCGGCCACAGGGGCCCACTCTGCGGCCGAGGAAGATGAA 


 




(SEQ ID NO: 110)


 2
TruncLo
TCATTATGATAGATTTCGGATGAAACCTTTTTTACA 


 




(SEQ ID NO: 111)


 3
ONrK1Bot
TATTGAAAAACCATCTATCGAAGCGTGCTCTG 


 




(SEQ ID NO: 112)


 4
ONrK1Top
CAGAGCACGCTTCGATAGATGGTTTTTCAATA 


 




(SEQ ID NO: 113)


 5
ONrK2Bot1
AAAAATATCTAGGATGAGCAGCTCGACAAGGACCAGGATCT 


 




(SEQ ID NO: 114)


 6
ONrK2To1
AGATCCTGGTCCTTGTCGAGCTGCTCATCCTAGATATTTTT 




(SEQ ID NO: 115)


 


 7
ONrK2B2
GTTTAGATTGCCCTCGCACCCAC 


 




(SEQ ID NO: 116)


 8
ONrK2T2
GTGGGTGCGAGGGCAATCTAAAC 


 




(SEQ ID NO: 117)


 9
ONrK3B1
GAAGAATCTGGGGAAAAAGGCTATACACGGTCDTCTGTCT


 




(SEQ ID NO: 118)


10
ONrK3T1
AGACAGAGGACCGTGTATAGCCTTTTTCCCCAGATTCTTC 


 




(SEQ ID NO: 119)


11
ONrK3B2
TCATTTCCCTGACATCCACCGTACTTAAATGG


 




(SEQ ID NO: 120)


12
ONrK3T2
ATTTAAGTACGGTGGATGTCAGGGAAATGA


 




(SEQ ID NO: 121)


13
ONrXbaILo
GGATTACTTCTAGATCATTATGATAGATTTCGGATGAAACCTTTTTTACA 
















TABLE 38







TABLE 4B of U.S. Pat. No. 6,583,108


Ki values for the inhibition of various 


proteases by refolded bikunin(7-64)












Protease
bikunin 

bikunin



(concen-
(7-64)
Aprotinin
(102-159)



tration)
Ki (nM)
Ki (nM)
Ki (nM)
















Trypsin (50 pM)
0.2
0.8
0.3



Human Plasma
0.7
19.0
0.7



Kallikrein 






(0.2 nM)






Human plasmin 
3.7
1.3
1.8



(50 pM)






F.XIIa
Not done
12,000
4,500



F.XIa(100 pM)
200
288
15



Human tissue 
2.3
0.004
0.13



kallikrein

















TABLE 39





AA sequence of bikunin from


U.S. Pat. No. 6,583,108















(SEQ ID NO: 122)


ADRERS


IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNNYLTKEE


CLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMFN


YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEA


CMLRCFRQgenpplplgskvvvlagAVS


 


(SEQ ID NO: 123)


         AGSFLAWlgslllsgvla


ADRERS


IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNNYLTKEE


CLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMFN


YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEA


CMLRCFRQgenpplplgskvvvlagAVS


 


(SEQ ID NO: 124)


    MLRAEADGVSRLlgslllsgvla


ADRERS


IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNNYLTKEE


CLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMFN


YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEA


CMLRCFRQgenpplplgskvvvlagLFVMVLILFLGASMVYLIRVARRNQ


ERALRTVWSSGDDKEQLVKNTYVL


 


(SEQ ID NO: 125)


MAQLCGLRRSRAFLALlgslllsgvla


ADRERS


IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNNYLTKEE


CLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMFN


YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEA


CMLRCFRQgenpplplgskvvvlaglfvmvlilflgasmvylirvarrng


eralrtvwsFGD
















TABLE 40







AA secuence of RaTFPKE04, a derivative of 


rabbit TFPI (SEQ ID NO: 126)









AAs
Function
AA sequence





 1-20
Linker 1
aaeedeftNitdikpplqkp


 


21-78
KuDom 1
thsfcamkvddgpGraRFDrWffnilthqceef




iyggVegnenrfesleeckekcard


 


79-91
Linker 2
Ypkmttkltfqkg


 


 92-149
KuDom 2
kpdfcfleedpgPGrAAHPryfynNqskqcerf




kyggVEgnlnnfesleeckntceNp


 


150-183
Linker 3
tsdfqvddhrtqlntvNntlinqptkaprrwaf




h


 


184-241
KuDom 3
gpswclppadrgPGIaFFPrffynaiigkcrpf




kyGgVQgnenNftskkacitackkg


 


242-247
Linker 4
firNls





N may be N-linked gycosylated.













TABLE 41







AA sequence of RaTFPKE05, a derivative of 


rabbit TFPI (SEQ ID NO: 127)









AAs
Function
AA sequence





 1-20
Linker 1
aaeedeftNitdikpplqkp


 


21-78
KuDom 1
thsfcamkvddgpGraRFDrWffnilth




qceefiyggVegnenrfesleeckekca




rd


 


79-91
Linker 2
Ypkmttkltfqkg


 


 92-149 
KuDom 2
kpdfcfleedpgPGrAAHPryfynNqsk




qcerfkyggVEgnlnnfesleeckntce




Np


 


150-183
Linker 3
tsdfqvddhrtqlntvNntlinqptkap




rrwafh


 


184-241
KuDom 3
gpswclppadrgPGIaFFPrffynaiig




kcrpfkyGgVQgnenNftskkacitack




kg


 


242-247
Linker 4
firNls


 


248-262
Linker 5
GGSGSSGSGGSGSSG


 


263-282
Linker 1
aaeedeftNitdikpplqkp


 


283-340
KuDom 1
thsfcamkvddgpGraRFDrWffnilth




qceefiyggVegnenrfesleeckekca




rd


 


341-353
Linker 2
Ypkmttkltfqkg


 


354-411
KuDom 2
kpdfcfleedpgPGrAAHPryfynNqsk




qcerfkyggVEgnlnnfesleeckntce




Np


 


436-469
Linker 3
tsdfqvddhrtqlntvNntlinqptkap




rrwafh


 


470-527
KuDom 3
gpswclppadrgPGIaFFPrffynaiig




kcrpfkyGgVQgnenNftskkacitack




kg


 


528-533
Linker 4
firNls
















TABLE 44





Amino acid sequence of DX-890 (SEQ ID NO: 128)















EACNLPIVRGPCIAFFPRWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECR


 


EYCGVP
















TABLE 45





 Amino acid sequence of DX-1000 (SEQ ID NO: 129)















MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEE


 


CKKMCTRD
















TABLE 50





hTFPImC1, an optimized, tagged w.t. TFPI minus Cterminal region.















!


   1    gccaCC


!           NcoI...


!           Pff1MI...


!


!       Signal seq


!        1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!       M   G   W   S   C   I   I   L   F   L   V   A   T   A   T


   7   ATG Gga TGG agc tgt atc atc ctc ttc ttg gTC GCG AcG GCC aca


!  NcoI.....                                    NruI.... SfiI.....


!     PflMI.......


!


!      Signal Mature protein His6 tag


!       16  17  18  19  20  21  22  23  24  25  26  27  28  29  30


!       G   A   H   S   E   G   R   P   G   H   H   H   H   H   H


  52   ggG GCC cac tcc gaa ggt cgt ccg GGT CAC Cac cat cat cat cat


!   SfiI......                         BstEII...


!      EcoO109I.


!       ApaI....


!


!             Thrombin cleavage site        Codon 2 for mature TFPI


!                                   |       |


!       31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!       G   G   S   S   L   V   P   R   G   S   E   E   D   E   E


  97   ggc ggt tct agt ctg gtc ccg cgt ggc tct gag gaa gat gaa gaa


!


!                                               Start first KuDom


!                                                               |


!       46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!       H   T   I   I   T   D   T   E   L   P   P   L   K   L   M


 142   cac aca att atc aca gat acg gag ttg cca cca ctg aaa ctt ATG


!                                                              NsiI...


!


!       61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!       H   S   F   C5  A   F   K   A   D   D   G   P  C14  K   A


 187   CAT tca ttt tgt gca ttc aag gcg gat gat ggc cca tgt aaa gca


!   NsiI..


!


!       76  77  78  79  80  81  82  83  84  85  86  87  88  89  90


!       I   M   K   R   F   F   F   N   I   F   T   R   Q  C30  E


 232   aTC ATG Aaa aga ttt ttc ttc aat att ttc act cga cag tgc gaa


!       BspHI...


!


!       91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!       E   F   I   Y   G   G  C38  E   G   N   Q   N   R   F   E


 277   gaa ttt ata tat ggg gga tgt gaa gga aat cag aAT CGA Ttt gaa


!                                                   BspDI...


!


!                                     End First KuDom


!                                                   |


!      106 107 108 109 110 111 112 113 114 115 116 117 118 119 120


!       S   L   E   E  C51  K   K   M  C55  T   R   D   N   A   N


 322   agt ctg gaa gag tgc aaa aaa atg TGT ACA aga gat aat gca aac


!                                      BsrGI..


!


!                              Start second KuDom


!                                               |


!      121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!       R   I   I   K   T   T   L   Q   Q   E   K   P   D   F   C5


 367   agg att ata aag aca aca ttg caa caa gaa aag cca gat ttc tgc


!


!      136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!       F   L   E   E   D   P   G   I  C14  R   G   Y   I   T   R


 412   ttt ttg gaa gaa gat cct gga ata tgt cga ggt tat att ACC AGG


!                                                          SexAI....


!


!                       *


!      151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!       Y   F   Y   N   N   Q   T   K   Q  C30  E   R   F   K   Y


 457   Tat ttt tat aac aat cag aca aaa cag tgt gaa cgt ttc aag tat


! SexAI.


!


!      166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!       G   G  C38  L   G   N   M   N   N   F   E   T   L   E   E


 502   ggt gga tgc ctg ggc aat atg aac aat ttt gag aca ctg gaa gaa


!


!                      End second KuDom


!                                   |


!      181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!      C51  K   N   I  C55  E   D   G   P   N   G   F   Q   V   D


 547   tgc aag aac att tgt gaa gat ggt ccg aat ggt ttc cag gtg gat


!


!                                           *


!      196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!       N   Y   G   T   Q   L   N   A   V   N   N   S   L   T   P


 592   aat tat gga acc cag ctc aat get gtg aat aac tcc ctg act ccg


!


!                                       Start third KuDom


!                                                       |


!      211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!       Q   S   T   K   V   P   S   L   F   E   F   H   G   P   S


 637   caa tca acc aag gtt ccc agc ctt ttt gaa ttt cac ggt ccc tca


!


!      226 227 228 229 230 231 232 233 234 235 236 237 238 239 240


!       W   C5  L   T   P   A   D   R   G   L  C14  R   A   N   E


 682   tgg tgt ctc aCT CCA Gca gac aga gga ttg tgt cgt gcc aat gag


!                   BpmI....


!


!      241 242 243 244 245 246 247 248 249 250 251 252 253 254 255


!       N   R   F   Y   Y   N   S   V   I   G   K  C30  R   P   F


 727   aac aga ttc tac tac aat tca gtc att ggg aaa tgc cgc cca ttt


!


!      256 257 258 259 260 261 262 263 264 265 266 267 268 269 270


!       K   Y   S   G  C38  G   G   N   E   N   N   F   T   S   K


 772   aag tac agt gga tgt ggg gga aat gaa aac aat ttt act tcc aaa


!


!      271 272 273 274 275 276 277 278 279 280 281


!       Q   E  C51  L   R   A  C55  K   K   G    (SEQ ID NO: 131).


 817   caa gaa tgt ctg agg gca tgt aaa aaa ggt taa


!


 850   tga TCTAGA ag  (SEQ ID NO: 130)


!          XbaI..


!
















TABLE 51





hTFPI[PpKhNE], a TFPI with plasmin, pKal, and hNe inhibition















!


   1    GCCACC


!           NcoI...


!           PflMI...


!


!       Signal sequence............................................


!         1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!        M   G   W   S   C   I   I   L   F   L   V   A   T   A   T


   7    ATG Gga Tgg agc tgt atc atc ctc ttc ttg gTC GCG AcG GCC aca


!     NcoI...                                    NruI.... SfiI........


!   PflMI..........


!


!Signal................  Cleavage..........  His6 purification tag..


!        16  17  18  19  20  21  22  23  24  25  26  27  28  29  30


!        G   A   H   S   E   G   R   P   G   H   H   H   H   H   H


  52    GGG GCC Cac tcc gaa ggt cgt ccg GGT CAC Cac cat cat cat cat


!    SfiI......                         BstEII...


!       EcoO109I


!        ApaI....


!


!                  Thrombin cleavage site    Codon 2 of mature TFPI


!                                       |    |


!        31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!        G   G   S   S   L   V   P   R   G   S   E   E   D   E   E


  97    ggc ggt tct agt ctg gtc ccg cgt ggt tct gag gaa gat gaa gaa


!


!                                                  Start 1st KuDom


!                                                                |


!        46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!        H   T   I   I   T   D   T   E   L   P   P   L   K   L   M


 142    cac aca att atc aca gat acg gag ttg cca cca ctg AAG CTT ATG


!                                                       HindIII NsiI...


!


!        61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!        H   S   F   C5  A   F   K   A   D   D   G   P  C14  R   A


 187    CAT tca ttt tgt gca ttc aag gcg gat gat ggc cca tgt aga gca


!     NsiI.


!


!        76  77  78  79  80  81  82  83  84  85  86  87  88  89  90


!        R   F   D   R   W   F   F   N   I   F   T   R   Q  C30  E


 232    cgc ttc gat aga tgg ttc ttc aat att ttc act cga cag tgc gaa


!


!        91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!        E   F   I   Y   G   G  C38  E   G   N   Q   N   R   F   E


 277    gaa ttt ata tat ggg gga tgt gaa gga aat cag aAT CGA Ttt gaa


!                                                    BspDI...


!


!                                     End of 1st KuDom


!                                                    |  Linker 2


!       106 107 108 109 110 111 112 113 114 115 116 117 118 119 120


!        S   L   E   E  C51  K   K   M  C55  T   R   D   N   A   N


 322    agt ctg gaa gag tgc aaa aaa atg TGT ACA aga gat aat gca aac


!                                       BsrGI..


!


!       Linker 2............................... Kudom 2...........


!       121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!        R   I   I   K   T   T   L   Q   Q   E   K   P   D   F   C5


 367    agg att ata aag aca aca ttg caa caa gaa aag cca gat ttc tgc


!


!       Kudom 2....................................................


!       136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!        F   L   E   E   D   P   G   P  C14  R   A   A   H   P   R


 412    ttt CTC GAG GAG GAT CCt gga ccg tgt cga get get cat ccc agg


!           XhoI...


!               BseRI..


!                     BamHI...


!


!                        *


!       151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!        W   F   Y   N   N   Q   T   K   Q  C30  E   R   F   I   Y


 457    tgg ttt tat aac aat cag aca aaa cag tgt gaa cgt ttc atc tat


!


!       166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!        G   G  C38  E   G   N   M   N   N   F   E   T   L   E   E


 502    ggt gga tgc gag ggc aat atg aac aat ttt gag aca ctg gaa gaa


!


!                          End KuDom 2


!                                    |  Linker 3...................


!       181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!       C51  K   N   I  C55  E   D   G   P   N   G   F   Q   V   D


 547    tgc aag aac att tgt gaa gat ggt ccg aat ggt ttc cag GTC GAC


!                                                           SalI...


!


!                                            *


!       196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!        N   Y   G   T   Q   L   N   A   V   N   N   S   L   T   P


 592    aat tat gga acc cag ctc aat get gtg aat aac tcc ctg act ccg


!


!                                                        KuDom 3....


!       211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!        Q   S   T   K   V   P   S   L   F   E   F   H   G   P   S


 637    caa tca acc aag gtt ccc agc ctt ttt gaa ttt cac ggt ccc tca


!


!       226 227 228 229 230 231 232 233 234 235 236 237 238 239 240


!        W   C5  L   T   P   A   D   R   G   P  C14  I   A   F   F 


 682    tgg tgt ctc act cCC GCG Gac aga gga ccg tgt att gcc ttt ttc


!                        SacII...


!


!       241 242 243 244 245 246 247 248 249 250 251 252 253 254 255


!        P   R   F   Y   Y   N   S   V   I   G   K  C30  R   P   F


 727    ccc aga ttc tac tac aat tca gtc att ggg aaa tgc cgc cca ttt


!


!                                                *


!       256 257 258 259 260 261 262 263 264 265 266 267 268 269 270


!        P   Y   G   G  C38  Q   G   N   E   N   N   F   T   S   K


 772    ccg tat ggc gga tgt cag gga aat gaa aac aat ttt act tcc aaa


!


!       271 272 273 274 275 276 277 278 279 280 281


!        Q   E  C51  L   R   A  C55  K   K   G   (SEQ ID NO: 133).


 817    caa gaa tgt ctg agg gca tgt aaa aaa ggt taa TGA


!


!


 853    TCTAGA AGCTCGCTGATC  (SEQ ID NO: 132)


!       XbaI..
















TABLE 52





PLA301B















!


   1   ggccaCC


!           NcoI...


!           PflMI....


!


!        1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!       M   G   W   S   C   I   I   L   F   L   V   A   T   A   T


   8   ATG Gga TGG tcc tgc atc atc ctg ttt ctg gTG GCC Acc gcc aca


!    NcoI...                                    MscI....


!    Pf1MI........


!


!       16  17  18  19  20  21  22  23  24  25  26  27  28  29  30


!       G   A   H   S   D   S   E   E   D   E   E   H   T   I   I


  53   GGC GCC cac agc gac agc gag gaa gat gag gaa cac acc atc atc


!KasI...


!


!       31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!       T   D   T   E   L   P   P   L   K   L   M   H   S   F   C


  98   acc gac acc gag ctg ccc ccc ctg AAG CTT atg cac agc ttc tgc


!                                      HindIII


!


!       46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!       A   F   K   A   D   D   G   P   C   R   A   R   F   D   R


 143   gcc ttc aag gcc gac gac ggc cCC TGC AGG gcc aga ttc gac aga


!                                   SbfI......


!


!       61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!       W   F   F   N   I   F   T   R   Q   C   E   E   F   I   Y


 188   tgg ttc ttc aac atc ttc acc agg cag tgc gag GAA TTC att tat


!                                                  EcoRI..


!


!       76  77  78  79  80  81  82  83  84  85  86  87  88  89  90


!       G   G   C   E   G   N   Q   N   R   F   E   S   L   E   E 


 233   gga ggc tgt gaa ggc aat cag aac cgg TTC GAA tct ctg gaa gaa


!                                          BstBI..


!


!       91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!       C   K   K   M   C   T   R   D   N   A   N   R   I   I   K 


 278   tgc aag aag atG TGC ACc agg gac aac gcc aac agg atc atc aag


!                    ApaLI...


!


!      106 107 108 109 110 111 112 113 114 115 116 117 118 119 120


!       T   T   L   Q   Q   E   K   P   D   F   C   F   L   E   E 


 323   acc acc ctg cag cag gag aag ccc gac ttc tgc ttc ctg gaa gaG


!                                                                BamHI..


!


!      121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!       D   P   G   P   C   R   A   R   F   D   R   W   F   Y   N 


 368   GAT CCt ggc ccc tgc aga gcc cgg ttt gac cgg tgg ttt tac aat


!    BamHI...


!


!       *


!      136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!       N   Q   T   K   Q   C   E   R   F   I   Y   G   G   C   E 


 413   aac cag acc aag cag tgt gAG CGC Ttt atc tat ggg gga tgt gaa


!                               AfeI....


!


!      151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!       G   N   M   N   N   F   E   T   L   E   E   C   K   N   I 


 458   gga aat atg aat aac ttc gag aca ctg gaa gag tgt aag aac atc


!


!      166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!       C   E   D   G   P   N   G   F   Q   V   D   N   Y   G   T 


 503   tgc gag gac ggc ccc aac ggc ttc cag gtg gac aac tac ggc acc


!


!                           *


!      181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!       Q   L   N   A   V   N   N   S   L   T   P   Q   S   T   K


 548   CAA TTG aac gcc gtg aac aac agc ctg acc ccc cag agc acc aag


!      MfeI...


!


!      196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!       V   P   S   L   F   E   F   H   G   P   S   W   C   L   T


 593   gtg ccc agc ctg ttc gag ttc cac ggc ccc agc tgg tgc ctg acc


!


!      211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!       P   A   D   R   G   P   C   R   A   R   F   D   R   W   Y 


 638   cca gcc gac agG GGC CCa tgc cgc gcc aga ttt gac agg tgg tac


!                    ApaI....


!


!      226 227 228 229 230 231 232 233 234 235 236 237 238 239 240


!       Y   N   S   V   I   G   K   C   R   P   F   I   Y   G   G


 683   tac aac tcc gtg atc ggc aaG TGC AGg ccc ttc atc tac ggc gga


!                                BsgI....


!


!                               *


!      241 242 243 244 245 246 247 248 249 250 251 252 253 254 255


!       C   E   G   N   E   N   N   F   T   S   K   Q   E   C   L


 728   tgt gag gga aac gag aac aac ttt ACT AGT aag cag gaa tgC CTG


!                                      SpeI...               Bsu36I..


!


!      256 257 258 259 260 261 262 263 264 265 266 267 268


!       R   A   C   K   K   G   F   I   Q   R   I   S   (SEQ ID NO: 135).


 773   AGG gcc tgc aag aag ggc ttc atc cag agg atc agc tga tga a


! Bsu36I..


!


 816   TCTAGAa  (SEQ ID NO: 134)


!      XbaI..
















TABLE 53





Vector encodingoptimized, tagged w.t. TFPI minus Cterminal region.















LOCUS      pFuse\with 5660 bp   DNA   circular               26 JAN. 2006


BASE COUNT    1376 a    1423 c     1421 g      1440 t


ORIGIN


(SEQ ID NO: 136)


    1  gtaccgaatt cacattgatt attgagtagt tattaatagt aatcaattac ggggtcatta


 


   61  gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc


 


  121  tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg


 


  181  ccaataggga ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg


 


  241  gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa


 


  301  tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac


 


  361  atctacgtgt tagtcatcgc tattaccata gtgatgcggt tttggcagta catcaatggg


 


  421  cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg


 


  481  agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca


 


  541  ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctttctgg


 


  601  ctaactagag aacccactgc ttactggcac gtggaaatta atacgacgtg gccaccatgg


 


  661  gatggagctg tatcatcctc ttcttggtcg cgacggccac aggggcccac tccgaaggtc


 


  721  gtccgggtca ccaccatcat catcatggcg gttctagtct ggtcccgcgt ggctctgagg


 


  781  aagatgaaga acacacaatt atcacagata cggagttgcc accactgaaa cttatgcatt


 


  841  cattttgtgc attcaaggcg gatgatggcc catgtaaagc aatcatgaaa agatttttct


 


  901  tcaatatttt cactcgacag tgcgaagaat ttatatatgg gggatgtgaa ggaaatcaga


 


  961  atcgatttga aagtctggaa gagtgcaaaa aaatgtgtac aagagataat gcaaacagga


 


 1021  ttataaagac aacattgcaa caagaaaagc cagatttctg ctttttggaa gaagatcctg


 


 1081  gaatatgtcg aggttatatt accaggtatt tttataacaa tcagacaaaa cagtgtgaac


 


 1141  gtttcaagta tggtggatgc ctgggcaata tgaacaattt tgagacactg gaagaatgca


 


 1201  agaacatttg tgaagatggt ccgaatggtt tccaggtgga taattatgga acccagctca


 


 1261  atgctgtgaa taactccctg actccgcaat caaccaaggt tcccagcctt tttgaatttc


 


 1321  acggtccctc atggtgtctc actccagcag acagaggatt gtgtcgtgcc aatgagaaca


 


 1381  gattctacta caattcagtc attgggaaat gccgcccatt taagtacagt ggatgtgggg


 


 1441  gaaatgaaaa caattttact tccaaacaag aatgtctgag ggcatgtaaa aaaggttaat


 


 1501  gatctagaag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt


 


 1561  tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa


 


 1621  taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg


 


 1681  gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatggc


 


 1741  ccgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac


 


 1801  gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct


 


 1861  acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg


 


 1921  ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt


 


 1981  gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca


 


 2041  tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga


 


 2101  ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa


 


 2161  gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac


 


 2221  gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag


 


 2281  gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc


 


 2341  ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata


 


 2401  gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg


 


 2461  cccctaggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag


 


 2521  ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg


 


 2581  ggaggtccac aatgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg


 


 2641  agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt


 


 2701  tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc


 


 2761  tgaatgaact ccaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt


 


 2821  gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag


 


 2881  tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg


 


 2941  ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag


 


 3001  cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg


 


 3061  atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc


 


 3121  gtatgcccga cggcgaggat ctcgtcgtga ctcatggcga tgcctgcttg ccgaatatca


 


 3181  tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc


 


 3241  gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg


 


 3301  ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct


 


 3361  atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc


 


 3421  gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg


 


 3481  cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct


 


 3541  ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa


 


 3601  tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc


 


 3661  caaactcatc aatgtatctt atcatgtctg tataccggat ctttccgctt cctcgctcac


 


 3721  tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt


 


 3781  aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca


 


 3841  gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc


 


 3901  ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact


 


 3961  ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct


 


 4021  gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg


 


 4081  ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtcca


 


 4141  cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa


 


 4201  cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc


 


 4261  gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag


 


 4321  aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg


 


 4381  tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca


 


 4441  gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc


 


 4501  tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag


 


 4561  gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata


 


 4621  tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat


 


 4681  ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg


 


 4741  ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc


 


 4801  tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc


 


 4861  aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc


 


 4921  gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc


 


 4981  gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc


 


 5041  ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa


 


 5101  gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat


 


 5161  gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata


 


 5221  gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca


 


 5281  tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag


 


 5341  gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgctccca actgatcttc


 


 5401  agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc


 


 5461  aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata


 


 5521  ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta


 


 5581  gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcag


 


 5641  atcgacggat cgggagatcg 
















TABLE 54





Vector for TFPI with plasmin, pKal, and hNe inhibition















LOCUS      TriKun\no\ 5660 bp   DNA   circular             19 DEC. 2005


BASE COUNT    1355 a    1442 c     1426 g      1437 t


ORIGIN


(SEQ ID NO: 137)


    1  gtaccgaatt cacattgatt attgagtagt tattaatagt aatcaattac ggggtcatta


 


   61  gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc


 


  121  tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg


 


  181  ccaataggga ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg


 


  241  gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa


 


  301  tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac


 


  361  atctacgtgt tagtcatcgc tattaccata gtgatgcggt tttggcagta catcaatggg


 


  421  cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg


 


  481  agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca


 


  541  ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctttctgg


 


  601  ctaactagag aacccactgc ttactggcac gtggaaatta atacgacgtg gccaccatgg


 


  661  gatggagctg tatcatcctc ttcttggtcg cgacggccac aggggcccac tccgaaggtc


 


  721  gtccgggtca ccaccatcat catcatggcg gttctagtct ggtcccgcgt ggttctgagg


 


  781  aagatgaaga acacacaatt atcacagata cggagttgcc accactgaag cttatgcatt


 


  841  cattttgtgc attcaaggcg gatgatggcc catgtagagc acgcttcgat agatggttct


 


  901  tcaatatttt cactcgacag tgcgaagaat ttatatatgg gggatgtgaa ggaaatcaga


 


  961  atcgatttga aagtctggaa gagtgcaaaa aaatgtgtac aagagataat gcaaacagga


 


 1021  ttataaagac aacattgcaa caagaaaagc cagatttctg ctttctcgag gaggatcctg


 


 1081  gaccgtgtcg agctgctcat cccaggtggt tttataacaa tcagacaaaa cagtgtgaac


 


 1141  gtttcatcta tggtggatgc gagggcaata tgaacaattt tgagacactg gaagaatgca


 


 1201  agaacatttg tgaagatggt ccgaatggtt tccaggtcga caattatgga acccagctca


 


 1261  atgctgtgaa taactccctg actccgcaat caaccaaggt tcccagcctt tttgaatttc


 


 1321  acggtccctc atggtgtctc actcccgcgg acagaggacc gtgtattgcc tttttcccca


 


 1381  gattctacta caattcagtc attgggaaat gccgcccatt tccgtatggc ggatgtcagg


 


 1441  gaaatgaaaa caattttact tccaaacaag aatgtctgag ggcatgtaaa aaaggttaat


 


 1501  gatctagaag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt


 


 1561  tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa


 


 1621  taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg


 


 1681  gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatggc


 


 1741  ccgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac


 


 1801  gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct


 


 1861  acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg


 


 1921  ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt


 


 1981  gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca


 


 2041  tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga


 


 2101  ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa


 


 2161  gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac


 


 2221  gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag


 


 2281  gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc


 


 2341  ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata


 


 2401  gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg


 


 2461  cccctaggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag


 


 2521  ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg


 


 2581  ggaggtccac aatgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg


 


 2641  agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt


 


 2701  tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc


 


 2761  tgaatgaact ccaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt


 


 2821  gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag


 


 2881  tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg


 


 2941  ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag


 


 3001  cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg


 


 3061  atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc


 


 3121  gtatgcccga cggcgaggat ctcgtcgtga ctcatggcga tgcctgcttg ccgaatatca


 


 3181  tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc


 


 3241  gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg


 


 3301  ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct


 


 3361  atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc


 


 3421  gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg


 


 3481  cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct


 


 3541  ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa


 


 3601  tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc


 


 3661  caaactcatc aatgtatctt atcatgtctg tataccggat ctttccgctt cctcgctcac


 


 3721  tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt


 


 3781  aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca


 


 3841  gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc


 


 3901  ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact


 


 3961  ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct


 


 4021  gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg


 


 4081  ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtcca


 


 4141  cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa


 


 4201  cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc


 


 4261  gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag


 


 4321  aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg


 


 4381  tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca


 


 4441  gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc


 


 4501  tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag


 


 4561  gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata


 


 4621  tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat


 


 4681  ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg


 


 4741  ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc


 


 4801  tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc


 


 4861  aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc


 


 4921  gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc


 


 4981  gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc


 


 5041  ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa


 


 5101  gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat


 


 5161  gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata


 


 5221  gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca


 


 5281  tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag


 


 5341  gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgctccca actgatcttc


 


 5401  agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc


 


 5461  aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata


 


 5521  ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta


 


 5581  gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcag


 


 5641  atcgacggat cgggagatcg 
















TABLE 55





Vector containing DXMMM















LOCUS      pFuse-dxmmm  5622 bp   DNA   circular            19 JUL. 2006


BASE COUNT    1321 a     1491 c     1446 g      1364 t


ORIGIN


(SEQ ID NO: 138)


    1  gtaccgaatt cacattgatt attgagtagt tattaatagt aatcaattac ggggtcatta


 


   61  gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc


 


  121  tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg


 


  181  ccaataggga ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg


 


  241  gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa


 


  301  tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac


 


  361  atctacgtgt tagtcatcgc tattaccata gtgatgcggt tttggcagta catcaatggg


 


  421  cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg


 


  481  agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca


 


  541  ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctttctgg


 


  601  ctaactagag aacccactgc ttactggcac gtggaaatta atacgacgtg gccaccatgg


 


  661  gatggtcctg catcatcctg tttctggtgg ccaccgccac aggcgcccac agcgacagcg


 


  721  aggaagatga ggaacacacc atcatcaccg acaccgagct gccccccctg aagcttatgc


 


  781  acagcttctg cgccttcaag gccgacgacg gcccctgcag ggccagattc gacagatggt


 


  841  tcttcaacat cttcaccagg cagtgcgagg aattcattta tggaggctgt gaaggcaatc


 


  901  agaaccggtt cgaatctctg gaagaatgca agaagatgtg caccagggac aacgccaaca


 


  961  ggatcatcaa gaccaccctg cagcaggaga agcccgactt ctgcttcctg gaagaggatc


 


 1021  ctggcccctg cagagcccgg tttgaccggt ggttttacaa taaccagacc aagcagtgtg


 


 1081  agcgctttat ctatggggga tgtgaaggaa atatgaataa cttcgagaca ctggaagagt


 


 1141  gtaagaacat ctgcgaggac ggccccaacg gcttccaggt ggacaactac ggcacccaat


 


 1201  tgaacgccgt gaacaacagc ctgacccccc agagcaccaa ggtgcccagc ctgttcgagt


 


 1261  tccacggccc cagctggtgc ctgaccccag ccgacagggg cccatgccgc gccagatttg


 


 1321  acaggtggta ctacaactcc gtgatcggca agtgcaggcc cttcatctac ggcggatgtg


 


 1381  agggaaacga gaacaacttt actagtaagc aggaatgcct gagggcctgc aagaagggct


 


 1441  tcatccagag gatcagctga tgaatctaga agctcgctga tcagcctcga ctgtgccttc


 


 1501  tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc


 


 1561  cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg


 


 1621  tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa


 


 1681  tagcaggcat gctggggatg gcccgggctc tatggcttct gaggcggaaa gaaccagctg


 


 1741  gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt


 


 1801  ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt


 


 1861  cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcggggcat


 


 1921  ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg


 


 1981  tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga


 


 2041  gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc


 


 2101  ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt taaaaaatga


 


 2161  gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt


 


 2221  ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc tcaattagtc


 


 2281  agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca


 


 2341  tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc


 


 2401  gcccagttcc gcccattctc cgcccctagg ctgactaatt ttttttattt atgcagaggc


 


 2461  cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct


 


 2521  aggcttttgc aaaaagctcc cgggaggtcc acaatgattg aacaagatgg attgcacgca


 


 2581  ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc


 


 2641  ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc


 


 2701  aagaccgacc tgtccggtgc cctgaatgaa ctccaggacg aggcagcgcg gctatcgtgg


 


 2761  ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg


 


 2821  gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct


 


 2881  gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct


 


 2941  acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa


 


 3001  gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa


 


 3061  ctgttcgcca ggctcaaggc gcgtatgccc gacggcgagg atctcgtcgt gactcatggc


 


 3121  gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt


 


 3181  ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct


 


 3241  gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc


 


 3301  gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg


 


 3361  ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg


 


 3421  ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc


 


 3481  tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt


 


 3541  ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac


 


 3601  tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgg


 


 3661  atctttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg


 


 3721  tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa


 


 3781  agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg


 


 3841  cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga


 


 3901  ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg


 


 3961  tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg


 


 4021  gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc


 


 4081  gctccaagct gggctgtgtc cacgaacccc ccgttcagcc cgaccgctgc gccttatccg


 


 4141  gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca


 


 4201  ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt


 


 4261  ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag


 


 4321  ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg


 


 4381  gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc


 


 4441  ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt


 


 4501  tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt


 


 4561  ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca


 


 4621  gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg


 


 4681  tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac


 


 4741  cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg


 


 4801  ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc


 


 4861  gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta


 


 4921  caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac


 


 4981  gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc


 


 5041  ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac


 


 5101  tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact


 


 5161  caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa


 


 5221  tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt


 


 5281  cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca


 


 5341  ctcgtgctcc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa


 


 5401  aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac


 


 5461  tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg


 


 5521  gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc


 


 5581  gaaaagtgcc acctgacgtc agatcgacgg atcgggagat cg 
















TABLE 56







AA sequence of hTFPImC1 (SEQ ID NO: 139)









Item
Extent
AA sequence





Signal
 1-19
MGWSCIILFLVATATGAHS


 


Cleaveage
20-24
EGRPG


 


His tag
25-30
HHHHHH


 


Thrombin
31-39
GGSSLVPRG


 


Linker 1
40-59
SEEDEEHTIITDTELPPLKL


 


KuDom 1
 60-117 
MHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD


 


Linker 2
118-130
NANRIIKTTLQQE


 




                        *


KuDom 2
131-188
KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG


 




                *


Linker 3
189-222
PNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFEFH


 




                                           *


KuDom 3
223-280
GPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG
















TABLE 57







AA sequence of hTFPI[PpKhNE] (SEQ ID NO: 140)









Function
Limits
AA sequence





Signal
 1-19
MGWSCIILFLVATATGAHS


 


Cleavage
20-24
EGRPG


 


His6
25-30
HHHHHH


 


Thrombin
31-39
GGSSLVPRG


 


Linker 1
40-59
SEEDEEHTIITDTELPPLKL


 


KuDom 1
 60-117
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD




              K IMK F  <- wild type


 


Linker 2
118-130
NANRIIKTTLQQE


 




                         *


KuDom 2
131-188
KPDFCFLEEDPGPCRAAHPRWFYNNQTKQCERFIYGGCEGNMNNFETLEECKNICEDG




            I  GYIT  Y            K    L   <- wild type


 




                *


Linker 3
189-222
PNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFEFH


 




                                           *


KuDom 4
223-280
GPSNCLTPADRGPCIAFFPRFYYNSVIGKCRPFPYGGCQGNENNFTSKQECLRACKKG




            L R NEN              K S  G   <- wild type
















TABLE 58







Amino-acid sequence of PLA301B (SEQ ID NO: 141)









Function
Limits
AA sequence





Signal
  1-19
MGWSCIILFLVATATGAHS


Linker 1
 20-40
DSEEDEEHTIITDTELPPLKL


 


KuDom 1
 41-98
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD




              K IMK F   <- wild type


 


Linker 2
 99-111
NANRIIKTTLQQE


 


KuDom 2
112-169
                        *




KPDFCFLEEDPGPCRARFDRWFYNNQTKQCERFIYGGCEGNMNNFETLEECKNICEDG




            I  GYIT Y            K  <- wild type


 


Linker 3
170-203
                *




PNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFEFH


 


KuDom 3
204-261
                                           *




GPSWCLTPADRGPCRARFDRWYYNSVIGKCRPFIYGGCEGNENNFTSKQECLRACKKG




            L   NEN F            K S  G   <- wild type


 


Linker 4
262-267
FIQRIS
















TABLE 60







Distributions of AA types in 103 KuDoms, including 19 human KuDoms









Res.
Different



Id.
AA types
Contents












−5
12
−58 21 T4 E4 S4 Q3 R3 P2 L D A K


 


−4
14
−56 21 A4 P3 D3 L3 F2 V2 W2 E2 N2 Q S G


 


−3
15
−52 20 Q5 K5 P5 E4 T2 F2 L2 A R D V M G


 


−2
16
−33 19 Q7 E7 K7 P5 A5 R4 F3 T3 H2 W2 G2 L2 S Y


 


−1
17
−24 19 R11 D7 Q6 T5 K5 P5 N4 V4 G3 E3 H2 L2 Z C S


 


 1
18
R31 K15 T12 V6 −6 G5 D4 I3 S3 A3 L3 P3 H2 Y2 M2 F N E


 


 2
18
P40 E11 R8 H7 V7 A6 K5 Q4 N3 D2 I2 −2 L M S T G F


 


 3
15
D37 S11 K9 E9 A8 T7 P6 R5 G4 Y2 L Q W N —


 


 4
13
F37 A12 I12 V8 S7 L6 Y6 D5 W3 P3 H2 M K


 


 5
3






 


 6
16
L25 N12 E11 S10 Q7 K7 AS I4 R4 F4 Y4 T3 D3 G2 V M


 


 7
13






 


 8
13






 


 9
16
P30 A17 K10 I8 V8 L6 Y6 E4 R4 F3 M2 Q H S N D


 


  9a
6
−96 G3 P Q S D


 


10
13
D23 E19 V15 Y15 R7 S7 N6 A5 K2 I Q W M


 


11
14
T31 Q12 P12 V8 R7 S6 E5 I4 K4 D4 A4 Y3 G2 H


 


12
6






 


13
11






 


14
1






 


15
14






 


16
12






 


17
18
R17 Y15 F13 S10 M10 K7 N6 H6 G4 L3 A2 P2 T2 Q2 D I V E


 


18
12






 


19
14
P23 I16 K13 R12 L8 S8 T7 Q6 E4 V2 H N F G


 


20
10






 


21
8






 


22
8






 


23
4






 


24
7






 


25
16
A31 P10 S10 G7 Q6 L6 N5 V5 W5 F4 T4 K3 R3 I2 D M


 


26
16
K27 A13 T13 V9 E8 R7 S7 Q5 D3 N2 I2 F2 Y2 H L G


 


 26a
3
−101 T N


 


 26b
2
−101 G2


 


 26c
4
−100 V Y S


 


27
12
A25 S24 T23 K9 Q5 L4 E4 13 D3 N R Y


 


28
10
G39 K23 N11 M9 H5 Q5 R5 E3 S2 Y


 


29
14
K31 Q19 L14 A11 R6 S6 T6 E2 F2 G2 H I M N


 


30
3






 


31
16






 


32
13
P21 T18 R11 L10 Q10 S8 E8 K6 A3 N2 G2 M2 V2


 


33
2






 


34
17
I24 V22 T10 K9 F6 N5 Q5 H4 L4 D3 W3 E2 P2 A R S Y


 


35
7






 


36
5






 


37
2






 


38
1






 


39
12
G32 R21 Q12 K8 E7 L7 M6 D5 P2 N H Y


 


40
5






 


41
3






 


42
12
A23 R17 G16 S10 E7 D7 N6 K5 H4 Q4 L2 M2


 


 42a
4
−100 N T I


 


 42b
3
−101 G E


 


 42c
2
−102 A


 


43
5






 


44
8






 


45
5






 


46
17
K31 E17 Y11 D7 V6 L6 S4 G4 R4 T3 Q2 N2 P2 I A H M


 


47
8






 


48
13
E21 I16 L16 A13 Q11 K8 R7 D3 W3 T2 V S Y


 


49
13






 


50
11






 


51
3






 


52
13
R26 M22 L15 K13 E11 Q5 N3 I2 F H D S V


 


53
15
R34 Q13 E13 K9 N8 A6 H4 C3 T3 G2 L2 D2 W2 M S


 


54
13
T45 A13 Y10 V7 K6 I5 F5 R5 E3 S L Q C


 


55
4






 


56
16
G32 R15 V12 I9 E7 K7 A6 S3 L3 P2 M2 H F D W —


 


57
16
G30 V11 T9 P8 A7 K6 −6 N5 D4 R4 S4 L3 I3 Q Y F


 


58
18
−23 A18 P13 V11 K7 G6 I4 R3 Y3 S3 N2 E2 Q2 D2 T M F C


 


59
17
−43 G11 A9 9 S4 E4 14 V3 Y3 F2 K2 P2 Q2 R2 N T L


 


60
16
−45 11 A6 D6 P5 I5 K5 G4 E4 N3 V3 S2 T Q R C


 


61
13
−53 11 G8 E5 P5 Q4 K4 A4 S2 T2 D2 R2 V


 


62
19
−53 12 D8 P4 R3 S3 K3 G3 Y2 E2 T2 A L M V W N I Q


 


63
13
−49 22 E10 Q3 R3 S3 G3 K3 I2 T2 V L P


 


64
12
−50 27 E6 S3 V3 K3 N3 I2 R2 L2 T D
















TABLE 61







PlaMatHep, a Modified Human TFPI inhibiting Plasmin, Matriptase, and


Hepsin (SEQ ID NO: 142)









AAs
Function
AA sequence





 1-21
Linker 1
DSEEDEEHTIITDTELPPLKL


 


22-79
KuDom 1
MHSFCAFKADDGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD



Inhibit




plasmin



 


80-92
Linker 2
NANRIIKTTLQQE


 


 93-150
KuDom 2
kpdfcfleedpgRcrgSFPrWfynnqtkqcKSfVyggclgnmnnfetleecknicedg



Inhibit




matriptase



 


151-184
Linker 3
PNGFQVDNYGTQLNAVnNSLTPQSTKVPSLFEFH


 


185-242
KuDom 3
gpswcltpadVgRcraSMPrWyynsvigkcrpfVyGgcggnSnnftsEqeclrackkg



Inhibit
1   5    1    1    2    2    3    3    4    4    5    5  5



hepsin
         0    5    0    5    0    5    0    5    0    5  8


 


243-248
Linker 4
FIQRIS





Possible sites of glycosylation are shown as “n”.






Other embodiments are within the following claims.

## Claims

1. An isolated protein comprising two to six engineered Kunitz domain sequences, wherein the protein comprises the amino acid sequence of SEQ ID NO:11, 54, 70, 71, 72, 73, 74, 102, 106, 107 or 127.

2. The isolated protein of claim 1, wherein the protein does not include a serum albumin moiety.

3. The protein of claim 1, wherein the protein comprises three Kunitz domains.

4. The protein of claim 1, wherein the protein comprises four Kunitz domains.

5. The protein of claim 1, wherein the protein comprises five Kunitz domains.

6. The protein of claim 1, wherein the protein comprises six Kunitz domains.

7. The protein of claim 1, wherein the protein is modified with two or more PEG moieties.