JP2003517269A - A novel serine protease capable of selectively cleaving insulin-like growth factor binding protein - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel nucleic acids and proteins having sequence identity to the ASP05 protease, and to the use of the compositions in assays and in the diagnosis and treatment of diseases.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION [0001] The present invention relates to novel serine proteases ("A" that are capable of selective cleavage of insulin-like growth factor binding protein ("IGFBP"), including nucleic acids, proteins and antibodies.
"SP05") and compositions. The present invention also provides an IG
It relates to the use of the novel ASP05 protease in an assay for the selective cleavage of FBP and a method of screening for modulators of this enzyme. The present invention relates to the use of antibodies against the novel ASP05 protein in the diagnosis of disease.

BACKGROUND OF THE INVENTION Insulin-like growth factor binding protein (IGFBP) plays an important role in IGF biology. The cellular protease that selectively cleaves IGFBP is
Mediates these activities. This proteolysis is known to alter the affinity of IGFBP for IGF and has been reported to agonize or antagonize IGF function in various systems.

Insulin-like growth factor-1 (IGF-I) and IGF-II are synthesized and secreted from various cells and act as autocrine and paracrine growth factors,
Regulates the differentiation, proliferation and function of many types of cells. Some of these cells are from the brain, blood, bone, liver, muscle, kidney and nervous system (Jones, J.
I et al. Endocrine Reviews 16 3-34 (1995)).

[0004] The IGF binding protein (IGFBP) is an essential component of the IGF system; by tightly binding to the IGF molecule, the IGF binding protein alters its bioavailability to target cells (Jones et al, supra, Cohen et al. al., Horm. Metab. Res
. 26 81-84 (1994)). The IGFBP family consists of 6 different proteins called IGFBP-1 to -6, each with high binding affinity for both IGF-1 and IGF-2 (Cohen et al. Supra, Oh et al. . Biol. Ch
em. 271 30322-30325 (1996), Kim et al. Proc. Natl. Acad. Sci. USA, 94 12
981-12986 (1997)). The amino acid sequence of IGFBP shows 40-60% similarity and encodes 11-18 highly conserved cysteine residues. Among the members of the IGFBP family, the most recently identified IGFBP7 has the least amino acid sequence similarity and the least conserved cysteine residues (Oh et al. Supra, Kim et al. Supra).

IGFBP metabolism affects the interaction with IGF and therefore alters IGF action. There are numerous reports describing the identification of proteases that cleave IGFBPs. Reportedly, IGFBP-1 is a potential physiological substrate for the matrix metalloproteinase stromelysin-3 (Manes et al. J. Biol.
Chem., 272 25706-25712 (1997); Protease activity against IGFBP-3 was revealed in human breast cancer cells MCF-7 (Salahifar et al., Endocr.
inology 138 1683-1690 (1997)); IGFBP-4 was found to be degraded in normal and transformed human fibroblasts (Conover et al.
al., J. Clin. Invest. 91 1129-1137 (1993)); and specific protease activity against IGFBP-3 and IGFBP-4 was found in sera from patients after major cardiac surgery (Hughes. et al., J. Endocrinology 135 135-145
(1992)). Human fibroblasts have been identified as a source of proteases that cleave IGFBP-5 (Nam et al., Endocrinology 135 1385-1391 (1994) Nam et al., En.
docrinology 137 5530-5536 (1996)). Proteases degraded IGFBP in the 150 kD complex during pregnancy, and it was also shown that the plasmin system is involved in dissociation of the IGFBP complex (Giudice et al., J. Clin. Endocrinology).
and Metabolism 71 806-816 (1990), Campbell et al., Am. J. Physiol. 273.
E996-E1004 (1997)). All of these reports show that IGFBP proteolysis
It has been shown to be an essential way to control the availability of GFBP and thereby regulate the function of the IGF system.

Despite a lot of work being done in this area over the last few years,
The identification of proteases that undergo such cleavage remains unclear. The sequence of ASP05 protease was also recently published by others (Japanese Application No. 9-1007).
980). However, attempts to express ASP05 protein from natural nucleic acids have been unsuccessful (Zumbrunn, J. & Trueb, B. FEBS Letters 398: (1996) p.
p.187-192).

Described herein is the identification and characterization of the IGFBP-specific serine protease “ASP05”. ASP05 is a member of the serine protease family and specifically cleaves IGFBPs. Illustrative, biologically active, IG
A nucleic acid sequence expressing a protein capable of specific cleavage of FBP (referred to as "ASP05") is displayed.

Accordingly, one object of the present invention is to provide ASP05, enzymatically active ASP05 and related molecules. Furthermore, an object of the present invention is to provide a recombinant nucleic acid encoding ASP05, as well as an expression vector and a host cell containing the nucleic acid encoding ASP05 and the like. Yet another object of the present invention is to provide a method of screening ASP05 protease and its antagonists.

SUMMARY OF THE INVENTION Consistent with the above objectives, the present invention provides the production of nucleic acids encoding IGFBP-specific serine proteins. This polypeptide is referred to as “ASP05” in this application.
Say

In one aspect, the invention provides an isolated nucleic acid molecule comprising DNA encoding ASP05. The present invention also provides ASP05 proteins and fragments thereof.

The present invention comprises IGFB comprising adding ASP05 protease to IGFBPs.
A method for P cleavage is provided. The present invention further provides a biologically active substance capable of inhibiting ASP05, a screening method for ASP05-like protein, and a cleavage method for IGFBP.

[0012] In another aspect, the invention provides a method of modulating cleavage of IGFBPs in a cell. This method involves providing cells with an exogenous compound that binds to a serine protease that is capable of selective cleavage of IGFBPs. This binding regulates the biological activity of the serine protease.

In another aspect, the invention provides a method of measuring the presence of ASP05 protease in an individual. The activity of the ASP05 in the tissue derived from the first individual is measured,
It can be compared to the activity of the ASP05 in a tissue from a second non-infected individual or a second tissue from a first individual. The first individual is associated with insulin metabolism when the activity of the ASP05 from the first individual is modified or different as compared to the ASP05 activity in tissue from the second individual or in the second tissue. You are at risk of disease.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a serine protease (“ASP05” that can selectively cleave IGFBPs.
Is designated as "."), Nucleotides 1-1443 (SEQ ID NO: 1) and amino acid sequences 1-480 (SEQ ID NO: 2). The nucleotides modified to express ASP05 are underlined. Arrows indicate signal cleavage sites. The IGFBP-like domain is underlined and the PSTI domain is double-lined. The catalysis domain is boxed and the histidine (H), aspartate (D) and serine (S) residues required for catalysis in the domain are circled. 2A and B show the nucleic acid sequence encoding the native protein (top; SEQ ID NO: 3).
) And a nucleic acid sequence encoding the synthetic ASP05 protease (bottom).
The modified nucleotides of the synthetic ASP05 protease are underlined. The amino acid sequences encoded by both sequences are also noted. FIG. 3 shows the evaluation results of the self-cleaving activity of ASP05. Western blot analysis of purified ASP05 subjected to SDS-PAGE, detection of anti-ASP05 peptide antibody bound to HRP-conjugated rabbit IgG antibody by visualization with a chemifluorophore.
Lane 1: protein purified by flag antibody affinity chromatography (0.5 μg) was subjected to 4-20% SDS-PAGE and stained with Commassie blue; lanes 2 and 3: 4-20 same amount of sample as lane 1. % SDS-P
It was subjected to AGE and blotted with anti-ASP05 peptide antibody. The sample in lane 2 was not incubated at 37 ° C and the sample in lane 3 was incubated for 15 minutes at 37 ° C before loading the gel ST: molecular weight standard protein marker.

FIG. 4A shows the selective cleavage of IGFBP-5. IGFBP-1 to -6 A
Incubated with SP05 and analyzed by Western blot with each antibody. "-" Is control without ASP05, "+" indicates with ASP05. FIG. 4B shows the selective cleavage of IGFBP-5. IGFBP-1 to -6 A
Incubated with SP05 and analyzed by Western Ligand Blot with ¹²⁵ 1-IGF-I. BP1-6 represents IGFBP1-6, respectively.
"IGFBP1-6 without ASP05 and" + "IGF with ASP05
BP1-6 is shown. FIG. 5 shows the effect of protease inhibitors on the degradation of IGFBP-5 by ASP05. ASP05 was incubated with protease inhibitors for 1 hour before the addition of IGFBP-5 and the products analyzed by Western blot with the IGFBP-5 antibody. Lane 1: No ASP05; Lane 2: AS
With P05; Lane 3: Aprotinin, 1.5 μM; Lane 4: Antipain,
370 μM; Lane 5: 3,4-DCI, 1 mM; Lane 6: E64, 140 μM;
Lane 7: Leupeptin, 5 μM; Lane 8: Pepstatin A, 5 μM; Lane 9
: PMSF, 5 mM; Lane 10: TLCK, 675 μM; Lane 11: TPCK
, 1.4 mM; Lane 12: EDTA, 10 mM. FIG. 6 shows the nucleotide sequence of the C-terminal domain of ASP05 and histidine tag useful for purification as shown in the examples. FIG. 7 shows the amino acid sequence of ASP05 C-terminal domain and histidine tag. FIG. 8 shows the selective cleavage of IGFBP-5 by the C-terminal fragment of ASP05.
IGFBP-1 to -6 were each incubated with the fragment for 2 hours at 37 ° C,
Analyzed by Western blot using the antibody shown at the top of each blot.
"-" Indicates samples that were not treated with fragments, "+" indicates samples that were treated with fragments.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel IGFBP-specific serine protease (called ASP05) capable of specifically cleaving IGFBP. The ASP05 protease of the present invention can be produced by recombinant or synthetic methods and isolated from a variety of sources. In particular, the wild-type ASP05 nucleotide sequence contains a GC-rich N-terminus,
Makes protein expression difficult. Therefore, the present invention reduces the G-C content,
In particular, ASP05 nucleic acids are provided that reduce the GC region at the N-terminal portion of the nucleic acid coding sequence. It completes the expression of functional (ie enzymatically active) ASP05. Accordingly, the present invention provides ASP05 proteins and nucleic acids.

The ASP05 protein of the invention may be identified in several ways. In this sense, “protein” includes proteins, polypeptides and peptides.
ASP05 nucleic acids or ASP05 proteins are first identified with substantial nucleic acid and / or amino acid sequence homology to the sequences shown in FIG. Such homology may be based on the entire nucleic acid or amino acid sequence.

Homology as used herein refers to sequence similarity or identity, with identity being preferred. Known standard techniques are used to measure this homology. For example, the Best Fit sequence program (Devereux et al., Nucl. Acid Res. 12: 387-395 (1984)), preferably the idle setting or BLASTX program (Altschul et al., J. Mo.
Biol. 215, 403-410). Equilibrium alignment involves the introduction of gaps in the sequences to be aligned. Furthermore, for sequences containing more or less amino acids than the proteins shown in Figures 1, 2 and 7, the homology is determined based on the number of homologous amino acids compared to the total number of amino acids. For example, as described below, homology for sequences shorter than that shown in the figure is determined using the number of amino acids in the short sequence.

For the measurement of similarity, the algorithm of Smith & Waterman (Adv.Appl.Math.2: 482 (1981)), the algorithm of Needleman & Wunsch (J.Mol.Biol.1970.48: 443), Pe.
arson & Lipman (1988.PNAS USA 85: 2444) similarity search method, computerized implementation of these algorithms (GAP, BESTFIT, FAST)
A and TFASTA, Wisconsin Genetics Software Package, Genetics Comp
uter Group, 575 Science Drive, Madison, WI), or known standard techniques, including but not limited to the Best Fit sequence program (described in Devereux et al. Nucl. Acid Res. 1984.12: 387-395). is not.

In a preferred embodiment, the percent identity or similarity is calculated by FastDB based on parameters such as a mismatch mismatch penalty of 1.0, a gap size penalty of 0.33, an association penalty of 30.0 (“ Current Methods in
Comparison and Analysis ”, Macromolecule Sequencing and Synthesis, Selec
ted Methods and Applications, pp127-149, 1998, Alan R. Liss, Inc.).

Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence equilibrium alignment from a group of related sequences using a progressive pairwise equilibrium alignment. It can also plot a tree showing the cluster relationships used to create the equilibrium alignment. In PILEUP, Feng and Doolittle (J. Mol. Evol.1987.35: 351-36
The simplification method of the progressive equilibrium alignment method of 0) is used. This method is based on Higgins and Shar.
Similar to the method reported by p. (1989.CABIOS 5: 151-153). Useful P
The ILEUP parameters have a default gap weight of 3.00, a default gap length weight of 0.10, and a final gap weighed.

Yet another example of a useful algorithm is Altschul et al. J. Mol. Biol. 1990.21.
5: 403-410 and BLAS described by Karlin et al., PNAS USA. (1993) 90: 5873-5787.
The T algorithm. A particularly useful BLAST program is Altschul et al.,
Methods in Enzymology. 1996.266: 460-480; [http: //blast.wustl/edu/blast/R
EADME.html] is a WU-BLAST-2 program. WU-
BLAST-2 uses several search parameters and sets most of them to default. Adjustable parameters are set with values such as overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself, which depends on the composition of the particular sequence and composition of the particular database in which the sequence of interest is searched. However, these values can be adjusted to increase sensitivity.

In another aspect, the percent amino acid sequence identity is measured manually. Percent identity calculations do not assign relative weights to sequence alterations, such as various expressions of insertions, deletions, substitutions, etc. By assessing only the identity positively (+1) and giving a value of "0" for all forms of sequence modification, the need for weighing scales or parameters described above for sequence similarity calculations is avoided. Therefore, percent identity represents a very precise method of sequence comparison.

Percent sequence identity can be calculated, for example, by dividing the number of identical residues paired by the total number of residues of the “short” sequence in the aligned region, and multiplying by 100. "Long" sequences are those that have mostly the actual residues in the alignment region.

The protein used herein is preferably (i) whose IGFBP binding domain corresponds to amino acids 30 to 104 of the sequence (SEQ ID NO: 2) shown in FIG.
If it has a general sequence identity of greater than or equal to 65%, typically greater than or equal to 80%, preferably greater than or equal to 90%, and / or (ii) its catalytic domain has the sequence shown in Figure 1 (SEQ ID NO: 2). 65% or more, typically 8 for amino acids 161-371 of
In the case of having a general sequence identity of 0% or more, preferably 90% or more, "ASP0
5 protein ”. In some variations, the sequence identity is about 90-95 or 98.
It can be as high as%.

The ASP05 protein can be identified by the presence of one or more important domains including the catalytic domain, the IGFBP domain, the PSTI-like domain and the PDZ-like domain, the last three of which are based on homology with known proteins. Can be identified.

In a preferred embodiment, the ASP05 protease comprises a serine protease catalytic domain. Expression of such catalytic domain produces a functional ASP05 protease. This domain contains amino acid 161 of the sequence shown in FIG.
Corresponding to ~ 371, it has a putative active site containing a histidine at position 220, an aspartic acid at position 250 and a serine at position 328. Thus, in a preferred embodiment, the ASP05 protein may be identified by at least about 65% identity to amino acids 161-371 of the sequence of Figure 1, and even at least about 8%.
0% is preferred and at least about 90% is especially preferred. In some embodiments, the identity may be as high as 90% -95% or 98%.

In a preferred embodiment, ASP05 protein may be identified by the enzymatic activity associated with the catalytic domain. That is, the present invention provides enzymatically active ASP05. Here, "enzymatic activity" means that ASP05 selectively cleaves IGFBP-5. Here, "selectively cleaves" means that IGFBP-5 is generally
It means that GFBP is cleaved under conditions that do not substantially cleave it. IGFBP-
A representative assay method for 5-cleavage is given in the examples.

In a preferred embodiment, ASP05 protein may be identified as comprising an N-terminal domain associated with IGFBPs. This domain corresponds to amino acids 30-104 of the sequence shown in FIG. That is, in a preferred embodiment, the ASP
05 protein is at least about 65% with amino acids 30-104 of the sequence of FIG.
They can be identified by their identity, with at least about 80% preferred, and at least about 90% especially preferred. In some variations, the identity is 90% to 95% or 9
It can be as high as 8%.

In a preferred embodiment, the ASP05 protein comprises a pancreatic secretory trypsin inhibitor (PSTI) -like domain. This PSTI-like domain corresponds to amino acids 105-160 of the sequence shown in FIG. That is, in a preferred embodiment, AS
The P05 protein also comprises a PSTI-like domain having at least about 65% identity with amino acids 105-160 of the sequence of Figure 2, where identity is preferably at least about 80%, particularly preferably at least about 90%. In some aspects,
Homology may be as high as 93% to 95% or 98%.

The ASP05 protein of the present invention may be shorter or longer than the amino acid sequence shown. That is, in a preferred embodiment, parts or fragments of the indicated sequences are also included within the definition of ASP05 protein. As outlined here,
ASP05 deletion mutants can be produced including, but not limited to, deletions of amino acids 106-480 and 1-105. A preferred ASP05 fragment is the catalytic domain of ASP05, as shown here, which selectively cleaves IGFBPs. A further preferred ASP05 fragment is the binding domain of ASP05,
5 Required for regulation of activity. A further preferred ASP05 fragment is the binding domain of ASP05, which binds to the catalytic domain of ASP05. More desirable AS
The P05 fragment is the PSTI-like protease inhibitory domain of ASP05
Required for regulation of P05 proteolytic activity. A further preferred ASP05 fragment is the C-terminal PDZ-like domain of ASP05. Fragments include the binding of either domain, and preferred embodiments may include the catalytic domain and the binding domain, at least the catalytic domain.

As further outlined herein, ASP05 fusion proteins can be produced, including but not limited to, heterologous sequences of amino acids 160-480, such as fusions to proteins such as signal sequences or purification tags. . An additional preferred fusion protein comprises the catalytic domain of ASP05 fused to a heterologous sequence domain.

In a preferred embodiment, the ASP05 protein is a derivative or variant of AS
P05 protein. Thus, as outlined in more detail below, the derivative ASP05 peptides contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. Amino acid substitutions, insertions or deletions,
It can occur at any residue within the ASP05 peptide. As outlined below, particularly preferred substitutions are made within the catalytic or binding domain of the ASP05 protein.

Furthermore, as more fully outlined below, the ASP05 protein may be produced as longer than that shown in the figure, eg by the addition of epitopes or purification tags, the addition of other fusion sequences, etc. .

In a preferred embodiment, the ASP05 protein is also an ASP05 protein as described below.
05 nucleic acid can be identified as encoded. That is, ASP05
The protein is encoded by a nucleic acid that hybridizes to the sequence shown in Figure 1 or its complement, as outlined herein.

In a preferred embodiment, when ASP05 protein is used for antibody production, ASP05 protein
The 05 protein must share at least one epitope or antigenic determinant with the full-length protein shown in FIG. By "epitope" or "antigenic determinant" herein is meant a portion of a protein that produces and / or binds an antibody. In most cases, antibodies raised against the small ASP05 protein will be able to bind the full length protein. In a preferred embodiment, the epitopes are unique, ie, antibodies raised against the unique epitope show little or no cross-reactivity. In a preferred embodiment, the production of antibody is A
This is done for the catalytic portion of the SP05 molecule, ie all or part of the region of amino acid numbers 106-480.

In a preferred embodiment, the antibody to ASP05 is A, as described below.
It may reduce or eliminate the biological function of SP05. That is, addition of anti-ASP05 antibody (polyclonal or preferably monoclonal) to cells or body fluids containing ASP05 protein may reduce or eliminate ASP05 activity. For example, cleavage of IGFBPs may be reduced or eliminated. That is, when ASP05 catalytic function is reduced or eliminated, all or some portion of IGFBP remains intact. Generally, at least a 20% reduction in activity is preferred, at least about 50% is particularly preferred, at least about 75% is more preferred and about 95-
A 100% reduction is especially preferred.

The ASP05 antibody of the present invention specifically binds to the ASP05 protein. Here, "specifically binds" means that the antibody binds to the protein with a binding constant of at least 10 ^-6 -10 ^-8 mol, preferably 10 ^-7 -10 ^-9 mol. .

In the case of nucleic acids, the overall homology of the nucleic acid sequences is equivalent to the amino acid homology, but the degeneracy in the genetic code and codon offset of different organisms should be taken into account. Thus, the homology of nucleic acid sequences can be lower or higher than the homology of protein sequences.

Regarding nucleic acid similarity, for example, BLASTN (Altschul et al. 1990. J. Mo.
l.Biol. 147: 195-197). BLASTN uses a simple scoring system that rates a proper match as +5 and a mismatch as -4. To achieve computational effectiveness, the lazy parameters are directly incorporated into the source code.

That is, (i) the nucleotide sequence encoding the IGFBP binding domain is 75% or more, typically 85% or more, with respect to nucleotides 90 to 314 of the polynucleotide sequence (SEQ ID NO: 1) shown in FIG. 1, preferably Has 95% or more overall sequence identity, and / or (ii) the nucleotide sequence encoding the catalytic domain is nucleotides 481-48 of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 2).
A nucleic acid is an "ASP05 nucleic acid" if it has a general sequence identity of greater than 75%, typically greater than 85%, preferably greater than 95% with 1113. In some embodiments, the identity may be as high as about 90-95 or 98%.

Accordingly, a preferred embodiment is the insulin-like growth factor binding protein (IGFBP
) A recombinant nucleic acid encoding a degrading protease, which comprises (i) a catalytic domain of an IGFBP degrading protease having a sequence identity of greater than 65% to amino acids 161-371 of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2). The encoding nucleic acid and / or (ii) amino acids 30 to 1 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2)
A nucleic acid encoding the IGFBP binding domain of an IGFBP-degrading protease having greater than 65% sequence identity to 04, and (iii) the first approximately 500 nucleotides of the nucleotide sequence of the nucleic acid having a total GC content of less than 75%. provide. In some embodiments, the identity may be as high as about 90-95 or 98%.

As outlined herein, wild-type or native nucleic acid sequences have an N-terminal GC-rich region. This has been shown to complicate expression. For example, Zumb
runn et al. (supra) report that their biological activity could not be confirmed because they could not express the ASP05 protein. The present invention allows the production of ASP05 proteins and especially enzymatically active proteins by reducing the G-C content of the gene.

Thus, in a preferred embodiment, in the case of nucleic acids, comparing the nucleotide sequence for ASP05 with the native nucleic acid sequence of FIG. 2, there is preferably a greater than 10% reduction in GC content and a greater than 20% reduction over the entire coding sequence. Is preferred,
Furthermore, reductions of more than 30% are particularly preferred. In a preferred embodiment, the reduction is in the N-terminal region of the gene. In addition, the nucleic acid consists of approximately the first 500 nucleotides of the nucleotide sequence.
An ASP05 nucleic acid having a total GC content of less than 75% nucleotides, typically less than 60% and preferably less than 50%.

In a preferred embodiment, the ASP05 nucleic acid encodes the ASP05 protein. As is recognized in the art, due to the degeneracy of the genetic code, very large numbers of nucleic acids can be produced, all of which encode the ASP05 protein of the invention. That is, once a specific amino acid sequence is identified, one skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of ASP05. .

In one aspect, nucleic acid identity is measured through hybridization tests. That is, for example, a nucleic acid that hybridizes with the nucleic acid sequence shown in FIG. 1 or its complement under high stringency conditions and has ASP05 biological activity (particularly preferable catalytic biological activity) is considered to be the ASP05 gene. Be done.

High stringency conditions are known in the art and are described, for example, in Maniatis et al., Mole.
cular Cloning: A Laboratory Manual, 2d Edition, 1989 and Short Protocol
s in Molecular Biology, ed. Ausubel, et al., Hames and Higgins, eds. Nu
cleic Acid Hybridization, A Practical Approach, IL Press, Washington, DC
, 1985; Berger and Kimmel eds. Methods in Enzymology, Vol.52, Guide to
Molecular Cloning Techniques, Academic press Inc., New York, NY; Bothwell, Yancopoulos and Alt, eds, Methods for Cloning and Analysis of
Eukaryotic Gene, Jones and Bartlett Publishers, Boston, Mass. 1990) (incorporated herein by reference).

The choice of hybridization conditions will be apparent to those of skill in the art and will generally be for the purpose of hybridization, the type of hybridization (DNA-
(DNA, DNA-RNA, RNA-RNA, oligonucleotide-DNA, etc.)
And the desired level of relatedness between the sequences. Hybridization methods are well established in the literature. For example, one of ordinary skill in the art should understand that increasing the number and proximity of mismatched bases decreases the stability of nucleic acid duplexes. That is, the stringency of hybridization can be used to maximize or minimize the stability of the above double helix.

The stringency of hybridization can be modified. For example, the temperature of the hybridization solution is adjusted, the percentage of helix destabilizing agent, such as formamide, in the hybridization solution is adjusted, and the temperature and salt concentration of the wash solution are adjusted. Generally, the stringency of hybridization is adjusted during post-hybridization washes by varying salt concentration and / or temperature. The stringency of hybridization can be increased. For example, by i) increasing the percentage of formamide in the hybridization solution, ii) increasing the temperature of the wash solution, or iii) decreasing the ionic strength of the wash solution.

High stringency conditions include high temperature hybridization (eg, in aqueous solution containing 4-6X SSC) combined with high temperature (eg, 5 ° C.-25 ° C. below Tm) and low salt concentration (eg, 0.1 × SSC) washes. 65 ° C.-68 ° C., or 42 ° C. in 50% formamide). Reduced stringency conditions include intermediate temperature (eg 40 ° C.-60 ° C.) washes with higher salt concentrations (eg 2-6 × SSC) as well as lower hybridization temperature (eg 35 ° C. in 20-50% formamide). 42
C.). Moderate stringency conditions include hybridization at temperatures between 50 ° C and 55 ° C and 0.1X SSC between 50 ° C and 55 ° C.
, 0.1% SDS wash (Maniatis and Ausubel, supra). In a preferred embodiment, the nucleic acid that hybridizes to this nucleic acid has the biological activity described herein.

The ASP05 proteins and nucleic acids of the invention are preferably recombinant. As used herein, "nucleic acid" means DNA or RNA, or a molecule containing both deoxyribonucleotides and ribonucleotides. Nucleic acid includes genomic DNA, cDNA and oligonucleotides, including sense and antisense nucleic acids. Further modifications of the ribose-phosphate backbone of the nucleic acid may increase the stability and half-life of the molecule in physiological environments.

Nucleic acid can be double-stranded, single-stranded, or comprises part of both double-stranded or single-stranded sequence. As is recognized in the art, also drawing one strand ("Watson") defines the sequence of the other strand ("click"). That is, the sequence depicted in Figure 1 also includes the complement of the sequence. As used herein, the term "recombinant nucleic acid" refers to a nucleic acid that was originally formed in vitro, in a form that is not normally found in nature by genetic engineering of the nucleic acid, generally by endonucleases. That is, both linearly separated ASP05 nucleic acids, or expression vectors formed in vitro by ligating DNA molecules that are not normally associated, are considered recombinants for the purposes of the present invention. Similarly, a fragment of the full length sequence is optionally linked to regulatory sequences and is considered recombinant. In addition, altered nucleic acid sequences are also recombinant, eg, sequences described herein that have altered nucleotide sequences (eg, low GC content). It will be appreciated that once the recombinant nucleic acid is produced and reintroduced into the host cell or organism, it replicates non-recombinantly, ie using the in vivo cellular machinery of the host cell rather than in vitro manipulation. However, once the nucleic acid is produced recombinantly, it is then non-recombinantly replicated and is still considered to be a recombinant for the purposes of the present invention.

Similarly, a “recombinant protein” is a protein produced using recombinant technology, that is, by expressing the above recombinant nucleic acid. Recombinant proteins are distinguished from native proteins by at least one or more characteristics. For example, the protein can be substantially pure because it can be separated or purified from some or all of the proteins and compounds normally associated with its wild-type host. For example, isolated protein is not associated with at least a portion of the substance with which it is normally associated in nature, which substance preferably is at least about 0. 0 by weight of total protein in a given sample. 5%, more preferably at least about 5%. Substantially pure protein comprises at least about 75% by weight of total protein, preferably at least about 80%, and further at least about 9%.
0% is particularly preferred. This definition includes the production of ASP05 protein from one organism in a different organism or host cell. In addition, the protein can be produced in high concentration by the use of inducible or highly expressed promoters such that the protein is produced in high concentration levels. In addition, the protein may be in a form not normally found in nature, with epitope tagging or amino acid substitutions, insertions and deletions, as discussed below. Furthermore, the recombinant protein may be enzymatically active.

Once identified, a polypeptide containing a bioactive sequence can be produced by conventional techniques, eg, synthesis (eg, using a Beckman model 990 peptide synthesizer or other commercially available synthesizer).

Amino acid sequence variants are also included within the definition of ASP05 protein of the invention. These variants are classified into three types, namely one or more of substitution, insertion or deletion variants. Conventional production of these variants is by site-directed mutagenesis of nucleotides in the DNA encoding the ASP05 protein. Following the use of cassette or PCR mutagenesis methods or other techniques known in the art, the DNA is expressed in the recombinant cell culture outlined above to produce the DNA encoding the mutant form. However, mutant ASP0 having about 100-150 or less residues
The 5 protein fragment may be produced by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by a predetermined nature of the mutation, namely AS
It has features that distinguish it from natural alleles or interspecies variations of the P05 protein amino acid sequence. Variants typically exhibit the same quality of biological activity as the natural analog, although variants with modified characteristics may also be selected as described in more detail below.

Even if the site or region where the amino acid sequence mutation is to be introduced is determined in advance, the mutation itself need not be determined in advance. For example, to optimize the performance of mutagenesis at a given site, random mutagenesis was induced at the target codon or region and expressed A
SP05 variants can be screened for the optimal combination of desired activities. Techniques for inducing substitution mutations at predetermined sites in DNA having a known sequence are well known. For example, M13 primer mutagenesis and PCR mutagenesis. Screening for variants can be performed using an assay for ASP05 protein activity, eg, in the case of binding domain mutations, the competitive binding test, eg, outlined in the Examples, can be performed.

Amino acid substitutions are typically by single residues and insertions are usually about 1 to 20 amino acids, although fairly large insertions are acceptable. Deletions range from about 1 to about 20 residues, although in some cases fairly large deletions are possible.
For example, a preferred variant is a deletion of the catalytic domain, leaving only the binding domain of ASP05. Additional preferred variants include a catalytic domain alone or a soluble receptor (ie, binding domain).

The final derivative may be reached by using substitutions, deletions, insertions or combinations thereof. Generally, these changes are made to a few amino acids to minimize the alteration of the molecule. However, large scale changes are acceptable in some circumstances. If minor modifications of the properties of the ASP05 protein are desired, substitutions are generally made according to the chart below.

[0059] Chart I

[Table 1]

[Table 2]

Substantial changes in functional or immunological identity can be made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions can be made that significantly affect the structure of the polypeptide backbone in the modified region, such as the alpha-helix or beta-sheet structure, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chains. Substitutions that are generally expected to result in maximal changes in the properties of the polypeptide include: If substituted (or by), (b) if cysteine or proline is substituted (or by) for any other residue, and (c) has an electropositive side chain A residue, for example lysyl, arginyl or histidyl, is substituted (or by) in place of an electronegative residue, for example glutamyl or aspartyl, or (d) a residue with a large side chain, for example phenylalanine A residue without a chain, eg substituted in place of (or by) glycine A.

Variants typically exhibit the same quality of biological activity as the natural analog and elicit the same immune response, although variants that optionally modify the characteristics of the ASP05 protein can be selected. Alternatively, variants can be designed to alter the biological activity of ASP05 protein. For example, glycosylation sites, and more particularly one or more O-
The linked or N-linked glycosylation sites may be modified or removed. Modification or removal of one or both of the transmembrane domains can produce soluble or secreted proteins, the extracellular domain.

For example, A, which desirably binds but does not cleave substrates or inhibitors
Make SP05 protein. In this way, active site sequence substitutions may be made for book or other purposes.

In another embodiment, the mutant library is created by a complete non-specific random mutagenesis method. This technique is known and does not require the selection of specific sites or regions to be modified. For example, the DNA shuffle method (Stemmer, Nature 370: 389-391)
(1994) and Stemmer, PNAS USA 91: 10747-10751 (1994)) can be used to create variants that are cloned, expressed and screened for the desired property. For example, the biochemical activity of ASP05 can be increased or decreased as needed.

Covalent modifications of the ASP05 polypeptide are also included within the scope of this invention. One type of covalent modification involves the targeted amino acid residue of an ASP05 polypeptide and an organic derivatizing agent capable of reacting with a selected side chain or N- or C-terminal residue of the ASP05 polypeptide. A reaction is included. As described in more detail below, for example, derivatization with a bifunctional agent is useful for cross-linking ASP05 to a water-insoluble support matrix or surface used in methods for purifying anti-ASP05 antibodies or screening assays. is there. Common crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imide esters, For example, disuccinimidyl esters such as 3,3 '
-Dithiobis (succinimidyl propionate), difunctional maleimides such as bis-N-maleimido-1,8-octane and methyl-3-[(p-azidophenyl) dithio] propioimidate reagents is there.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, lysine, Arginine and histidine side chain “-amino group methylation [TECreighton, Pro
teins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of the C-terminal carboxyl group.

Other types of covalent modifications of the ASP05 polypeptide are included within the scope of this invention, including altering the native glycosylation pattern of the polypeptide. For purposes herein, "altering the native glycosylation pattern" refers to the deletion of one or more carbohydrate moieties found in the native sequence ASP05 polypeptide, and / or native sequence ASP.
05 refers to the addition of one or more glycosylation sites not present in the polypeptide.

Addition of glycosylation sites to the ASP05 polypeptide may be accomplished by modifying its amino acid sequence. This modification can be made, for example, by adding or substituting one or more serine or threonine residues to the native sequence ASP05 polypeptide (for O-linked glycosylation sites). The ASP05 amino acid sequence may be modified, optionally through modification at the DNA level, by mutating the DNA encoding the ASP05 polypeptide with preselected bases to generate codons that specifically translate to the desired amino acid.

Another means of increasing the number of carbohydrate moieties on the ASP05 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. This method is described in the literature, for example WO 87/05330, published September 11, 1987, and Aplin and Wriston, CRC Crit. Rev. Biochem., 259-306 (198).
Reported in 1).

Removal of carbohydrate moieties present on the ASP05 polypeptide may be accomplished by chemical or enzymatic methods or by mutated substitution of codons encoding amino acid residues used as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin et al., Arch. Biochem. Biophys., 259: 52 (1).
987) and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties in polypeptides has been reported by Thotakura.
Et al., Meth. Enzymol., 138: 350 (1987), can be achieved by the use of various endo- and exo-glycosidases.

Another type of ASP05 covalent modification involves attachment of the ASP05 polypeptide to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes. This is described in U.S. Pat.
192 or 4179337.

The ASP05 polypeptides of the present invention may also be modified in a manner to form chimeric molecules comprising the ASP05 polypeptide fused to another heterologous (including synthetic) polypeptide or amino acid sequence. In one aspect, such a chimeric molecule is an ASP with a labeled polypeptide that provides an epitope to which an anti-labeled antibody can be selectively bound.
05 polypeptide fusions. The epitope tag is generally located at the amino- or carboxyl-terminus of the ASP05 polypeptide. The presence of the above epitope-tagged forms of ASP05 polypeptide can be detected using an antibody against the labeled polypeptide. Also, by providing an epitope tag, ASP05 polypeptides can be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In another embodiment, the chimeric molecule may comprise a fusion of an ASP05 polypeptide with an immunoglobulin or a particular region of an immunoglobulin. In the case of the divalent form of the chimeric molecule, such a fusion may be to the Fc region of an IgG molecule or a GST fusion. Similarly, a chimeric nucleic acid encoding a chimeric protein can be made as described herein.

Various labeled polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) labels, flu HA labeled polypeptides and their antibodies 12C.
A5 [Field et al., Mol. Cell Biol., 8: 2159-2165 (1988)], c.
-Myc label and 8F9, 3C7, 6E10, G4, B7 and 9 for it
E10 antibody [Evan et al., Molecular and Cellular Biology, 5: 3610-361.
6 (1985)], and herpes simplex virus glycoprotein D (gD) labeling and its antibodies [Paborsky et al., Protein Engineering 3 (6): 547-553.
(1990)]. Other labeled polypeptides include flag-peptide [Hopp
Et al., Bio Technology, 6: 1204-1210 (1988)], KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)], tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266]. : 1516
3-15166 (1991)], and T7 gene 10 protein peptide labeling [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-639.
7 (1990)].

Also included in the definition of ASP05 protein are other ASP05 proteins belonging to the ASP05 family, and ASP05 proteins from other organisms, which are cloned and expressed as outlined below. That is, by using probes or degenerate polymerase chain reaction (PCR) primer sequences, other related ASP05 proteins from humans or other organisms can be found. Particularly useful probes and / or PCs as recognized in the art
The R primer sequence contains a unique region of the ASP05 nucleic acid sequence. Thus, useful probe or primer sequences are all or part of the sequence of the immunoglobulin V-like ASP05 domain, all or part of the only extracellular domain spanning approximately amino acids 18-253, or sequences outside the coding region. Can be designed against. As is generally known in the art, preferred PCR primers are about 15 to about 35 nucleotides in length, preferably about 20 to about 30, and may optionally include inosine. The conditions for the PCR reaction are well known in the art.

Once identified, the ASP05 nucleic acid can be cloned and, if necessary, its components recombined to form the entire ASP05 nucleic acid. Once isolated from its natural source, eg, encapsulated in a plasmid or other vector or excised as a linear nucleic acid segment therefrom, the recombinant ASP05 nucleic acid is further used as a probe to identify other ASP05 nucleic acids. And can be isolated.
It can also be used as a "precursor" nucleic acid to produce modified or mutated ASP05 nucleic acids and proteins.

A variety of expression vectors are produced using the nucleic acids of the invention encoding the ASP05 protein. The expression vector can be an autonomously replicating extrachromosomal vector or a vector that integrates into the host genome. Generally, these expression vectors are
05 transcriptional and translational regulatory nucleic acid operably linked to nucleic acid encoding the 05 protein. The term "regulatory sequence" includes DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DN for precursor sequences or secretory leaders
A is operably linked to the DNA for a polypeptide when expressed as a preprotein involved in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. The ribosome binding site is operably linked to the coding sequence when it is in a position that facilitates translation. Generally, "operably linked" is contiguous to the DNA sequences being linked, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Coupling is accomplished by a ligation reaction at convenient restriction sites. In the absence of the above sites, synthetic oligonucleotide adapters or linkers can be used routinely. Transcriptional and translational regulatory nucleic acids are generally compatible with the host cell used to express ASP05 protein. For example, transcription and translation regulatory nucleic acid sequences from Bacillus are preferably ASP0 in Bacillus.
Used for the expression of 5 proteins. Various types of suitable expression vectors and suitable regulatory sequences are known in the art for different host cells.

In general, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start / stop sequences, translational start / stop sequences, and enhancer or activator sequences. . In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

The promoter sequence encodes a constitutive or inducible promoter. The promoter can be a natural promoter or a hybrid promoter. Hybrid promoters, which combine elements of multiple promoters, are also known in the art and are useful in the present invention.

In addition, the expression vector may contain additional elements. For example, an expression vector may have more than one replication system and thus be maintained in more than one organism, for example for expression in mammalian or insect cells and cloning and amplification in prokaryotic hosts. Furthermore, in the case of an integrative expression vector, the expression vector comprises at least one sequence homologous to the host cell genome, and preferably two homologous sequences flanking the expression construct. Integrating vectors can be directed to specific loci in the host cell by selecting the appropriate homologous sequences for inclusion in the vector. Constructs for integrating vectors are well known in the art.

Further, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

Preferred expression vector systems are retroviral vector systems such as PCT / US
97/01019 and PCT / US97 / 01048, both of which are incorporated herein by reference.

The ASP05 protein of the present invention is produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the ASP05 protein under conditions suitable for inducing or inducing the expression of the ASP05 protein. Suitable conditions for ASP05 protein expression will vary with the choice of expression vector and host cell,
It will be readily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in expression vectors requires optimization of host cell growth and amplification, and the use of inducible promoters requires growth conditions suitable for induction. Furthermore, in some cases, the timing of collection may be important. For example, the baculovirus system used in insect cell expression is a lytic virus, so the choice of harvest time can be critical to product yield.

The ASP05 protein of the invention is “expressible” in a suitable host cell, making a biologically active protein. In a preferred embodiment, ASP05 is expressed in baculovirus-infected insect cells. Proteins are expressed using the Bac-to-Bac baculovirus expression system (GibcoBRL, Gaiterburg, MD). Flag
The ASP05 cDNA labeled with an epitope (Eastman Kodak, New Haven. CT)
It is ligated into the FASTBac1 vector and transformed into DH1OABac cells to make a recombinant bacmid. Bacmid DNA is transduced into Spodoptera frugiperda ("Sf9") insect cells (ATCC CRL 1711) to produce recombinant baculovirus. High titer stocks are used to infect Sf9 insect cells and the expressed proteins are purified by affinity chromatography. This uses an agarose-linked antibody specific for the flag epitope tag incorporated into the C-terminus of expressed ASP05. Fractions containing the eluted and labeled ASP05 polypeptide were collected and
Dialyze against ES or HEPES buffer.

Suitable host cells include yeast, bacterial, archaeal, fungal and insect and animal cells such as mammalian cells and primate cells, including but not limited to stem cells including bone marrow stem cells. Of particular interest is Drosophila melangusta (Dros
ophila melangaster) cells, Saccharomyse
cerevisiae) and other yeasts, E. coli, Bacillus
Subtilis (Bacillus subtilis), SF9 cells, C129 cells, 29
3 cells, Neurospora, BHK, CHO, CO
S and HeLa cells, fibroblasts, schwannoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jukat cells, human cells and other primate cells.

Alternatively, known chromatographic techniques can be used to purify IgG-labeled (or Fc-labeled) ASP05 polypeptides.

In a preferred embodiment, the ASP05 protein is expressed in mammalian cells. Mammalian expression systems are also known in the art and include retroviral systems. The mammalian promoter is any DN capable of binding mammalian RNA polymerase and initiating the downstream (3 ′) transcription of the coding sequence for ASP05 protein into mRNA.
It may be an A array. The promoter has a transcription initiation region usually located proximal to the 5'end of the coding sequence, and a TATA box with 25-30 base pairs located upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to direct RNA synthesis at the correct site. Mammalian promoters also contain upstream promoter elements (enhancer elements), which are typically located within 100 to 200 base pairs upstream of the TATA box. Upstream promoter elements determine the rate of transcription initiation and can act in either direction. Of particular utility as mammalian promoters are those derived from mammalian viral genes, as viral genes are often highly expressed and have a wide host range. Examples include SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, CM
There is a V promoter, a retrovirus LTR promoter, a mouse Maloney leukemia virus LTR or pBabeMN.

Typically, the transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 ′ to the transcription stop codon and, in co-presence with a promoter element, are flanked by coding sequences. Adjacent to. The 3'end of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminators and polyadenylation signals include those derived from SV40.

Methods for introducing foreign nucleic acid into mammalian and other hosts are well known in the art and will vary with the host used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of polynucleotide (s) in liposomes, and direct microinjection of DNA into the nucleus. .

In a preferred embodiment, the ASP05 protein is expressed in bacterial systems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter binds bacterial RNA polymerase and induces mRNA
Any nucleic acid sequence capable of initiating the downstream (3 ′) transcription of the ASP05 protein coding sequence into Bacterial promoters usually have a transcription initiation region located proximal to the 5'end of the coding sequence. This transcription start region typically includes an RNA polymerase binding site and a transcription start site. From the sequence encoding the metabolic pathway enzyme,
Particularly useful promoter sequences are provided. Examples are promoter sequences derived from sugar-metabolizing enzymes such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage can also be used and are known in the art. In addition, synthetic and hybrid promoters are also useful. For example, the tac promoter is a hybrid of the trp and lac promoter sequences. In addition, bacterial promoters can include native promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functional promoter sequence, an efficient ribosome binding site is desirable. In the case of E. coli, the ribosome binding site is called the Shine-Delgarno (SD) sequence and has a start codon and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the start codon.

The expression vector may also include a signal peptide sequence that directs secretion of the ASP05 protein in bacteria. The signal sequence typically encodes a signal peptide composed of hydrophobic amino acids that direct secretion of the protein from the cell, as is well known in the art. Proteins are secreted into the growth medium (Gram-positive bacteria) or into the periplasmic space between the inner and outer membranes of cells (Gram-negative bacteria).

The bacterial expression vector may also contain a selectable marker gene to allow the selection of transformed bacterial strains. Suitable selection genes include genes that confer to bacteria resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes such as those in the histidine, tryptophan and leucine biosynthetic pathways.

These components are constructed into an expression vector. Expression vectors for bacteria are well known in the art, and in particular Bacillus subtilis (Bacillus subti
lis), Escherichia coli (E.coli), Streptococcus cremoris (Str
eptococcus cremoris) and Streptococcus lividans (Streptococcus
lividans).

The bacterial expression vector is transformed into a bacterial host cell using techniques well known in the art such as calcium chloride treatment, electroporation and the like.

In a preferred embodiment, the ASP05 protein is produced in yeast cells. Yeast expression systems are well known in the art and include Saccharomyces cerevisiae (Sacc
haromyces cerevisiae), Candida albicans and C. maltosa, Hansenula polymorpha (Hansenula po)
lymorpha), Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. Pastoris, Schizosaccharomyces poncharces Schizosacomy pombe) and Yarrowia
There is an expression vector for Yarrowia lipolytica. Preferred promoters for expression in yeast include the inducible GAL1,10 promoter, alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, phosphofructokinase, 3-phospho. There are promoters from the glycerate mutase, pyruvate kinase and acid phosphatase genes. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1 and ALG7 (
It confers resistance to tunicamycin), the neomycin phosphotransferase gene confers resistance to G418, and the CUP1 gene that allows yeast to grow in the presence of copper ions.

ASP05 protein may also be produced as a fusion protein using techniques well known in the art. Thus, for example, to produce a monoclonal antibody, the ASP05 protein may be fused to a carrier protein to form an immunogen if the desired epitope is small. Alternatively, the ASP05 protein may be produced as a fusion protein for enhanced expression or other reasons.
For example, when the ASP05 protein is an ASP05 peptide, the nucleic acid encoding the peptide can be linked to other nucleic acids for expression purposes. In a preferred embodiment, the nucleic acid encoding the protease cleavage site is a nucleic acid retention protein and AS.
Place between P05 polypeptides and in frame. Protease cleavage sites are used to facilitate purposes such as, for example, purification of the expressed ASP05 polypeptide. Preferably, the protease cleavage site is not in the ASP05 polypeptide or the ASP05 polypeptide is not substantially digested by a protease that binds and digests at the cleavage site.

In one aspect, the ASP05 nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant attached to a compound with at least one moiety, isotope or chemical compound to allow detection of the compound. Generally, the sign is 3
They are classified into types: a) isotope labels that can be radioactive or heavy isotopes, b) immunolabels that can be antibodies or antigens, and c) colored or fluorescent dyes. The label may be incorporated at any position on the compound.

In a preferred embodiment, the ASP05 protein is purified or isolated after expression. A
SP05 protein may be isolated or purified by a variety of methods well known to those of skill in the art depending on what other components are present in the sample. Standard purification methods include electrophoresis, molecules,
There are immunological and chromatographic techniques, such as ion exchange, hydrophobicity,
Includes affinity and reverse phase HPLC chromatography and chromatofocusing. For example, ASP05 protein can be purified using a standard anti-ASP05 antibody column. It is also useful to use ultrafiltration and diafiltration techniques in conjunction with protein concentration. See Scopes, R., Protein Purification, Springer-Verlag, New York (1982) for general guidance on suitable purification techniques. The required degree of purification depends on the application of ASP05 protein. In some cases, no purification is necessary.

When expressed and purified as needed, ASP05 protein and nucleic acid
Useful in many applications.

In a preferred embodiment, cells containing modified ASP05 and modified protein are produced. In one aspect, non-human animals (preferably transgenic) containing modified ASP05 are produced. Similarly, "knockout" animal models containing inducible promoters and ASP05 transgenic animals can be produced.

In a preferred embodiment, ASP05 proteins, nucleic acids, modified ASP05 proteins and cells containing native or modified ASP05 proteins are used in screening assays. The identification of this important serine protease is ASP05
Allows the design of drug screening assays to find compounds that modulate biological activity (proteolytic activity, binding, etc.).

The screen can be designed first to find candidate substances that can bind to ASP05, and then to use these substances in assays that assess the ability of the candidate substances to modulate ASP05 activity. Thus, as will be apparent to those of skill in the art, there are many different assays that proceed, such as binding assays followed by activity assays. Of particular importance in these embodiments is that the catalytic portion of ASP05 is
It is mainly responsible for the selective cleavage of IGFBPs. Furthermore, I
GFBP may be present in body fluids and tissues in vivo. Thus, a candidate substance can be added directly to the cell during the cleavage assay without the need to target it. Therefore, the catalytic domain of ASP05 can be used independently as the basis for bioassays.

Thus, in a preferred embodiment, the method comprises combining ASP05 protein with a candidate bioactive agent and determining the binding of the candidate agent to ASP05 protein. A preferred embodiment is the human ASP05 protein (
Alternatively, as outlined herein, for example utilizing a moiety such as a catalytic domain or a binding domain), but in any case rodents (mouse, rat, hamster, Other mammalian ASP05 proteins, including guinea pigs) and primates are also available. These latter embodiments are preferred in developing animal models of human disease. As outlined herein, in certain embodiments, ASP0 containing the aforementioned deleted ASP05 protein.
Variants or derivatives of 5 proteins can be used.

Furthermore, the ASP05 protein portion is included in the definition of ASP05 protein. That is, either the full length protein or a functional portion thereof can be used. In a preferred embodiment, the catalytic domain of ASP05 can be used with or without a binding region. In a further preferred embodiment, the binding domain of ASP05 can be used with or without a catalytic region. In addition, the assays described herein can use either isolated ASP05 protein or cells containing ASP05 protein, as required for a particular assay.

In general, in a preferred embodiment of the method of the present invention, ASP05 and the candidate substance are co-located in the sample receiving area, one of which is bound to an insoluble support (eg microtiter plate, array, etc.). Insoluble support is ASP
05 or a composition to which the candidate substance can bind, or is compatible with all steps of the screening method. The surface of those supports can be solid or porous of any convenient shape. Examples of suitable insoluble supports are microtiter plates, arrays, membranes and beads and the like. These are typically made of glass, plastic (eg polyethylene), polysaccharides, nylon or nitrocellulose, Teflon ™, etc. Microtiter plates and arrays are particularly convenient because multiple assays can be performed simultaneously with small amounts of reagents and samples. The particular method of binding the composition is not limiting as long as it is compatible with the reagents and all steps of the invention, maintains the activity of the composition and is non-diffusible. Preferred methods of attachment include antibodies, which are those that do not sterically block the protease sequences when the ASP05 protein binds to the support, "sticky" or direct attachment to ionic supports, chemistry. Crosslinking, surface synthesis of ASP05 protease, and the like. Following binding of ASP05 protein, excess unbound material is removed by washing. The sample receiving area is then blocked by incubating with innocuous proteins such as bovine serum albumin (BSA), casein or other moieties.

Candidate bioactive agents are added to the assay as described below. The new binding substance is
Includes specific antibodies, non-natural binding agents identified in chemical library screens, peptide analogs, and the like. Particularly advantageous is a screening assay for substances that have low toxicity to human cells. A wide variety of assays can be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, protein binding immunoassays, functional assays such as phosphorylation assays. ) And similar methods.

The terms “candidate bioactive agent” and “exogenous compound” as used herein, directly or indirectly, of the ASP05 protein in response to or without ligand binding. It refers to all molecules with the ability to regulate biological activity, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc. Generally, multiple assay mixtures are run in parallel at different reagent concentrations so that a differential response is obtained corresponding to different concentrations. Typically, one of each of these concentrations is a negative control, ie, zero concentration or below the measurement limit.

Candidate substances encompass a wide variety of chemicals, but are typically organic molecules, preferably small organic compounds having a molecular weight of 100 or more and about 2500 daltons or less.
Candidates are functional groups required for structural interactions with proteins, especially hydrogen bonds, and typically at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two chemical functional groups. Contains a sexual group. Candidate agents often include cyclical carbon or heterocyclic structures substituted with one or more of the above functional groups, and / or aromatic or polyaromatic structures. Candidate agents are also found in biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs or combinations thereof.
Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, a variety of means can be employed for the synthesis of indiscriminate or directed organic compounds and biomolecules, including randomized oligonucleotide expression. Alternatively, a library of natural compounds can be obtained or readily produced in the form of bacterial, fungal, plant and animal extracts. In addition, naturally or synthetically obtained libraries and compounds can be readily modified by conventional chemical, physical and biochemical means. Known pharmacological agents can also be subjected to directed or random chemical modifications, for example acylated, alkylated, esterified, amidated to produce structural analogs.

In a preferred embodiment, the candidate bioactive agent is a protein. As used herein, “protein” means at least two covalently linked amino acids, a protein,
Includes polypeptides, oligopeptides and peptides. The protein may be composed of naturally occurring amino acids, peptide bonds, or synthetic pseudo-peptide structures. Similarly, "amino acid" or "
"Peptide residue", as used herein, refers to both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains can be in the (R) or (S) configuration. In a preferred embodiment, the amino acids are in the (S) or L configuration. If you use a non-naturally occurring side chain,
Non-amino acid substituents can be used, for example, to prevent or delay degradation in vivo.

In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cell extracts containing proteins or random or directional digests of live protein extracts of proteins can be used. In this way libraries of prokaryotic and eukaryotic proteins can be prepared for screening against ASP05. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral and mammalian proteins, the latter being preferred and human proteins being especially preferred.

In a preferred embodiment, the candidate bioactive agent is a peptide of about 5 to about 30 amino acids, with about 5 to about 20 amino acids being preferred and about 7 to about 15 being especially preferred. The peptides can be random peptides or "biased" random peptides that are digests of naturally occurring proteins, as described above. "Randomized" or synonyms thereof, as used herein, mean that each nucleic acid and peptide is composed essentially of random nucleotides and amino acids, respectively. Generally, since these random peptides (or nucleic acids described below) are chemically synthesized, they introduce any nucleotide or amino acid into any position. The synthetic process can be designed to produce randomized proteins and nucleic acids, forming all or most of the possible combinations over the entire length of the sequence, thus creating randomized candidate bioactivities. A library of substances can be formed.

In one embodiment, the library is fully randomized and has no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. It is whether several positions in the sequence are either constant or selected from a limited number of possibilities. For example, in a preferred embodiment, these nucleotides or amino acid residues are linked to the creation of cysteine, due to cross-linking, proline to SH-3 main, serine to phosphorylation sites, threonine, tyrosine or histidine, etc. Or, for purines and the like, they are randomized within defined classes, eg, hydrophobic amino acids, hydrophilic residues, sterically biased (small or large) residues.

In a preferred embodiment, the candidate bioactive agent is a nucleic acid. "Nucleic acid" or "oligonucleotide" or synonyms thereof, as used herein, mean at least two nucleotides that are covalently linked to each other. The nucleic acids of the invention generally contain phosphodiester bonds, but in some cases, as described below,
Includes nucleic acid analogs. They include, for example, those having another skeleton: phosphoramide (Beaucage, et.al., Tetrahedron, 49, (10); 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35: 3800 (1970); Sprinzl, et. Al.,
Eur. J. Biochem. 81: 579 (1977); Letsinger, et. Al., Nucl. Acids Res. ,
14: 3487 (1986); Sawai, et.al., Chem. Lett . 805 (1984), Letsinger, et.
al., J. Am. Chem. Soc. , 110: 4470 (1988); and Pauwels et. al., Chemica S cripta , 26: 141 (1986)), phosphorothioate (Mag, et. al., Nucl. Acids R es, 19:. 1437 ( 1991); and US Patent No. 5644048), phosphorodithioate (..... Briu, et al, J. Am Chem Soc, 111: 2321 (1989)), O- Methyl phosphoramidite bond (Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid backbones and bonds (Egholm, J. Am. Chem. Soc. , 114: 1895 (1992); Meier, et. Al., Chem. Int. Engl. , 31: 1008 (1992); Nielsen, Nature , 365: 566 (1993); Carlsson
, et. al., Nature , 380: 207 (1996)), which are incorporated herein by reference.

[0116]   Other similar nucleic acids have a positive backbone (Denpcy, et. Al.,Proc. Natl. Acad. Sci. USA , 92: 6097 (1995)); Nonionic skeleton (U.S. Patent Nos.
.5386023; 5637684; 5602240; 5216141; and 4469863; Kiedrowshi, et. Al., Angew. Chem. Intl. Ed. English , 30: 423 (1991); Letsinger, et. Al.,J. A m. Chem. Soc. , 110: 4470 (1988); Letsinger, et. Al., Nucleotide, 13: 1597
(1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modific
ationsin Antisense Research "Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeke
r, et. al.,Bioorganic & Medical Chem Lett., 4: 395 (1994); Jeffs, et. A
l.,J. Biomolecular NMR, 34: 17 (1994); Tetrahedron Lett., 37: 743 (1996
)) And non-ribose skeletons including those described below (U.S. Patent Nos. 52350
33 and 5034506 and Chapters 6 and 7, ASC Symposium Series 580, "Carbohy
drate Modificationsin Antisense Research "Ed. Y. S. Sanghui and P. Dan C
ook) is included. Nucleic acids containing one or more carbocyclic sugars are also nuclear.
Included within the definition of acid (Jenkins, et. Al.,Chem. Soc. Rev., (1995) pp. 169-
176). Several nucleic acid analogs have been published (Rawls, C & E News, June 2, 1997, page 35).
)It is described in. These are incorporated herein by reference.

Modifications of these ribose-phosphate backbones can facilitate the addition of additional moieties, such as labels, and also increase the stability and half-life of those molecules in the physical environment. In addition, mixtures of naturally occurring nucleic acids and analogs can be prepared. Alternatively, a mixture of different nucleic acid analogs and a mixture of naturally occurring nucleic acids and analogs can be prepared. These nucleic acids can be single-stranded or double-stranded, and can contain portions of both double-stranded or single-stranded sequences. Nucleic acid may be DNA, both chromosomal and cDNA, RNA or hybrid, in which the nucleic acid includes any combination of deoxyribo- and ribo-nucleotides and uracil, adenine, thymine, cytosine. , Guanine, inosine, xatatanine, hypoxatanine, isocytosine, isoguanine and the like.

As described generally above for proteins, nucleic acid candidate bioactive agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, prokaryotic or eukaryotic chromosomal digests can be used as described above for proteins.

In a preferred embodiment, the candidate bioactive agents are various types of organic chemical moieties available in the literature.

Assays for binding of candidate bioactive agents to ASP05 can be performed in a number of ways. In a preferred embodiment, the candidate bioactive agent is labeled and binding is measured directly. For example, this may involve binding all or part of ASP05 to a solid support, adding a labeled candidate (eg, a fluorescent label), washing off excess reagent, and determining if the label is present on the solid support. It can be carried out by measuring. A variety of blocking and washing steps can be used, as is known in the art.

“Labeling”, as used herein, refers to a signal, such as radioactivity, fluorescence, an enzyme, an antibody, a particle such as a magnetic particle, to which the compound is directly or indirectly detectable. It is meant to be labeled with a label that provides a chemiluminescent agent, or a specific binding molecule, or the like. Specific binding molecules include pairs of biotin and streptavidin, digoxin and antidigoxin, and the like. For specific binding members, the complementary member is labeled with a detection-imparting molecule according to conventional, well-known procedures, as described above.
The label directly or indirectly provides a detectable signal.

In some embodiments, only one of the components is labeled. For example, the ASP05 protein (or proteinaceous candidate) can be labeled with ¹²⁵ I or a fluorophore at the tyrosine position. Alternatively, two or more components are labeled with different labels, eg ASP0.
¹²⁵ I can be used for 5 proteins and a fluorophore for candidate substances.

In a preferred embodiment, binding of the candidate bioactive agent is measured using a competitive binding assay. In this embodiment, the competitor is a binding moiety known to bind the target molecule, eg, an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding, such as between the bioactive agent and the binding moiety, that replaces the binding moiety bioactive agent.

In some embodiments, the candidate bioactive agent is labeled. Either the candidate bioactive agent or the competitor, or both, are first added to ASP05 for a time sufficient for binding to occur, if any. Incubations are performed at any temperature that facilitates optimal activity, typically 4-40 ° C. The incubation time is chosen for optimal activity, but can also be optimized to facilitate rapid and advanced screening. Typically 0.1 to 1 hour will be sufficient. Excess reagent is typically removed or washed off. Then, when the second component is added, the presence or absence of the labeled component appears as an index of binding.

In a preferred embodiment, the competitor component is added first, followed by the candidate bioactive agent. The replacement of the competing component is such that the candidate bioactive agent is bound to the ASP05 protein and thus capable of binding to the ASP05 protein, and ASP0
It is an indicator that the activity of 5 protein may be regulated. In this embodiment, any component can be labeled. Therefore, for example, when a competitive component is labeled, the presence of the label in the washing solution indicates that the substance was replaced. Alternatively,
When the candidate bioactive agent is labeled, the presence of the label on the carrier indicates displacement.

[0126] In another embodiment, the candidate bioactive agent is added first, incubated, washed, and then the competitor components are added. The absence of binding by the competing component indicates that the bioactive agent binds to the ASP05 protein with high affinity. Therefore, when labeling a candidate bioactive substance, the presence of the label on the support, in combination with the absence of binding of the competing component, indicates that the candidate substance can bind to the ASP05 protein.

Alternatively, a preferred embodiment utilizes a differential screen to identify drug candidates that bind the native ASP05 protease but not the modified ASP05 protease. The structure of the ASP05 protease can be modeled and used in theoretical drug design to synthesize substances that interact at that site. Drug candidates that modulate cleavage are also identified by screening agents for their ability to enhance or reduce specific cleavage by ASP05.

Positive and negative controls can be used in these assays.
Preferably, all control and test samples are performed with at least 3 samples each to obtain statistically significant results. Incubation of all samples is ASP
05. Allow sufficient time for binding of substance to protease. Following incubation, all samples are washed free of non-specifically bound material and the amount of generally labeled material bound is determined. For example, when a radioactive label is used, the sample is counted with a scintillation counter to measure the amount of bound compound.

Screening for substances that modulate the activity of ASP05 can also be performed. In a preferred embodiment, the method of screening for bioactive agents capable of modulating the activity of ASP05 protein comprises adding a candidate bioactive agent to a sample of ASP05 and measuring changes in the bioactivity of ASP05, as described above. Including stages.
"Modulate activity of ASP05" includes increased activity, decreased activity, altered type or type of activity present. Thus, in this embodiment, as described herein, the candidate agent binds ASP05 (although this is not necessary) and alters its biological or biochemical activity. There must be.
These methods generally include in vitro screening methods as described above,
Both in vivo cell screening for changes in the presence, distribution, activity or amount of ASP05.

Thus, in this embodiment, the method of the invention comprises combining the ASP05 protein with a candidate bioactive agent and assaying the ASP05 biological activity as known to those of skill in the art to determine the ASP05 activity of the agent. This includes assessing the effects. "ASP
"05 activity" or synonyms thereof, as used herein, refers to one of the putative identified functions of ASP05, including but not limited to the ability of ASP05 to selectively cleave IGFBP, IGFBP. It includes the ability of ASP05 to bind, the ability of ASP05 to alter IGF-mediated cell function.

In certain embodiments, such modulation may result in a response to the catalytic domain of ASP05, corresponding to increased or decreased selective cleavage. In a preferred embodiment,
The activity of the catalytic domain of ASP05 is reduced. Thus, in some embodiments bioactive agents that are antagonists (ie, increased activity of ASP05 protein) are preferred, and in other embodiments bioactive agents that are agonists are preferred. For example, a substance that binds to ASP05 protein can be an antagonist. Moreover, substances that bind ASP05 increase the specific cleavage by the ASP05 protein and thus act as agonists.

In other embodiments, such modulation may result in a response to the binding domain of ASP05, corresponding to increased or decreased binding. In a preferred embodiment, ASP
The activity of the 05 binding domain is reduced. Thus, in some embodiments bioactive agents that are antagonists (ie, increased activity of ASP05 protein) are preferred, and in other embodiments bioactive agents that are agonists are preferred. For example, a substance that binds to ASP05 protein can be an antagonist. Moreover, substances that bind ASP05 increase the specific cleavage by the ASP05 protein and thus act as agonists.

In a preferred embodiment, the present invention provides a method for screening bioactive agents capable of modulating the serine protease activity of ASP05 protease. In this method, the above-mentioned candidate bioactive substance is converted into ASP05 protein or A
Adding to cells containing the SP05 catalytic domain. Suitable cell types include SF
9, E. coli, and mammalian cells, but are not limited thereto. The cells are recombinant nucleic acids encoding ASP05 protease, namely ASP05
Including cells expressing. In a preferred embodiment, the library of candidate substances is tested in multiple assays for different IGFBPs. For example, investigate the effect of a candidate substance on selective cleavage. When ASP05 acts, ie, in the absence of antagonistic substances, IGFBPs are not selectively cleaved, as evidenced by Westen blot analysis with detection of antibody or ligand. On the other hand, in the presence of an antagonistic substance, cleavage does not occur.

Cleavage detection is performed as done by one of skill in the art. In some embodiments, the purified AS
P05 includes various IGs including, but not limited to, IGFBP-1 to IGFBP-6.
Incubate with FBP. The reaction products to be analyzed are run on an SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. In a further embodiment, the membrane is incubated with an IGFBP-specific antibody, incubated with a second antibody, such as a HRP-conjugated mouse IgG antibody, and then visualized with a chemiluminophore. In another embodiment, detection is by incubation with ¹²⁵ IGF-1 (or ¹²⁵ IGF-1) followed by autoradiography.

In addition, assays using fluorescent synthetic peptides as substrates can also be performed as would be performed by one of skill in the art, including competitive assays.

A variety of other reagents can be included in the screening assay. It includes reagents such as salts, albumin, neutral proteins, detergents, etc. that may facilitate optimal protein-protein binding and / or may be used to reduce non-specific or background interactions. is there. Reagents that can improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., can also be used.
Mixtures of ingredients can be added for the required binding.

Bioactive substances are identified in this way. Compounds having pharmacological activity are ASP
05 promotes or inhibits the activity of the protein. A compound having the desired pharmacological activity can be administered to the host in a physiologically acceptable carrier, as described above. These substances can be administered orally, parenterally such as subcutaneous injection, intraperitoneal injection and intravascular injection, and various other methods. The compound can be formulated into various forms depending on the kind of administration method. The concentration of therapeutically active ingredient in the formulation is about 0.1-1.
It can vary from 00% by weight.

The pharmaceutical composition can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, dispersants, ointments, lotions and other similar forms. Pharmaceutical grade organic or inorganic carriers, and / or diluents suitable for oral or topical use can be used to prepare compositions containing the therapeutically effective compounds. Diluents known to the art include aqueous media, vegetable or animal oils and fats. Stabilizers, wetting and dispersing agents, salts for changing the osmotic pressure or buffers for maintaining a suitable pH value, and skin penetration enhancers can be used as auxiliaries.

Without being bound by theory, ASP05 is a key enzyme involved in the regulation, differentiation, proliferation and function of many types of cells. These cells include those from the brain, blood, bones, liver, muscles, kidneys and nervous system. Therefore, mutated or variant ASP
05-based disorders or concentrations in various body fluids or tissues can be measured. In one aspect, the invention provides methods for identifying cells that contain a variant form or concentration of ASP05. This involves measuring the type and concentration of exogenous ASP05 in body fluids and tissues. As will be appreciated by those in the art, the measurement can be done by many commonly used techniques. The method involves comparing the type or concentration of ASP05 with a known type or concentration of ASP05 in different biological sources.

In a preferred embodiment, the presence of a difference in the type or concentration of ASP05 in a patient is indicative of a disease state or tendency thereof, as described herein. The discovery of the role of ASP05 in the selective cleavage of IGFBPs provides a method for screening inhibitors of such cleavage.

In a preferred embodiment, the ASP05 protein and especially fragments thereof are IGF
Are useful in the study and treatment of conditions mediated by, ie, in diagnosing, treating and preventing IGF-mediated disorders. That is, "IGF-mediated disorder" or "disease state"
Includes conditions involving IGF-mediated disorders or cellular processes and conditions with inappropriate IGF metabolism. Thus, IGF-mediated disorders include, but are not limited to, cancer (
Proliferative disorders such as various types), neoarterial intimal hyperplasia associated with restenosis, angiogenesis associated with tumor growth, neovascularization disorders of the eye. This eye disease involves cumulative muscle degeneration (
AMD), diabetic retinopathy, premature retinopathy (ROP), ischemic retinopathy, and neovascular glaucoma. Other IGF-mediated disorders include, but are not limited to, disorders of bone metabolism such as osteoporosis and osteoarthritis, and disorders of the muscle, brain, ovary, uterus, placenta.

Accordingly, in certain embodiments, a method of inhibiting IGFBP comprises selective antagonism of IGF in using the inhibitor in the treatment of human disease. In some embodiments, the method comprises administering to the cells an anti-ASP05 antibody that reduces or eliminates the biological activity of endogenous ASP05. Alternatively, this method
Administering a recombinant nucleic acid encoding SP05 to a cell or organism.
As will be apparent to those skilled in the art, this can be done in any number of ways. In a preferred embodiment, for example, by utilizing known gene therapy techniques, by inhibiting the expression of endogenous ASP05 or by administering a gene encoding ASP05,
The amount of ASP05 in the cell is reduced to reduce ASP05 activity. In a preferred embodiment, the gene therapy technique involves introducing an exogenous gene.

In one embodiment, the invention provides a method of diagnosing an IGF-mediated disorder in an individual. The method involves measuring ASP05 activity and expression in tissue from the individual or patient, which involves measuring the amount and specific activity of ASP05. This activity is quantified and compared to the activity of ASP05 obtained either from unaffected second individuals or from unaffected tissues of the first individual. If the activities differ,
The first individual is at risk of an IGF-mediated disorder. In this way, for example, ASP
By monitoring the 05 level, various disease states can be monitored. Similarly, ASP05 levels may be correlated with the listed diseases and conditions.

In one aspect, by using the ASP05 protein of the invention
Polyclonal and monoclonal antibodies to the catalytic or binding domains of proteins can be produced and are useful as described herein. Similarly,
ASP05 protein can be bound to an affinity chromatography column using standard techniques. Then, by using these columns, ASP05
The antibody may be purified. In a preferred embodiment, the antibody is raised against an epitope unique to the ASP05 protein. That is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in many applications. For example, the ASP05 protein can be purified by using the ASP05 antibody bound to a standard affinity chromatography column. Antibody is AS
Since it specifically binds to the P05 protein, it can also be used for blocking the above polypeptides.

In one aspect, a therapeutically effective amount of ASP05 or an inhibitor thereof is administered to a patient. As used herein, "therapeutically effective amount" means the dose that produces the effect for which it is administered. The exact dose depends on the purpose of the treatment and can be ascertained by one skilled in the art using known techniques. Regulation of ASP05 degradation, systemic versus local delivery, and rate of de novo protease synthesis, as well as age, weight, general health, as known in the art,
Gender, diet, time of administration, drug interaction and severity of the condition may be needed and can be ascertained by one of ordinary skill in the art in routine trials.

“Patient” for the purposes of the present invention includes both humans and other animals, especially mammals and organisms. That is, these methods can be applied to both human medical and veterinary indications. In the preferred embodiment, the patient is a mammal, and in the most preferred embodiment the patient is human.

Administration of the ASP05 protein of the invention and its inhibitors can be carried out by various routes, such as the oral, subcutaneous, intravenous, intranasal, transdermal, intraperitoneal, intramuscular, pulmonary, vaginal, rectal or intraocular routes. However, it is not limited to these. In some cases,
For example, in the treatment of injury and inflammation, ASP05 can be applied directly as a solution or spray.

The pharmaceutical composition of the invention comprises ASP05 or an inhibitor thereof in a form suitable for administration to a patient. In a preferred embodiment, the pharmaceutical composition is present in a water-soluble form, eg as pharmaceutically acceptable salts, which shall include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" includes salts that retain the biological effectiveness of the free base and are biologically or otherwise acceptable,
Inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, Citric acid, benzoic acid, cinnamic acid, mandelic acid,
It is formed of methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

“Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. is there. Particularly preferred is
Such as ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary and tertiary amines, substituted amines such as naturally substituted amines, cyclic amines and basic ion exchange resins such as iro. There are salts of propylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine.

These pharmaceutical compositions also include the following substances: carrier proteins such as serum albumin, buffers, fillers such as microcrystalline cellulose, lactose, corn and other starches, binders, sweeteners. And one or more of other seasonings, colorants, and polyethylene glycols. Additives are well known in the art and are used in various formulations.

The following is a more detailed description of the method of use of the invention, including examples, and further illustrates the best method envisioned for practicing the various aspects of the invention. These examples are not intended to limit the true scope of the invention in any way, but are presented for illustrative purposes only. All references cited herein are incorporated herein by reference.

Example 1 Cloning of full length ASP05 cDNA A synthetic 5'end of ASP05 cDNA was created using a combined strategy of annealing, ligation and polymerase chain reaction (PCR). This synthetic sequence contained the first 156 nucleotides in the coding region, but did not modify any of the encoded amino acids. The full-length cDNA contains 1443 nucleotides (including the stop codon). The complete native cDNA sequence and the sequence used to express the active protein are shown in FIG. 1 used for annealing
The two oligonucleotides are:

[Chemical 1]

[Chemical 2]

Complementary oligonucleotides (50 pmol each) were added to 10 mM Tris-Cl, pH 7.
4, mixed with 150 mM NaCl, then heated at 95 ° C. for 3 minutes and slowly cooled to room temperature. Then mix all six annealed DNA fragment pairs,
Ligation was performed using Rapid DNA Ligation Reagent (Boehringer Mannheim Corp., Indianapolis, IN).

[0154]   In the PCR reaction, this ligated cDNA was used as a template to prepare a primer.

[Chemical 3] Used together to amplify the complete fragment. The PCR method was carried out by using cDNA fragments and primers (each 100 pmol), dNTPs each 0.2 mM, Taq DNA polymerase (Perki).
n Elmer, Branchburg, NJ) 0.1 U, 50 mM KCl, 1.75 mM MgCl ₂ and 10 mM Tris-HCl, pH 8.4 in 100 μl of solution. PCR
The method was performed for 25 cycles with an amplification profile as follows: denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 1 minute, followed by extension at 72 ° C for 1 minute. This P
The CR product was cloned into a TA cloning vector (Invitrogen, Carlsbad, CA).

The 3'-870 bp end of ASP05 was labeled with human placental cDNA (Clo
ntech, Palo Alto, CA) and sequences

[Chemical 4] (SEQ ID NO: 18) 5'terminal primer and sequence

[Chemical 5] It was cloned by PCR using the 3'terminal primer of (SEQ ID NO: 19).
This fragment was amplified and cloned as above.

This 5 ′ end fragment was excised from the vector using the restriction enzymes BamHI and HindIII and separated from the vector on a 1% agarose gel. DNA bands of the expected size were extracted with Quick Gel Extraction Reagent (Qiagen, Inc., Chatsworth, CA). The purified DNA fragment was ligated with the 3'terminal fragment of the HindIII site using a rapid ligation reagent. The full-length ASP05 cDNA is obtained from the ABI377 DNA sequencer (
Complete sequencing in both directions using the ABI Division, Perkin Elmer, Foster City, CA).

Example 2 Expression of ASP05 and Protein Purification in Baculovirus Proteins were expressed using the Bac-to-Bac baculovirus expression system (GibcoBRL, Gaithersburg, MD). In short, flag epitope labeling (Eastman Kodak, N
ew Haven. CT) -attached ASP05 cDNA was ligated with pFASTBac1 vector and transformed into DHI OBac competent cells to obtain recombinant bacmid (bacm).
id) was created. This bacmid DNA was transfected into sf9 insect cells to produce recombinant baculovirus. High titer stocks were used to infect sf9 insect cells (multiplicity of infection> 5) cultured in SF-900II SFM. The medium was collected 48 hours after infection. The expressed protein was purified by affinity chromatography using an agarose-conjugated antibody specific for the flag epitope tag inserted at the C-terminus of expressed ASP05. In short, 90ml of 4ml flag agarose
After incubating with 0 ml of medium at 4 ° C. for 2 hours, the agarose medium slurry was injected into a 10 ml column. This gel was washed with 20 gels of phosphate buffered saline (PBS, pH 7.2) to bind the bound ASP05 with 4 gels of Tri.
It was eluted with s-glycine solution (pH 2.5). Immediately add this eluate to 10x PBS.
It was neutralized with and concentrated 20 times with microsep 10K (Filtron, Northborough, MA). The concentrated protein was dialyzed against BPS at 4 ° C overnight. Affinity purified ASP05 is a BCA protein quantification reagent (Pierce, Rockf
ord, IL).

Example 3 Determination of the N-terminal amino acid sequence of ASP05 Purified ASP05 (2 μg) was run on a 4-20% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. did. The membrane was stained with Coomassie blue in 50% methanol for 5 minutes and destained with 10% acetic acid and 10% methanol. The correct molecular weight protein bands were cut out and sent to the Protein Structures Laboratory at the University of California, Davis for sequence analysis.

[0159]   The N-terminal sequences of insect cells expressing mature ASP05 are as follows:

[Chemical 6] .

Example 4 Assay for Proteolytic Activity of ASP05 Purified ASP05 (200 nM) was added to 25 mM Hepes (pH 7.4), 25
Incubated with 125 nM IGFBF-5 in 20 μl of mM CHES, 50 mM NaCl and 0.025% Tween 20 (assay buffer) for 2 hours at 37 ° C. The reaction products to be analyzed were run on a 4-20% gradient of sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane which was then loaded with 10 mM Tris-HCl, pM.
1% in H8.0, 150 mM NaCl, and 0.05% Tween (TBST)
Blocking was performed with bovine serum albumin (BSA) for 1 hour. After that, this film is
Incubated with FBP-5 antibody (Autral Biologicals) for 1 hour. T
After washing 3 times with BST (15 minutes each), the membrane was washed with HRP-conjugated mouse I.
Incubated with gG antibody for 1 hour. The PVDF membrane was washed 3 times again for 15 minutes each time and the protein was labeled with ECL chemiluminophore (Amersham, Arlington Heights., IL)
Visualized with. At the time of the test, ASP05 is replaced with IGBT-1, IGBT-2, and IGBT.
When cleaved by -3, IGBT-4 or IGBT-6, 125 nM of each was added to the reaction instead of IGFBP-5, and the analysis was performed as described above (see FIG. 4A).

Example 5 Ligand Blot Analysis for IGFBP Cleavage Activity of ASP05 Purified ASP05 (200 nM) was added to 20 ml of 25 mM Hepes (pH 7.4).
), 25 mM CHES, 50 mM NaCl and 0.025% Tween 20 (assay buffer), IGFBP-1, IGFBP-2, IGFBP-3, IG.
Each was incubated with 125 nM each of FBP-4, IGFBP-5 and IGFBP-6 at 37 ° C. for 2 hours. The reaction product was run on a non-reducing 4-20% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was washed with 0.15M NaCl, 0.01M Tris-HCl, p.
Incubated in H7.4 (TS) and 3% NP40 for 30 minutes at 4 ° C. (all steps were done at 4 ° C. hereafter), then added 1% BSA in TS for 2 hours followed by Tween20 The membrane was rinsed for 10 minutes in TS, 10 minutes. Then 3 ml
TS, 1% BSA and 0.1% Tween 20, 400,000 I ¹²⁵ I
Membranes were incubated overnight with GF-I (or ¹²⁵ IGF-II). TS, 0.
After washing twice in 1% Tween 20 for 15 minutes each and once in TS for 15 minutes, the membranes were air dried and exposed to X-ray film overnight. The film was developed with Konika QX-130A self film developer (Figure 4B).

Example 6 Inhibitory Effect of Protease Inhibitors on the Proteolytic Activity of ASP05 To analyze the inhibitory effect of protease inhibitors on the proteolytic activity of ASP05, each individual inhibitor was assayed at 37 ° C. together with ASP05 in assay buffer. Incubate for 1 hour, then add IGFBP-5 as above. The reaction was further incubated at 37 ° C for 2 hours. The protease inhibitors (and concentrations used) analyzed were: aprotinin (1.5 μM)
), Antipain (370 μM), 3,4-DCI (1 mM), E64 (140 μM)
), Leupeptin (5 μM), pepstatin A (5 μM), PMSF (5 mM), L
-1-Chloro-3- (4-tosylamide) -7-amino-2-heptanone-HC
I (TLCK) (675 μM), L-1-chloro-3- (4-tosylamide)-
4-phenyl-2-butanone (TPCK) (1.4 mM) and EDTA (10 mM)
). Results are shown in FIG. ASP05 was inhibited only by the serine protease inhibitors 3,4 dichloroisocoumarin (lane 5) and PMSF (lane 9) of the inhibitors tested.

Example 7 Evaluation of Autocleavage Activity of ASP05 Purified ASP05 (200 nM) was added to 25 mM Hepes (pH 7.4), 25
Incubated for 15 hours at 37 ° C. in 20 μl of mM CHES, 50 mM NaCl and 0.025% Tween 20 (assay buffer). 10 μl of the reaction product was added to 4-20% sodium dodecyl sulfate / polyacrylamide gel (SDS-PAG).
E) and then transferred to a polyvinylidene difluoride (PVDF) membrane, which is then 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and
Blocking with 1% bovine serum albumin (BSA) in 05% Tween (TBST) for 1 hour. Next, this membrane was used together with ASP05 peptide antibody (Zymed, Inc)
Incubated for hours. After washing with TBST three times for 15 minutes each, the membrane was washed with H
Incubated with RP-conjugated rabbit IgG antibody for 1 hour. P
The VDF membrane was washed 3 times again for 15 minutes each time to allow the protein to bind to the ECL chemiluminophore (Amer
sham, Arlington Heights., IL) (Fig. 3).

Example 8 Construction of ASP05 C-Terminal Fragment Expression Vector A cDNA encoding the ASP05 C-terminal fragment was prepared using the polymerase chain reaction (PCR). The two oligonucleotides used in the PCR method were as follows; oligo 1:

[Chemical 7] . Using these two oligonucleotides as primers, full-length synthetic ASP05
This fragment was amplified using the cDNA as a template in a PCR reaction. PCR
The method is as follows: template and primer (100 pmol each), dNTP 0.2 mM each, TaqD.
NA polymerase (Perkin Elmer, Branchburg, NJ) 0.1 U, 50 mM KCl,
It was carried out in 100 μl of a solution containing 1.75 mM MgCl ₂ and 10 mM Tris-HCl, pH 8.4. The PCR method was performed for 25 cycles with an amplification profile as follows: 95 ° C for 1 min denaturation, 55 ° C for 1 min annealing, followed by 72 ° C for 1 min extension. The PCR product was extracted with phenol-chloroform,
It was precipitated with ethanol. This DNA pellet was added to 10 mM Tris-HCl, p
Resuspended in H7.6, 1 mM EDTA (TE).

The amplified cDNA fragment was treated with restriction enzymes BamHI and PstI at 37 ° C. for 1 hour and purified on a 1% agarose gel. A DNA band of the expected size was excised from the gel and extracted with a quick gel extraction reagent (Qiagen, Inc., Chatsworth, CA).
). The purified DNA fragment was treated with BamH in the same manner as the amplified cDNA fragment.
I and PstI treated agarose gel purified pQE-30 vector (
Qiagen, Inc., Chatsworth, CA). A restriction digest was used to confirm that the cDNA was inserted into the vector in the correct orientation.

Plasmid (0.2μ) was added to 100μl of SG13009 competent cells (Qiag
en, Inc., Chatsworth, CA), the mixture was placed on ice for 20 minutes and then the mixture was heated at 42 ° C. for 90 minutes. After cooling on ice for 2 minutes, 10 mM MgC
SOC medium 500μl containing l ₂ was added to the mixture and shaken vigorously for one hour the cells at 37 ° C.. Cells were spread on LB agar plates containing 100 mg / ml ampicillin and 10 mg / ml kanamycin and grown overnight at 37 ° C.

Example 9 Expression of ASP05 C-Terminal Fragment in E. coli A single colony of transformed E. coli was picked from the plate and 100μ
37 in LB broth containing g / ml ampicillin and 10 mg / ml kanamycin
Grow overnight with vigorous shaking at ° C. The following morning, the culture was 1:50 with LB broth.
Diluted and grown at 37 ° C. to an OD600 value of 0.5. Isopropyl (3-D-thiogalactopyrronoside (IPTG) was added to a final concentration of 2 mM and the culture was added to 3 more volumes.
Continued to grow for hours. The cells were harvested by centrifugation and the cell pellet 20
Resuspended in 0 μl deionized water. 100 μl of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) sample buffer was added to lyse the cells and heated at 95 ° C. for 2 hours. Reduce SDS-PAG with 5 μl of each sample
Separated by E, the expressed ASP05 C-terminal fragment was visualized by Coomassie blue staining.

Example 10 Scale-Up Expression and Purification of ASP05 C-Terminal Fragment Transformed E. coli colonies were grown in 3 ml LB containing 100 μmg / ml ampicillin and 10 mg / ml kanamycin with vigorous infiltration at 37 ° C. overnight. . After 15 hours, the culture was transferred to 150 ml of the same medium which had been pre-warmed to obtain OD60.
It was grown to a 0 value of 0.6. IPTG was added to a final concentration of 2 mM and the culture was continued for another 5 hours. The cells were harvested by centrifugation and frozen at -80 ° C. Next morning, 6M Guanidine-HCl, 0.1M Sodium Phosphate, and 0.01M Tris
The cells were thawed in 20 ml of HCl, pH 8.0 (GuHCl). The cells were stirred at room temperature for 1 hour and centrifuged at 10,000 g for 15 minutes at 4 ° C. This supernatant is
Ni-NTA column (Qiagen, In) pre-equilibrated with 10 column of GuHCl
c., Chatsworth, CA). Then, the column is loaded with 10 column amounts of GuHCl.
Wash with buffer, then 8M urea, 0.1m sodium phosphate, and 0.1M.
01M Tris-HCl, pH 8.0, then 10 column volumes of 8M urea, 0.0.
Wash with 1m sodium phosphate, and 0.01M Tris-HCl, pH 6.3. The bound protein was eluted with 5 column volumes of 8M urea, 0.1m sodium phosphate, and 0.01M Tris-HCl, pH 4.5. The protein was dialyzed against 10 mM Mes buffer, pH 5.5. After dialysis, the final urea concentration was less than 10 ^-7 M.

Example 11 Analysis of Proteolytic Activity of C-Terminal Fragment of ASP05 Purified ASP05 C-terminal fragment was added to 25 mM Hepes (pH 7.4), 25 mM.
Incubated with 125 nM IGFBP-5 in 20 μl of CHES, 50 mM NaCl and 0.025% Tween 20 (assay buffer) for 2 hours at 37 ° C. The reaction product was then run on a 4-20% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane, which was then 10 mM.
Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween.
Blocked with 1% bovine serum albumin (BSA) in 20 (TBST) for 1 hour. Next, this membrane was treated with IGFBP-5 antibody (Autral Biologicals, San Ramon, CA).
And incubated for 1 hour. After washing 3 times with TBST for 15 minutes each,
Membranes were incubated for 1 hour with HRP-conjugated mouse IgG antibody. The PVDF membrane was washed three times again for 15 minutes each time to allow the proteins to be washed with the ECL chemiluminophore (
Amersham, Arlington Heights., IL).

This fragment is derived from other IGFBPs, namely IGFBP-1, IGFBP-2,
It was also tested for its ability to cleave IGFBP-3, IGFBP-4 or IGFBP-6. For these reactions, IGFBP125nM was added to each IGFBP
Instead of -5, added to reaction as above.

[0171]

[Sequence list]

      SEQUENCE LISTING <110> Axys Pharmaceuticals, Inc. <120> Novel Serine Protease Capable of Selective Cleavage of                Insulin-like Growth Factor Binding Protein <130> FP66065-1 / RMS / DSS <140> PCT / US99 / 09224 <141> 1999-04-28 <150> 60 / 083,321 <151> 1998-04-28 <160> 24 <170> PatentIn Ver. 2.0 <210> 1 <211> 1443 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (1) .. (1443)          <220> <223> Description of Artificial Sequence: synthetic <400> 1 atg cag att cca aga gct gca ttg tta cct ctt ctg tta tta ctg ctc 48 Met Gln Ile Pro Arg Ala Ala Leu Leu Pro Leu Leu Leu Leu Leu   1 5 10 15 gca gct cct gca tct gca caa ctt tct cga gct gga aga tct gct cca 96          Ala Ala Pro Ala Ser Ala Gln Leu Ser Arg Ala Gly Arg Ser Ala Pro              20 25 30          tta gct gct gga tgt cct gat aga tgt gag cca gct aga tgt cct cca 144          Leu Ala Ala Gly Cys Pro Asp Arg Cys Glu Pro Ala Arg Cys Pro Pro          35 40 45 caa cct gaa cat tgc gaa ggt ggt aga gct aga gat gca tgc gga tgt 192 Gln Pro Glu His Cys Glu Gly Gly Arg Ala Arg Asp Ala Cys Gly Cys      50 55 60          tgc gaa gtt tgc gga gct cct gaa gga gct gct tgt gga tta caa gag 240   Cys Glu Val Cys Gly Ala Pro Glu Gly Ala Ala Cys Gly Leu Gln Glu  65 70 75 80 ggt cct tgt gga gaa ggt cta caa tgc gta gtt cca ttc gga gta cca 288 Gly Pro Cys Gly Glu Gly Leu Gln Cys Val Val Pro Phe Gly Val                  85 90 95 gct tca gca aca gta aga cga agg gcc caa gct ggt tta tgt gta tgc 336 Ala Ser Ala Thr Val Arg Arg Arg Ala Gln Ala Gly Leu Cys Val Cys             100 105 110 gcg agt tca gaa cca gta tgt ggc tct gat gca aat aca tac gca aac 384 Ala Ser Ser Glu Pro Val Cys Gly Ser Asp Ala Asn Thr Tyr Ala Asn         115 120 125 tta tgc caa ttg aga gct gct tct aga cgt agt gaa aga cta cat aga 432 Leu Cys Gln Leu Arg Ala Ala Ser Arg Arg Ser Glu Arg Leu His Arg     130 135 140 ccg cct gtt ata gtc ctg caa cgg gga gcc tgc ggc caa ggg cag gaa 480          Pro Pro Val Ile Val Leu Gln Arg Gly Ala Cys Gly Gln Gly Gln Glu 145 150 155 160 gat ccc aac agt ttg cgc cat aaa tat aac ttt atc gcg gac gtg gtg 528 Asp Pro Asn Ser Leu Arg His Lys Tyr Asn Phe Ile Ala Asp Val Val                 165 170 175 gag aag atc gcc cct gcc gtg gtt cat atc gaa ttg ttt cgc aag ctt 576          Glu Lys Ile Ala Pro Ala Val Val His Ile Glu Leu Phe Arg Lys Leu             180 185 190 ccg ttt tct aaa cga gag gtg ccc gtg gct agt ggg tct ggg ttt att 624 Pro Phe Ser Lys Arg Glu Val Pro Val Ala Ser Gly Ser Gly Phe Ile         195 200 205 gtg tcg gaa gat gga ctg atc gtg aca aat gcc cac gtg gtg acc aac 672 Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala His Val Val Thr Asn     210 215 220 aag cac cgg gtc aaa gtt gag ctg aag aac ggt gcc act tac gaa gcc 720 Lys His Arg Val Lys Val Glu Leu Lys Asn Gly Ala Thr Tyr Glu Ala 225 230 235 240 aaa atc aag gat gtg gat gag aaa gca gac atc gca ctc atc aaa att 768   Lys Ile Lys Asp Val Asp Glu Lys Ala Asp Ile Ala Leu Ile Lys Ile                 245 250 255 gac cac cag ggc aag ctg cct gtc ctg ctg ctt ggc cgc tcc tca gag 816 Asp His Gln Gly Lys Leu Pro Val Leu Leu Leu Gly Arg Ser Ser Glu             260 265 270 ctg cgg ccg gga gag ttc gtg gtc gcc atc gga agc ccg ttt tcc ctt 864 Leu Arg Pro Gly Glu Phe Val Val Ala Ile Gly Ser Pro Phe Ser Leu         275 280 285 caa aac aca gtc acc acc ggg atc gtg agc acc acc cag cga ggc ggc 912    Gln Asn Thr Val Thr Thr Gly Ile Val Ser Thr Thr Gln Arg Gly Gly     290 295 300 aaa gag ctg ggg ctc cgc aac tca gac atg gac tac atc cag acc gac 960 Lys Glu Leu Gly Leu Arg Asn Ser Asp Met Asp Tyr Ile Gln Thr Asp 305 310 315 320 gcc atc atc aac tat gga aac tcg gga ggc ccg tta gta aac ctg gac 1008 Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu Val Asn Leu Asp                 325 330 335 ggt gaa gtg att gga att aac act ttg aaa gtg aca gct gga atc tcc 1056 Gly Glu Val Ile Gly Ile Asn Thr Leu Lys Val Thr Ala Gly Ile Ser             340 345 350 ttt gca atc cca tct gat aag att aaa aag ttc ctc acg gag tcc cat 1104 Phe Ala Ile Pro Ser Asp Lys Ile Lys Lys Phe Leu Thr Glu Ser His         355 360 365 gac cga cag gcc aaa gga aaa gcc atc acc aag aag aag tat att ggt 1152 Asp Arg Gln Ala Lys Gly Lys Ala Ile Thr Lys Lys Lys Tyr Ile Gly     370 375 380 atc cga atg atg tca ctc acg tcc agc aaa gcc aaa gag ctg aag gac 1200   Ile Arg Met Met Ser Leu Thr Ser Ser Lys Ala Lys Glu Leu Lys Asp 385 390 395 400 cgg cac cgg gac ttc cca gac gtg atc tca gga gcg tat ata att gaa 1248 Arg His Arg Asp Phe Pro Asp Val Ile Ser Gly Ala Tyr Ile Ile Glu                 405 410 415 gta att cct gat acc cca gca gaa gct ggt ggt ctc aag gaa aac gac 1296          Val Ile Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp             420 425 430 gtc ata atc agc atc aat gga cag tcc gtg gtc tcc gcc aat gat gtc 1344 Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Ser Ala Asn Asp Val         435 440 445 agc gac gtc att aaa agg gaa agc acc ctg aac atg gtg gtc cgc agg 1392 Ser Asp Val Ile Lys Arg Glu Ser Thr Leu Asn Met Val Val Arg Arg     450 455 460 ggt aat gaa gat atc atg atc aca gtg att ccc gaa gaa att gac cca 1440 Gly Asn Glu Asp Ile Met Ile Thr Val Ile Pro Glu Glu Ile Asp Pro 465 470 475 480 tag 1443          <210> 2 <211> 480 <212> PRT <213> Artificial Sequence          <400> 2 Met Gln Ile Pro Arg Ala Ala Leu Leu Pro Leu Leu Leu Leu Leu Leu   1 5 10 15 Ala Ala Pro Ala Ser Ala Gln Leu Ser Arg Ala Gly Arg Ser Ala Pro              20 25 30 Leu Ala Ala Gly Cys Pro Asp Arg Cys Glu Pro Ala Arg Cys Pro Pro          35 40 45 Gln Pro Glu His Cys Glu Gly Gly Arg Ala Arg Asp Ala Cys Gly Cys      50 55 60 Cys Glu Val Cys Gly Ala Pro Glu Gly Ala Ala Cys Gly Leu Gln Glu  65 70 75 80   Gly Pro Cys Gly Glu Gly Leu Gln Cys Val Val Pro Phe Gly Val Pro                  85 90 95 Ala Ser Ala Thr Val Arg Arg Arg Ala Gln Ala Gly Leu Cys Val Cys             100 105 110 Ala Ser Ser Glu Pro Val Cys Gly Ser Asp Ala Asn Thr Tyr Ala Asn         115 120 125 Leu Cys Gln Leu Arg Ala Ala Ser Arg Arg Ser Glu Arg Leu His Arg     130 135 140 Pro Pro Val Ile Val Leu Gln Arg Gly Ala Cys Gly Gln Gly Gln Glu 145 150 155 160 Asp Pro Asn Ser Leu Arg His Lys Tyr Asn Phe Ile Ala Asp Val Val                 165 170 175 Glu Lys Ile Ala Pro Ala Val Val His Ile Glu Leu Phe Arg Lys Leu             180 185 190 Pro Phe Ser Lys Arg Glu Val Pro Val Ala Ser Gly Ser Gly Phe Ile         195 200 205 Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala His Val Val Thr Asn     210 215 220 Lys His Arg Val Lys Val Glu Leu Lys Asn Gly Ala Thr Tyr Glu Ala 225 230 235 240 Lys Ile Lys Asp Val Asp Glu Lys Ala Asp Ile Ala Leu Ile Lys Ile                 245 250 255 Asp His Gln Gly Lys Leu Pro Val Leu Leu Leu Gly Arg Ser Ser Glu             260 265 270 Leu Arg Pro Gly Glu Phe Val Val Ala Ile Gly Ser Pro Phe Ser Leu         275 280 285 Gln Asn Thr Val Thr Thr Gly Ile Val Ser Thr Thr Gln Arg Gly Gly     290 295 300 Lys Glu Leu Gly Leu Arg Asn Ser Asp Met Asp Tyr Ile Gln Thr Asp 305 310 315 320 Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu Val Asn Leu Asp                 325 330 335          Gly Glu Val Ile Gly Ile Asn Thr Leu Lys Val Thr Ala Gly Ile Ser             340 345 350     Phe Ala Ile Pro Ser Asp Lys Ile Lys Lys Phe Leu Thr Glu Ser His         355 360 365          Asp Arg Gln Ala Lys Gly Lys Ala Ile Thr Lys Lys Lys Tyr Ile Gly     370 375 380      Ile Arg Met Met Ser Leu Thr Ser Ser Lys Ala Lys Glu Leu Lys Asp 385 390 395 400 Arg His Arg Asp Phe Pro Asp Val Ile Ser Gly Ala Tyr Ile Ile Glu                 405 410 415 Val Ile Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp             420 425 430 Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Ser Ala Asn Asp Val         435 440 445 Ser Asp Val Ile Lys Arg Glu Ser Thr Leu Asn Met Val Val Arg Arg     450 455 460 Gly Asn Glu Asp Ile Met Ile Thr Val Ile Pro Glu Glu Ile Asp Pro 465 470 475 480 <210> 3 <211> 1443 <212> DNA <213> Homo sapiens <400> 3 atgcagatcc cgcgcgccgc tcttctcccg ctgctgctgc tgctgctggc ggcgcccgcc 60          tcggcgcagc tgtcccgggc cggccgctcg gcgcctttgg ccgccgggtg cccagaccgc 120          tgcgagccgg cgcgctgccc gccgcagccg gagcactgcg agggcggccg ggcccgggac 180          gcgtgcggct gctgcgaggt gtgcggcgcg cccgagggcg ccgcgtgcgg cctgcaggag 240          ggcccgtgcg gcgaggggct gcagtgcgtg gtgcccttcg gggtgccagc ctcggccacg 300          gtgcggcggc gcgcgcaggc cggcctctgt gtgtgcgcca gcagcgagcc ggtgtgcggc 360          agcgacgcca acacctacgc caacctgtgc cagctgcgcg ccgccagccg ccgctccgag 420          aggctgcacc ggccgccggt catcgtcctg cagcgcggag cctgcggcca agggcaggaa 480          gatcccaaca gtttgcgcca taaatataac tttatcgcgg acgtggtgga gaagatcgcc 540          cctgccgtgg ttcatatcga attgtttcgc aagcttccgt tttctaaacg agaggtgccg 600          gtggctagtg ggtctgggtt tattgtgtcg gaagatggac tgatcgtgac aaatgcccac 660          gtggtgacca acaagcaccg ggtcaaagtt gagctgaaga acggtgccac ttacgaagcc 720          aaaatcaagg atgtggatga gaaagcagac atcgcactca tcaaaattga ccaccagggc 780          aagctgcctg tcctgctgct tggccgctcc tcagagctgc ggccgggaga gttcgtggtc 840          gccatcggaa gcccgttttc ccttcaaaac acagtcacca ccgggatcgt gagcaccacc 900          cagcgaggcg gcaaagagct ggggctccgc aactcagaca tggactacat ccagaccgac 960          gccatcatca actatggaaa ctcgggaggc ccgttagtaa acctggacgg tgaagtgatt 1020          ggaattaaca ctttgaaagt gacagctgga atctcctttg caatcccatc tgataagatt 1080          aaaaagttcc tcacggagtc ccatgaccga caggccaaag gaaaagccat caccaagaag 1140          aagtatattg gtatccgaat gatgtcactc acgtccagca aagccaaaga gctgaaggac 1200          cggcaccggg acttcccaga cgtgatctca ggagcgtata taattgaagt aattcctgat 1260          accccagcag aagctggtgg tctcaaggaa aacgacgtca taatcagcat caatggacag 1320          tccgtggtct ccgccaatga tgtcagcgac gtcattaaaa gggaaagcac cctgaacatg 1380          gtggtccgca ggggtaatga agatatcatg atcacagtga ttcccgaaga aattgaccca 1440          tag 1443          <210> 4 <211> 85 <212> DNA <213> Artificial Sequence          <220> <223> Description of Artificial Sequence: synthetic          <400> 4 atgcagattc caagagctgc attgttacct cttctgttat tactgctcgc agctcctgca 60          tctgcacaac tttctcgagc tggaa 85          <210> 5 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 5 agcagatctt ccagctcgag aaagttgtgc agatgcagga gctgcgagca gtaataacag 60      aagaggtaac aatgcagctc ttggaatctg cat 93          <210> 6 <211> 103 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic     <400> 6 gatctgctcc attagctgct ggatgtcctg atagatgtga gccagctaga tgtcctccac 60          aacctgaaca ttgcgaaggt ggtagagcta gagatgcatg cgg 103          <210> 7 <211> 103 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic   <400> 7 cgcaacatcc gcatgcatct ctagctctac caccttcgca atgttcaggt tgtggaggac 60          atctagctgg ctcacatcta tcaggacatc cagcagctaa tgg 103          <210> 8 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 8 atgttgcgaa gtttgcggag ctcctgaagg agctgcttgt ggattacaag agggtccttg 60          tggagaaggt ctacaatgcg tagttccatt 90          <210> 9 <211> 91 <212> DNA <213> Artificial Sequence          <220> <223> Description of Artificial Sequence: synthetic <400> 9 ggtactccga atggaactac gcattgtaga ccttctccac aaggaccctc ttgtaatcca 60          caagcagctc cttcaggagc tccgcaaact t 91          <210> 10 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic          <400> 10 cggagtacca gcttcagcaa cagtaagacg aagggcccaa gctggtttat gtgtatgcgc 60          gagttcagaa ccagtatgtg gctctgatgc aaatacata 99          <210> 11 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 11 agtttgcgta tgtatttgca tcagagccac atactggttc tgaactcgcg catacacata 60          aaccagcttg ggcccttcgt cttactgttg ctgaagct 98          <210> 12 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic          <400> 12 cgcaaactta tgccaattga gagctgcttc tagacgtagt gaaagactac atagaccgcc 60          tgttatagtc ctgcaacggg gagcctgcgg ccaagg 96          <210> 13 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 13 tcttcctgcc cttggccgca ggctccccgt tgcaggacta taacaggcgg tctatgtagt 60          ctttcactac gtctagaagc agctctcaat tggcata 97          <210> 14 <211> 103 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 14 gcaggaagat cccaacagtt tgcgccataa atataacttt atcgcggacg tggtggagaa 60          gatcgcccct gccctggttc atatcgaatt gtttcgcaag ctt 103          <210> 15 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic          <400> 15 aagcttgcga aacaattcga tatgaaccac ggcaggggcg atcttctcca ccacgtccgc 60          gataaagtta tatttatggc gcaaactgtt ggga 94          <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 16 actatgcaga ttccaagagc tg 22          <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 17 gtctaaagct tgcgaaacaa ttcg 24 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic          <400> 18 aattgtttcg caagcttccg ttttctaaac g 31 <210> 19 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 19 agtctagaat tccatgaagt ccagctcatg cctctgccta tgg 43          <210> 20 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 20 Met Gln Ile Pro Arg Ala Ala Leu Leu Pro Leu Leu Leu Leu Leu Leu   1 5 10 15 Ala Ala Pro Ala Ser Ala Gln Leu Ser Arg Ala Gly Arg Ser Ala Pro              20 25 30 Leu Ala Ala Gly Cys Pro Asp Arg Cys Glu Pro Ala Arg Cys Pro Pro          35 40 45          Gln Pro Glu His Cys Glu Gly Gly Ser Ala Pro Leu Ala Ala Gly      50 55 60 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 21 gatctacgga tcccaagctg gtttatgtgt atgc 34          <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 22 gatctacctg cagtgggtca atttcggcgg gaatc 35          <210> 23 <211> 1185 <212> DNA <213> Homo sapiens <400> 23 atgagaggat cgcatcatca tcatcatcat ggatcccaag ctggtttatg tgtatgcgcg 60          agttcagaac cagtatgtgg ctctgatgca aatacatacg caaacttatg ccaattgaga 120          gctgcttcta gacgtagtga aagactacat agaccgcctg ttatagtcct gcaacgggga 180          gcctgcggcc aagggcagga agatcccaac agtttgcgcc ataaatataa ctttatcgcg 240          gacgtggtgg agaagatcgc ccctgccgtg gttcatatcg aattgtttcg caagcttccg 300          ttttctaaac gagaggtgcc ggtggctagt gggtctgggt ttattgtgtc ggaagatgga 360          ctgatcgtga caaatgccca cgtggtgacc aacaagcacc gggtcaaagt tgagctgaag 420          aacggtgcca cttacgaagc caaaatcaag gatgtggatg agaaagcaga catcgcactc 480          atcaaaattg accaccaggg caagctgcct gtcctgctgc ttggccgctc ctcagagctg 540          cggccgggag agttcgtggt cgccatcgga agcccgtttt cccttcaaaa cacagtcacc 600          accgggatcg tgagcaccac ccagcgaggc ggcaaagagc tggggctccg caactcagac 660          atggactaca tccagaccga cgccatcatc aactatggaa actcgggagg cccgttagta 720          aacctggacg gtgaagtgat tggaattaac actttgaaag tgacagctgg aatctccttt 780          gcaatcccat ctgataagat taaaaagttc ctcacggagt cccatgaccg acaggccaaa 840          ggaaaagcca tcaccaagaa gaagtatatt ggtatccgaa tgatgtcact cacgtccagc 900          aaagccaaag agctgaagga ccggcaccgg gacttcccag acgtgatctc aggagcgtat 960          ataattgaag taattcctga taccccagca gaagctggtg gtctcaagga aaacgacgtc 1020          ataatcagca tcaatggaca gtccgtggtc tccgccaatg atgtcagcga cgtcattaaa 1080          agggaaagca ccctgaacat ggtggtccgc aggggtaatg aagatatcat gatcacagtg 1140          attcccgaag aaattgaccc actgcagcca agcttaatta gctga 1185          <210> 24 <211> 394 <212> PRT <213> Homo sapiens <400> 24 Met Arg Gly Ser His His His His His His Gly Ser Gln Ala Gly Leu   1 5 10 Cys Val Cys Ala Ser Ser Glu Pro Val Cys Gly Ser Asp Ala Asn Thr              20 25 30          Tyr Ala Asn Leu Cys Gln Leu Arg Ala Ala Ser Arg Arg Ser Glu Arg          35 40 45          Leu His Arg Pro Pro Val Ile Val Leu Gln Arg Gly Ala Cys Gly Gln      50 55 60 Gly Gln Glu Asp Pro Asn Ser Leu Arg His Lys Tyr Asn Phe Ile Ala  65 70 75 80 Asp Val Val Glu Lys Ile Ala Pro Ala Val Val His Ile Glu Leu Phe                  85 90 95 Arg Lys Leu Pro Phe Ser Lys Arg Glu Val Pro Val Ala Ser Gly Ser             100 105 110 Gly Phe Ile Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala His Val         115 120 125 Val Thr Asn Lys His Arg Val Lys Val Glu Leu Lys Asn Gly Ala Thr     130 135 140 Tyr Glu Ala Lys Ile Lys Asp Val Asp Glu Lys Ala Asp Ile Ala Leu 145 150 155 160 Ile Lys Ile Asp His Gln Gly Lys Leu Pro Val Leu Leu Leu Gly Arg                 165 170 175   Ser Ser Glu Leu Arg Pro Gly Glu Phe Val Val Ala Ile Gly Ser Pro             180 185 190 Phe Ser Leu Gln Asn Thr Val Thr Thr Gly Ile Val Ser Thr Thr         195 200 205 Arg Gly Gly Lys Glu Leu Gly Leu Arg Asn Ser Asp Met Asp Tyr Ile     210 215 220 Gln Thr Asp Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu Val 225 230 235 240 Asn Leu Asp Gly Glu Val Ile Gly Ile Asn Thr Leu Lys Val Thr Ala                 245 250 Gly Ile Ser Phe Ala Ile Pro Ser Asp Lys Ile Lys Lys Phe Leu Thr             260 265 270 Glu Ser His Asp Arg Gln Ala Lys Gly Lys Ala Ile Thr Lys Lys Lys         275 280 285          Tyr Ile Gly Ile Arg Met Met Ser Leu Thr Ser Ser Lys Ala Lys Glu     290 295 300 Leu Lys Asp Arg His Arg Asp Phe Pro Asp Val Ile Ser Gly Ala Tyr 305 310 315 320          Ile Ile Glu Val Ile Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu Lys                 325 330 335 Glu Asn Asp Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Ser Ala             340 345 350 Asn Asp Val Ser Asp Val Ile Lys Arg Glu Ser Thr Leu Asn Met Val         355 360 365 Val Arg Arg Gly Asn Glu Asp Ile Met Ile Thr Val Ile Pro Glu Glu     370 375 380 Ile Asp Pro Leu Gln Pro Ser Leu Ile Ser 385 390

[Brief description of drawings]

FIG. 1 is a serine protease capable of selectively cleaving IGFBP (
(Named "ASP05") nucleotide sequence cDNA, nucleotide 1-1
443 (SEQ ID NO: 1) and amino acid sequence 1-480 (SEQ ID NO: 2). A
The nucleotides modified to express SP05 are underlined. Arrows indicate signal cleavage sites. The IGFBP-like domain is underlined and the PSTI domain is double-lined. The catalysis domain is boxed and the histidine (H), aspartate (D) and serine (S) residues required for catalysis in the domain are circled.

2A and B show an alignment of the nucleic acid sequence encoding the native protein (top; SEQ ID NO: 3) with the nucleic acid sequence encoding the synthetic ASP05 protease (bottom). The modified nucleotides of the synthetic ASP05 protease are underlined. The amino acid sequences encoded by both sequences are also noted.

FIG. 3 shows the evaluation results of the self-cleaving activity of ASP05. SDS-
Western blot analysis of purified ASP05 subjected to PAGE, detection of anti-ASP05 peptide antibody bound to HRP-conjugated rabbit IgG antibody by visualization with a chemifluorophore. Lane 1: protein purified by flag antibody affinity chromatography (0.5 μg) was subjected to 4-20% SDS-PAGE, Commass
ie blue stained; lanes 2 and 3: same amount of lane 1 as sample 4-20
% SDS-PAGE and blotted with anti-ASP05 peptide antibody. The sample in lane 2 was not incubated at 37 ° C and the sample in lane 3 was gel ST
: Incubated for 15 minutes at 37 ° C before loading with molecular weight standard protein markers.

FIG. 4A shows selective cleavage of IGFBP-5. IGFBP-
1 to -6 were incubated with ASP05 and westernized with each antibody
Analyzed by blot. “−” Is control without ASP05, “+” is AS
Indicates that there is P05.

FIG. 4B shows selective cleavage of IGFBP-5. IGFBP-
1 to -6 were incubated with ASP05 and analyzed by Western ligand blot with ¹²⁵ 1-IGF-I. BP1-6 represents IGFBP1-6, respectively, "-" is IGFBP1-6 without ASP05 and "+" is ASP0.
5 shows IGFBP1-6 with 5 present.

FIG. 5 shows the effect of protease inhibitors on the degradation of IGFBP-5 by ASP05. ASP05 was incubated with protease inhibitors for 1 hour before the addition of IGFBP-5 and the products analyzed by Western blot with the IGFBP-5 antibody. Lane 1: Without ASP05; Lane 2: With ASP05; Lane 3: Aprotinin, 1.5 μM; Lane 4: Antipain, 370 μM; Lane 5: 3,4-DCI, 1 mM; Lane 6: E64.
, 140 μM; Lane 7: leupeptin, 5 μM; Lane 8: pepstatin A, 5
μM; Lane 9: PMSF, 5 mM; Lane 10: TLCK, 675 μM; Lane 1
1: TPCK, 1.4 mM; Lane 12: EDTA, 10 mM.

FIG. 6 shows the C05 terminal domain of ASP05 and histidine-tagged nucleotide sequences useful for purification, as shown in the Examples.

FIG. 7 shows the amino acid sequence of ASP05 C-terminal domain and histidine tag.

FIG. 8 shows selective cleavage of IGFBP-5 by the C-terminal fragment of ASP05. IGFBP-1 to -6 were each incubated with the fragments for 2 hours at 37 ° C and analyzed by Western blot using the antibody shown at the top of each blot. "-" Indicates samples that were not treated with fragments, "+" indicates samples that were treated with fragments.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12N 9/64 Z 4H045 5/10 C12Q 1/37 9/64 G01N 33/15 Z C12Q 1 / 37 33/50 Z G01N 33/15 33/53 D 33/50 C12N 15/00 ZNA 33/53 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Jinjao How United States 92130 Sterling Grove Rain 5075 F term, San Diego, CA (reference) 2G045 AA28 AA40 BB20 CB01 DA20 DA36 DA77 FB02 4B024 AA11 BA14 BA80 CA04 DA02 DA06 EA02 GA11 HA11 HA15 4B050 CC03 DD07 LL01 LL08 QB3QAQ QQ36 QR66 QS33 QX02 QX07 4B065 AA01X AA26X AA57X AA72X AA90X AA90Y AB01 BA02 CA33 CA46 4H045 AA10 AA11 AA30 BA10 CA40 DA76 DA89 EA50 FA72 FA74 GA10 GA26

Claims (33)

[Claims] 1. Nucleotide 481-1 of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1)
A recombinant comprising a nucleic acid which hybridizes to 113 or its complement under high stringency conditions and which has at least a 10% reduction in GC content compared to the wild-type ASP05 nucleic acid sequence of FIG. 2 (SEQ ID NO: 3). Nucleic acid. 2. The recombinant nucleic acid of claim 1, which hybridizes to the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 3) under high stringency conditions. 3. The amino acid sequence 161-3 of the amino acid sequence of FIG. 1 (SEQ ID NO: 2).
A recombinant nucleic acid comprising a nucleic acid sequence encoding 71, of which the nucleic acid sequence has at least a 10% reduction in GC content compared to the native nucleic acid sequence of FIG. 4. The recombinant nucleic acid of claim 3, which encodes the ASP05 protein of FIG. 1 (SEQ ID NO: 2). 5. The recombinant nucleic acid of claim 1, comprising the nucleotide sequence of SEQ ID NO: 1 or its complement. 6. Nucleotides 481-11 of the nucleotide sequence of SEQ ID NO: 1
The recombinant nucleic acid of claim 1, comprising 13 or its complement. 7. The recombinant nucleic acid of claim 1, consisting essentially of a nucleic acid that hybridizes to nucleotides 481-1113 of FIG. 1 (SEQ ID NO: 1) under high stringency conditions. 8. Amino acid residue 161-3 of the amino acid sequence of FIG. 1 (SEQ ID NO: 2)
The recombinant nucleic acid of claim 1, consisting essentially of a nucleic acid encoding 71. 9. Amino acid residue 30-10 of the amino acid sequence of FIG. 1 (SEQ ID NO: 2)
The recombinant nucleic acid of claim 1, consisting essentially of a nucleic acid encoding 4. 10. An expression vector comprising the nucleic acid of claim 1. 11. An expression vector comprising the nucleic acid of claim 3. 12. A host cell comprising the nucleic acid of claim 1. 13. A host cell containing the nucleic acid of claim 3. 14. A host cell containing the expression vector of claim 10. 15. A host cell containing the expression vector of claim 11. 16. A method for producing ASP05 protein, which comprises: (a) culturing the host cell of claim 12 under conditions suitable for expression of said ASP05 protein; (b) obtaining said ASP05 protein from a cell culture medium. A method comprising: 17. Amino acids 161-37 of the amino acid sequence of FIG. 1 (SEQ ID NO: 2)
An enzymatically active ASP05 protein containing 1. 18. A method comprising amino acids 1-481 of FIG. 1 (SEQ ID NO: 2).
7. Enzymatically active ASP05 protein of 7. 19. Nucleotide 481-of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1)
Encoded by a nucleic acid which hybridizes to 1113 or its complement under high stringency conditions and which has at least a 10% reduction in GC content relative to the sequence of the wild-type ASP05 nucleic acid of Figure 3 (SEQ ID NO: 3). Recombinant ASP05 protein. 20. The recombinant protein of claim 19, encoded by a nucleic acid that hybridizes to the nucleic acid sequence shown in Figure 1 (SEQ ID NO: 3) under conditions of high stringency. 21. The recombinant protein of claim 19, which comprises the amino acid sequence of SEQ ID NO: 2. 22. Amino acids 161- of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2)
21. The recombinant protein of claim 19, consisting essentially of 371. 23. A chimeric molecule comprising the catalytic domain of ASP05 protein fused to a heterologous amino acid sequence. 24. An antibody that specifically binds to the ASP05 protein. 25. A method for the selective cleavage of insulin-like growth factor binding protein (IGFBP) comprising about amino acid 161- of the amino acid sequence of Figure 1 (SEQ ID NO: 2).
A method comprising combining an enzymatically active ASP05 protein containing 371 with IGFBPs. 26. The method of claim 25, further comprising determining that the insulin-like growth factor binding protein is cleaved. 27. A method for screening a bioactive substance capable of modulating the activity of ASP05 protein, which comprises: (a) combining a candidate bioactive substance with a protein containing a catalytic domain of ASP05; (b) Determining whether the substance modulates the activity of the ASP05 protein. 28. The method of claim 27, wherein the ASP05 protein comprises a full length ASP05 protein. 29. A method for screening a bioactive substance capable of modulating serine protease activity capable of selectively cleaving IGFBP, comprising the steps of:
(a) a candidate bioactive agent is added to a cell containing a recombinant nucleic acid encoding an ASP05 protein, where the ASP05 protein is expressed; (b) the agent generally lacks the activity of the ASP05 protein and lacks the expressed ASP05 protein. Determining whether to regulate relative to cells. 30. Adding a library of candidate bioactive substances to a plurality of cells,
30. The method of claim 29. 31. A method of modulating the cleavage of IGFBPs in a cell comprising administering to said cell an exogenous compound that modulates the biological activity of ASP05 protein. 32. The method of claim 31, wherein the modulation is a reduction or elimination of biological activity of the ASP05 protein. 33. A method of diagnosing an IGF-related condition in a first individual, comprising: (a) measuring the activity of ASP05 protein in the first tissue of the first individual; (b) the first ASP05 protein. The activity is compared to the activity of the ASP05 protein in the non-infected tissue of the second non-infected individual or the first individual; (wherein the activity of the ASP05 protein from the first individual is the second individual or the second individual An alteration compared to the activity of the ASP05 protein in the tissue,
The first individual is at risk of an IGF-related condition).